CN101816629B - Dual target liposome and preparation method and application thereof - Google Patents
Dual target liposome and preparation method and application thereof Download PDFInfo
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- CN101816629B CN101816629B CN200910078359XA CN200910078359A CN101816629B CN 101816629 B CN101816629 B CN 101816629B CN 200910078359X A CN200910078359X A CN 200910078359XA CN 200910078359 A CN200910078359 A CN 200910078359A CN 101816629 B CN101816629 B CN 101816629B
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Abstract
The invention discloses a dual target liposome and a preparation method and application thereof. The target liposome provided by the invention consists of liposome and modifiers on the surface of the liposome, wherein the modifiers on the surface of the liposome comprise p-aminophenyl-alpha-D-manno-pyranoside and transferrin. The invention also discloses a medicament-loaded liposome, which is obtained by wrapping daunorubicin by using the target liposome. The obtained target liposome has good capability of crossing the blood brain barrier, targets brain glioma, and can be used as a medicament carrier. The medicament-loaded liposome can target the medicament to a brain glioma site after crossing the blood brain barrier so as to greatly increase the concentration of the medicament at a tumor site and improve the effect of chemotherapy. The target liposome provides a new measure for brain glioma chemotherapy, contributes to the research of non-invasive therapy of the brain glioma, and has important theoretical meaning and clinical meaning.
Description
Technical field
The present invention relates to a kind of dual target liposome and its production and application.
Background technology
Primary brain tumor has become one of ten big causes of the death of cancer.100,000 philtrum just has 5 to 10 people to suffer from malignant glioma [Wrensch M, Minn Y, Chew T, Bondy M, Berger MS.Epidemiology of primarybrain tumors:current concepts and review of the literature.Neuro Oncol.2002 Oct; 4 (4): 278-99.Behin A, Hoang-Xuan K, Carpentier AF, Delattre JY.Primary brain tumours in adults.Lancet.2003 Jan 25; 361 (9354): 323-31.].The invasive brain glioblastoma cell can soak into and destroy normal cerebral tissue's structure apace, thereby can not be by the complete tumor resection of operation.Although use excision, radiation and chemotherapy therapeutic alliance, malignant glioma patient's mean survival time is no more than 1 year [Astner ST, Pihusch R, Nieder C, Rachinger W, LohnerH, Tonn JC, Molls M, Grosu AL.Extensive local and systemic therapy inextraneural metastasized glioblastoma multiforme.Anticancer Res.2006Nov-Dec; 26 (6C): 4917-20.Nieder C, Grosu AL, Astner S, Molls M.Treatmentof unresectable glioblastoma multiforme.Anticancer Res.2005Nov-Dec; 25 (6C): 4605-10.].The main cause of embolic chemotherapy failure is that anticarcinogen passes through can not arrive brain essence behind the intravenously administrable.Blood brain barrier is made up of inner hypophloeodal single-layer cell, astrocyte and perithelial cells, and it separates brain essence and blood, and stops medicine to permeate to the central nervous system.Blood brain barrier stops picked-up [the Pardridge WM.BBB-Genomics:creating new openings forbrain-drug targeting.Drug Discov Today.2001 Apr 1 of the small-molecule drug more than macromole and 98%; 6 (8): 381-383].The obstacle of another of chemotherapy makes chemotherapeutic keep high concentration and not diffuse into normal structure [Minchinton AI, Tannock IF.Drug penetration in solid tumours.Nat Rev Cancer.2006 Aug at tumor locus exactly; 6 (8): 583-92.Ohlfest JR, Demorest ZL, Motooka Y, VengcoI, Oh S, Chen E, Scappaticci FA, Saplis RJ, Ekker SC, Low WC, Freese AB, LargaespadaDA.Combinatorial antiangiogenic gene therapy by nonviral gene transferusing the sleeping beauty transposon causes tumor regression and improvessurvival in mice bearing intracranial human glioblastoma.Mol Ther.2005Nov; 12 (5): 778-88].Therefore, concerning researcher, study a kind of new drug-supplying system and can make medicine see through blood brain barrier to become a urgent task by the targeting cerebral glioma again.
Cerebral glioma is divided into following a few class according to the type of cell: (ventricles of the brain) ependymoma (ependymocyte), glioblastoma (astrocyte), oligodendroglia (cell) tumor (oligodendrocyte), mixed glioma (dissimilar neuroglia).Low cerebral glioma well differentiated (cell can not be replied than initial condition) and be benign can well be diagnosed.The severe glioma is not broken up (cell reply than initial condition) and is virulent, and very difficult quilt is well diagnosed.According to the hierarchy system of WHO to glioma, the diagnosis of 4 grades of gliomas is the most difficult, and patient's mean survival time only is 12 months.In general, almost not having patient to survive can be above 3 year.Almost can not cure glioblastoma (glioma) by operation and/or radiotherapy.Mainly be that following two big reasons have limited the effect that chemotherapy can play: antineoplastic agent can not see through blood brain barrier and very poor to the penetrating power of tumor tissues after seeing through blood brain barrier.Can keep a major challenge that high treatment concentration has become chemotherapy at cerebral tumor position.
Summary of the invention
The purpose of this invention is to provide a kind of dual target liposome and its production and application.
Dual target liposome provided by the invention, form by liposome and its surperficial trim, it is characterized in that: the trim of described surface of liposome is p-aminophenyl-α-D-manno-pyranoside (p-nitrophenyl-α-D-mannopyranose glycosides, MAN) and transferrins (transferrin, TF).
Described p-aminophenyl-α-D-manno-pyranoside can be connected on the amino of described liposome; Described transferrins can be connected on the carboxyl of described liposome.The amino of described liposome can be by DSPE-Polyethylene Glycol-NH
2(DSPE-PEG-NH
2) provide; The carboxyl of described liposome can be provided by DSPE-Polyethylene Glycol-COOH (DSPE-PEG-COOH).
Specifically: during described liposome preparation, can in raw material, add DSPE-Polyethylene Glycol-NH
2And DSPE-Polyethylene Glycol-COOH, DSPE-Polyethylene Glycol-NH
2And DSPE-Polyethylene Glycol-COOH is attached to surface of liposome, is provided for connecting the amino and the carboxyl of p-aminophenyl-α-D-manno-pyranoside and transferrins.
The concrete available lecithin of described liposome, cholesterol, Polyethylene Glycol-DSPE (DSPE-PEG2000), DSPE-Polyethylene Glycol-NH
2(DSPE-PEG-NH
2) and DSPE-Polyethylene Glycol-COOH (DSPE-PEG-COOH) obtain as feedstock production.Lecithin, cholesterol, Polyethylene Glycol-DSPE, DSPE-Polyethylene Glycol-NH
2, DSPE-Polyethylene Glycol-COOH mass ratio can be (104.5-105.5): (44.5-45.5): (28-28.5): (3.4-3.6): (3.4-3.6).Lecithin, cholesterol, Polyethylene Glycol-DSPE, DSPE-Polyethylene Glycol-NH
2, DSPE-Polyethylene Glycol-COOH concrete mol ratio can be 52: 43: 4: 0.5: 0.5.
In the described target liposomes, the mass ratio of described transferrins and described liposome is (4-5): 15, and the mass ratio of described p-aminophenyl-α-D-manno-pyranoside and described liposome is (1.5-2): 15.
In the described target liposomes, described transferrins can be 300-400 μ g transferrins/μ mol phospholipid with the ratio of lecithin.In the described target liposomes, described p-aminophenyl-α-D-manno-pyranoside and described DSPE-Polyethylene Glycol-NH
2Mol ratio can be (3-10): 1.
Described Polyethylene Glycol-DSPE specifically can be DSPE-PEG2000.Described DSPE-Polyethylene Glycol-NH
2Specifically can be DSPE-PEG2000-NH
2Described DSPE-Polyethylene Glycol-COOH specifically can be DSPE-PEG2000-COOH.
The present invention also provides a kind of preparation method of target liposomes, comprises the steps:
1) with lecithin, cholesterol, Polyethylene Glycol-DSPE, DSPE-Polyethylene Glycol-NH
2, DSPE-Polyethylene Glycol-COOH has the liposome of amino and carboxyl for the feedstock production surface;
2) p-aminophenyl-α-D-manno-pyranoside is connected with the amino of liposome; Transferrins is connected with the carboxyl of liposome;
Obtain target liposomes.
In the described step 1), the method for preparing liposome specifically can be ammonium sulphate gradient.
Described step 2) in, available glutaraldehyde connects agent, with the amino of p-aminophenyl-α-D-manno-pyranoside and liposome (by DSPE-Polyethylene Glycol-NH
2Provide) amino connect.
Described step 2) in, available EDCI connects agent, and transferrins is connected with the carboxyl (being provided by DSPE-Polyethylene Glycol-COOH) of liposome.
In the described step 1), lecithin, cholesterol, Polyethylene Glycol-DSPE, DSPE-Polyethylene Glycol-NH
2, DSPE-Polyethylene Glycol-COOH mass ratio can be (104.5-105.5): (44.5-45.5): (28-28.5): (3.4-3.6): (3.4-3.6).Lecithin, cholesterol, Polyethylene Glycol-DSPE, DSPE-Polyethylene Glycol-NH
2, DSPE-Polyethylene Glycol-COOH concrete mol ratio can be 52: 43: 4: 0.5: 0.5.
Described Polyethylene Glycol-DSPE specifically can be DSPE-PEG2000.Described DSPE-Polyethylene Glycol-NH
2Specifically can be DSPE-PEG2000-NH
2Described DSPE-Polyethylene Glycol-COOH specifically can be DSPE-PEG2000-COOH.
Described target liposomes can be applicable to prepare pharmaceutical carrier.
The present invention also protects a kind of pharmaceutical carrier, and its active component is described target liposomes.
The present invention also protects a kind of drug-loaded liposome, obtains with described target liposomes bag medicine carrying thing.
Described medicine specifically can be daunorubicin.
The present invention also protects a kind of medicine for the treatment of the cerebral tumor, and its active component is the drug-loaded liposome that bag carries daunorubicin.
The described cerebral tumor can be cerebral glioma, specifically can be the C6 glioma.
The drug-loaded liposome that described bag carries daunorubicin can be applicable to prepare the medicine for the treatment of the cerebral tumor.
The described cerebral tumor can be cerebral glioma, specifically can be the C6 glioma.
Liposome can be made the liposome transmembrane transport pass through blood brain barrier by the GLUT1 on the blood brain barrier (glucose transporter 1) identification after MAN modifies; Liposome can initiatively be discerned brain glioblastoma cell by TfR (TfR) after striding blood brain barrier after TF modifies, thereby improve the drug level of tumor locus, increases the interior transmission efficiency of cell of cancer therapy drug; Therefore bring into play the dual-target effect.Mechanism may be relevant with following two aspects: have TfR to express on the blood brain barrier, have GLUT1 to express on the tumor cell surface; The most cerebral tumor all is a solid tumor, and the no vasculature part of solid tumor becomes the major obstacle of effective control tumor growth.The daunorubicin liposome preparation that mannose derivative and transferrins are modified and the outline figure of secondary targeting thereof see Fig. 1: glucose transporter (GLUT1, the one-level targeting) blood brain barrier, the mediated targeted C6 glioma cell of transferrins (secondary targeting) are then striden in mediation.
The particle diameter of liposome can significance the interior medicine dynamics character, toxicity and the anti-tumor activity thereof that influence liposome.With regard to particle diameter itself, the liposome of mean diameter about 100nm has the longest plasma half-life.The bag that the present invention obtains carries the mean diameter of liposome of daunorubicin near 120nm, polydispersity coefficient is about 0.13-0.25, it is the dispersion that size is suitable, be evenly distributed, circulation time in blood is long, make liposome vesicle have more opportunity pass through on the tumor locus new capillary vessel endothelial tissue " hole " and to the tumor tissues enrichment, thereby realize better anti-tumor activity.Zeta potential is an one of parameter of weighing the physical stability of liposome or lipid nanoparticle monodisperse system.The zeta current potential that the bag that the present invention obtains carries the liposome of daunorubicin shows slightly negativity, illustrates to have an amount of electrostatic repulsion forces between the liposome vesicle, and the liposome bin stability is better, is difficult for taking place coagulation.The daunorubicin liposome that the present invention obtains has the obvious suppression effect to the C6 glioma cell.
The present invention is with MAN and surface of liposome DSPE-PEG-NH
2Connecting is increased strides the blood brain barrier ability, and transferrins is connected with the targeting cerebral glioma with the DSPE-PEG-COOH of surface of liposome, has obtained dual target liposome.The antineoplastic agent daunorubicin is encapsulated in the liposome, obtained a kind of brand-new anti-malignant glioma dual target liposome, can make medicine after striding across blood brain barrier, be targeted to the cerebral glioma position, thereby the drug level of tumor locus increases greatly, improve the effect of chemotherapy.The present invention provides a new countermeasure for the cerebral glioma chemotherapy, and helps the research to the noninvasive laser therapy of cerebral glioma, has important significance for theories and clinical meaning.
Description of drawings
Fig. 1 is dual target liposome preparation and dual-target effect outline figure.
Fig. 2 is the BSA standard curve.
Fig. 3 is the form of drug-loaded liposome A1 and drug-loaded liposome C1.
Fig. 4 is the HRP standard curve.
Fig. 5 is the penetrating rate of HRP different time.
Fig. 6 is the external blood brain barrier transhipment rate of striding of daunorubicin in measuring through the blood brain barrier ability.
Fig. 7 is the external blood brain barrier transhipment rate of striding of daunorubicin in the MAN competitive assay.
After Fig. 8 was 48 hours, drug-loaded liposome was to the antiproliferative effect of C6 glioma cell.
Fig. 9 is for using different drug-loaded liposomes, the situation of C6 glioma cell picked-up daunorubicin.
Figure 10 is in the experiment of TF receptor competition, and in the different disposal, the C6 glioma cell absorbs the situation of daunorubicin.
Figure 11 detects cell to the picked-up of different drug-loaded liposomes and in intracellular distribution for the laser co-focusing method.
Figure 12 is the suppression ratio to the C6 glioma after striding external blood brain barrier of different drug-loaded liposomes in the BMVEC/C6 co-culture experiments.
Figure 13 is tumor sphere volume rate in the different disposal of glioma cell ball experiment.
Figure 14 is the meningeal-colloid cancer volume suppression ratio of the C6 glioma rat of different drug-loaded liposomes processing.
Figure 15 is the kaplan-Meier survival curve of the C6 glioma rat of different drug-loaded liposomes processing.
The specific embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is conventional method.Used test material among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
The preparation of embodiment 1, liposome
(polyethyleneglycol-distearoylphosphatidylethanolamine is PEG2000-DSPE) available from NOF Corp (Japanese NOF company) for lecithin (EPC) and Polyethylene Glycol-DSPE; Cholesterol (cholesterol) is available from Haidian District, Beijing City microbiological culture media products factory (Beijing bispin microbiological culture media products factory); DSPE-PEG2000-COOH, DSPE-PEG2000-NH
2, p-aminophenyl-α-D-manno-pyranoside (MAN), transferrins (molecular weight is 80,000 roads) (holo-transferrin, TF) available from Sigma-Aldrich Corporation, Beijing local agent, China; Shephedex G-100, trinitrobenzene sulphonic acid (TNBS) be available from Sigma-Aldrich company, Beijing local agent, China;
1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDCI) is available from Shanghai Medpep Co., Ltd., China; N-hydroxysuccinimide (NHS) is available from BeijingSansheng Tengda Technology Co., Ltd., China; The BCA test kit is available from PierceCorporation, Beijing local agent, China; Poly-carbon ester filter (aperture 400,200nm) available from Millipore (Bedford, MA, USA); Daunomycin hydrochloride is available from the many chemical reagents corporations in Nanjing celestial worthy pool.HP1100 type high performance liquid chromatograph (Agilent, the U.S.).Nano Series Zen 4003 ZetaSizer (Malvern instruments, Britain).SPI3800N series SPA-400 type atomic force microscope (Japan).
One, the preparation of blank liposome
1, the preparation of blank liposome
With lecithin, cholesterol, DSPE-PEG2000, DSPE-PEG2000-COOH, DSPE-PEG2000-NH
252: 43: 4 in molar ratio: be dissolved in chloroform at 0.5: 0.5; Rotary evaporation is removed chloroform, and 40 ℃, 40rpm revolve to steam and form thin film in eggplant-shape bottle; Add 10ml 250mM ammonium sulfate; first ultrasonic 5min in water-bath transfers to then that further ultrasonic in JY92-2D type ultrasonic cell disruptor in the serum bottle (the ultrasonic working time is 10s, and the intermittent time is 10s; the omnidistance time is 10min, and the protection temperature is 35 ℃).Obtain liposome A.
2, the preparation of the liposome of blank MAN modification
Connect agent by glutaraldehyde, the MAN chemistry is connected to liposome A goes up DSPE-PEG2000-NH
2Amino on, concrete steps are: 8ml liposome A (7.5mg/ml) is mixed with 2mg MAN; Slowly add glutaraldehyde then, making the glutaraldehyde final concentration is 15mM; Hatch 5min under the room temperature; MAN and the glutaraldehyde that does not connect removed in dialysis in PBS (pH7.4).Obtain liposome B.
3, the preparation of the liposome of blank MAN and TF modification
Connect agent by EDCI, the amino of transferrins is connected with the carboxyl that liposome B goes up DSPE-PEG-COOH, concrete steps are: get 2ml liposome B (7.5mg/ml), add 1.0mg EDCI and 1.44mg NHS (DSPE-PEG-COOH: EDCI: NHS=0.063: 2.5: 6.3; Mol ratio), adds the 5mg transferrins behind the mixing, slowly stir and incubated at room 3h.Cross Shephedex G-100 post (with the PBS balance of pH7.4), remove free transferrins, obtain liposome C.
4, the preparation of the liposome of blank TF modification
Connect agent by EDCI, the amino of transferrins is connected with the carboxyl that liposome A goes up DSPE-PEG-COOH, step is with step 3.Obtain liposome D.
Two, the mensuration of the preparation of drug-loaded liposome and envelop rate
The quality of medicine fat ratio=daunomycin hydrochloride: the quality sum of lecithin and cholesterol in the liposome.
1, the preparation of drug-loaded liposome
The medicine fat of daunomycin hydrochloride with 1: 12 is mixed than with liposome A, and jolting 20min in 40 ℃ of water-baths dialyses then, removes free daunomycin hydrochloride, obtains drug-loaded liposome A1.
The medicine fat of daunomycin hydrochloride with 1: 12 is mixed than with liposome B, and jolting 20min in 40 ℃ of water-baths dialyses then, removes free daunomycin hydrochloride, obtains drug-loaded liposome B1.
The medicine fat of daunomycin hydrochloride with 1: 12 is mixed than with liposome C, and jolting 20min in 40 ℃ of water-baths dialyses then, removes free daunomycin hydrochloride, obtains drug-loaded liposome C1.
The medicine fat of daunomycin hydrochloride with 1: 12 is mixed than with liposome D, and jolting 20min in 40 ℃ of water-baths dialyses then, removes free daunomycin hydrochloride, obtains drug-loaded liposome D1.
2, the mensuration of envelop rate
Drug-loaded liposome is placed the 10mL volumetric flask, with the methanol destruction of 10 times of amounts, and use the mobile phase standardize solution, the HPLC method is measured daunorubicin content.
The content of daunorubicin in envelop rate (%)=drug-loaded liposome/(content of daunorubicin in the drug-loaded liposome+free daunorubicin content) * 100%.
The envelop rate of the different drug-loaded liposomes of table 1
In prepared drug-loaded liposome A1, B1, C1, D1, the envelop rate of daunorubicin is all greater than 90%.
The performance measurement of embodiment 2, liposome
One, transferrins modification rate is measured (BCA method)
1, the preparation of standard curve
Adopt BCA test kit (Pierce Corporation, Beijing local agent, China), with BSA is that standard is measured, BSA with PBS configuration 0,25,125,250,500,750 and 1000 μ g/ml, hatch 30min under 37 ℃ behind the adding working solution, be cooled to room temperature and measure light absorption value in 540nm.Standard curve is seen Fig. 2.
2, modification rate
Press the liposome C of BCA test kit description mensuration embodiment 1 preparation and the transferrin content among the liposome D, and calculating modification rate (μ g TF/ μ mol phospholipid) (modification rate=be connected to the transferrins quality on the liposome: the amount of substance of lecithin), the results are shown in Table 2.
Transferrin content in table 2 liposome and transferrins modification rate (n=3)
Two, MAN modification rate is measured
DSPE-PEG-NH on the liposome
2Amino and trinitrobenzene sulfuric acid reaction, detect respectively on liposome A, the liposome B of embodiment 1 preparation and the liposome C can with the amino quantity of trinitrobenzene sulfuric acid reaction.The MAN modification rate of liposome B is: (on the 1-liposome B can with the amino quantity/liposome A of trinitrobenzene sulfuric acid reaction on can with the amino quantity of trinitrobenzene sulfuric acid reaction) * 100%.The MAN modification rate of liposome C is: (on the 1-liposome C can with the amino quantity/liposome A of trinitrobenzene sulfuric acid reaction on can with the amino quantity of trinitrobenzene sulfuric acid reaction) * 100%.
On the liposome can be: in the 1ml liposome solutions, add 1ml 4%NaHCO with the detection method of the amino quantity of trinitrobenzene sulfuric acid reaction
3With 1ml 10% sodium lauryl sulphate (sodium dodecyl sulphate, SDS).Mix back 30 ℃ and react 20min down, and then adding 1ml 0.1%TNBS solution, react 2h down at 40 ℃ again, add 0.5ml 1M HCl cessation reaction at last, 320nm measures absorbance (Mitra M down, Mandal AK, Chatterjee TK, Das N.Targeting of mannosylated liposome incorporated benzylderivative of Penicillium nigricans derived compound MT81 toreticuloendothelial systems for the treatment of visceral leishmaniasis.J Drug Target.2005 Jun; 13 (5): 285-93).
The MAN modification rate (n=3) of table 3 liposome B and liposome C
Three, particle diameter and potential measurement
(Malvern instruments, Ltd UK) measure drug-loaded liposome A1, the B1 of embodiment 1 preparation, particle diameter, polydispersity coefficient and the Zeta potential of C1, D1 to use Nano Series Zen 4003 Zeta Sizer.
(polydispersity index, PDI) measurement result with zeta current potential (zeta potential) sees Table 4 for the particle diameter (particle size) of different drug-loaded liposomes, polydispersity coefficient.
The particle diameter of the different drug-loaded liposomes of table 4, polydispersity coefficient and zeta current potential (n=3)
The liposome particle size distribution is even, and particle diameter is about 120nm.Each drug-loaded liposome is all slightly electronegative, illustrates to have an amount of electrostatic repulsion forces between the liposome vesicle, and bin stability is better, is difficult for taking place coagulation.
Five, atomic force microscope (AFM)
Adopt atomic force microscope that drug-loaded liposome A1 and the drug-loaded liposome C1 that embodiment 1 prepares carried out morphological observation, method is as follows: using deionized water to be diluted to daunorubicin concentration is 100 μ g/mL, with 0.2 μ m filtering with microporous membrane, getting 10 μ L liposome solutions is applied on the silicon chip, drying at room temperature is measured with atomic force microscope.The form of drug-loaded liposome A1 and drug-loaded liposome C1 is seen Fig. 3, among Fig. 3, and A: drug-loaded liposome A1; B: drug-loaded liposome C1.The result shows: medicine liposome A1 surface is smooth shape, the visible little spherical structure in drug-loaded liposome C1 surface.
Mus C6 glioma cell line is available from medicine institute of the Chinese Academy of Medical Sciences; Ham ' s F10 culture medium is acted on behalf of Beijing available from GIBCO/BRL company (Germany) Beijing and is stepped into Science and Technology Ltd.; Hyclone (FBS) is available from sino-america joint-venture Lanzhou people's marine growth Engineering Co., Ltd; Horse serum is available from Beijing Baeyer enlightening bio tech ltd; SRB acts on behalf of Baeyer enlightening bio tech ltd available from Sigma Beijing; (Horseradishperoxidase, HRP molecular weight are 40000 to horseradish peroxidase, enzymatic activity＞250Umg
-1), the Mus brain endothelial cell (brainmicrovascular endothelial cells, BMVEC) and culture fluid of endothelial cell (cndothclialcell culture medium is ECCM) all available from clinical research institute of China-Japan Friendship Hospital; Microplate reader (BIO-RADModel 680, Chinese Shanghai Bio-Rad laboratory equlpment company), and the cell inserter (Corning, USA); Resistance determinator, (EVOMk, Word Precision Instruments, USA).Adopt the drug-loaded liposome of embodiment 1 preparation to carry out following test.
One, strides blood brain barrier research
1, the cultivation of BMVEC cell
(it consists of DMEM to the Mus brain endothelial cell at Mus brain endothelial cell culture fluid, 20% hyclone, the 100U/ml penicillin, 100 μ g/ml streptomycins, 2mmol/L 1-glutamine, 100 μ g/ml endothelial cell growth factor (ECGF) (ECGF), 40U/ml heparin) cultivation of going down to posterity in, condition of culture is 37 ℃, 5% carbon dioxide.
2, external blood brain barrier is set up
Cell inserter (Corning, NY, USA; 0.4 μ m aperture, 12mm diameter, 1.12cm
2Surface area) with behind 2% the gelatin bag quilt, plants 37,500 Mus brain endothelial cells in each cell inserter, cultivated 5 days, culture fluid of replacing in per two days.
3, external blood brain barrier model evaluation
(1) the blood brain barrier external model is striden the mensuration of membrane resistance
Judge the tightness degree that BBB forms by the resistance of measuring monolayer blood brain barrier (BBB).Resistance is higher than 250 Ω cm
2Can be used for experiment.The membrane resistance of striding of blood brain barrier external model is 276～282 Ω cm.
(2) foundation of HRP content measuring standard curve
Compound concentration is 100 μ gml
-1HRP solution, it is 12.5,6.25,3.125,1.56 and 0.78ngml that doubling dilution obtains concentration
-1The standard serial solution of HRP, respectively get 50 μ l and add 96 orifice plates, add 50 μ l colour developing liquid A and B (purchasing clinical research institute) successively in China-Japan Friendship Hospital, add 2M H behind the colour developing 10min
2SO
4Cessation reaction in the absorbance of 450nm working sample, is set up the standard curve (see figure 4) of HRP assay.
(3) the BBB permeability is measured
After blood brain barrier forms, add the culture medium 500 μ l that contain 500ngHRP in the upper storage reservoir of cell inserter, the culture medium that adds 1000 μ l in the lower storage reservoir, it is equal to keep the inside and outside pond of cell inserter liquid level, in 0,1.0,2.0,4.0,6.0,8.0 with get the lower storage reservoir liquid of 50 μ l during 24.0h, measure the amount that sees through blood brain barrier HRP, the sampling back replenishes isopyknic blank culture medium, to keep inside and outside level balance, calculate the penetrating rate of HRP according to following formula.
Penetrating rate=C
T, lower storage reservoir/ C
0, upper storage reservoir
C
T, lower storage reservoir: the concentration of HRP in the t moment lower storage reservoir;
C
0, upper storage reservoir: the concentration of HRP in the upper storage reservoir during experiment beginning.
The penetrating rate of HRP different time is seen Fig. 5.
4, each preparation sees through the mensuration of blood brain barrier ability
On 18 cell inserters, inoculate the Mus brain endothelial cell respectively, when the resistance measurement demonstration all forms complete tight connection, be divided into six groups (every group of 3 cell inserters), the serum-free DMEM culture medium that all adds 500 μ l in every group the upper storage reservoir, the serum-free DMEM culture medium of adding 1000 μ l in the lower storage reservoir.First group upper storage reservoir culture medium is done blank, contain the free daunorubicin of 5 μ g in second group the upper storage reservoir, contain drug-loaded liposome B1 (containing 5 μ g daunorubicins) in the 3rd group the upper storage reservoir, contain drug-loaded liposome D1 (containing 5 μ g daunorubicins) in the 4th group the upper storage reservoir, contain drug-loaded liposome C1 (containing 5 μ g daunorubicins) in the 5th group the upper storage reservoir, contain drug-loaded liposome A1 (containing 5 μ g daunorubicins) in the 6th group the upper storage reservoir.The cell inserter places on 12 orifice plates, in incubator, cultivate, in 0,2,4,8 and 24h take out solution 0.5ml in the hole, mend the 0.5ml fresh medium again, daunorubicin content in the HPLC working sample calculates daunorubicin according to following formula and strides external blood brain barrier transhipment rate.
C
T, lower storage reservoir: the concentration of daunorubicin in the t moment lower storage reservoir;
C
0, upper storage reservoir: the concentration of daunorubicin in the upper storage reservoir during experiment beginning.
Fig. 6 strides external blood brain barrier transhipment rate for daunorubicin.It is 37.18 times of free daunorubicin that drug-loaded liposome C1 strides the BBB ability, 1.50 times of drug-loaded liposome B1,2.76 times of drug-loaded liposome D1,2.94 times of drug-loaded liposome A1.The result shows, daunorubicin liposome is after modifying through MAN and TF, and that has improved daunorubicin greatly strides the blood brain barrier turn-over capacity.
5, MAN competitive assay
On 12 cell inserters, inoculate the Mus brain endothelial cell respectively, when the resistance measurement demonstration all forms complete tight connection, be divided into four groups (every group of 3 cell inserters), the serum-free DMEM culture medium that all adds 500 μ l in every group the upper storage reservoir, the serum-free DMEM culture medium of adding 1000 μ l in the lower storage reservoir.Add 200 μ g MAN in the upper storage reservoir of the 3rd group and the 4th group, with saturated GLUT1 receptor.MAN added after 30 minutes, added drug-loaded liposome B1 (containing 5 μ g daunorubicins) respectively at first group and the 3rd group, added drug-loaded liposome C1 (containing 5 μ g daunorubicins) respectively at second group and the 4th group.The cell inserter places on 12 orifice plates, in incubator, cultivate, 0,2,4,8 with from lower storage reservoir, get 500 μ l samples during 24h, mend fresh medium 500 μ l after the sampling immediately, the content of daunorubicin in the HPLC method working sample calculates daunorubicin and strides external blood brain barrier transhipment rate (the same step 4) of formula.
The result behind the adding MAN, combines because free MAN and GLUT1 are competitive as shown in Figure 7, makes the blood brain barrier ability of striding of drug-loaded liposome B1, drug-loaded liposome C1 reduce greatly.This result proves that the daunorubicin liposome that MAN and TF modify combines with GLUT1 identification by the MAN mediation, significantly improves thereby stride the blood brain barrier turn-over capacity.
Two, targeting C6 glioma research
1, the C6 glioma cell is cultivated
Mus C6 glioma cell is grown in the F10 culture fluid (containing 5% hyclone, 15% horse serum, 100U/ml penicillin and 100 μ g/ml streptomycins), condition of culture: 37 ℃, 5% carbon dioxide.
2, C6 glioma cell cellulotoxic experiment
1. Mus C6 glioma cell is inoculated in the 96 porocyte culture plates, put in the CO2 gas incubator in 37 ℃ in 2500 in every hole (the culture fluid volume is every hole 180 μ L), hatches 24h under the condition of relative humidity 95%.
2. the cell on the culture plate is divided into 5 groups, every group is provided with 5 concentration, and each concentration is established four multiple holes.
The 1st group: after the cell attachment growth, add the free daunorubicin of 20 μ L serum-free mediums preparation respectively in the cell culture hole, the final concentration of daunorubicin is respectively: 0.25,0.5,1,2.5,5 μ g/L.
The 2nd group: after the cell attachment growth, add the drug-loaded liposome B1 of 20 μ L serum-free mediums preparation respectively in the cell culture hole, the final concentration of daunorubicin is respectively: 0.25,0.5,1,2.5,5 μ g/L.
The 3rd group: after the cell attachment growth, add the drug-loaded liposome D1 of 20 μ L serum-free mediums preparation respectively in the cell culture hole, the final concentration of daunorubicin is respectively: 0.25,0.5,1,2.5,5 μ g/L.
The 4th group: after the cell attachment growth, add the drug-loaded liposome C1 of 20 μ L serum-free mediums preparation respectively in the cell culture hole, the final concentration of daunorubicin is respectively: 0.25,0.5,1,2.5,5 μ g/L.
The 5th group: after the cell attachment growth, add the drug-loaded liposome A1 of 20 μ L serum-free mediums preparation respectively in the cell culture hole, the final concentration of daunorubicin is respectively: 0.25,0.5,1,2.5,5 μ g/L.
Other establishes solvent control hole (4 multiple holes).
3. Tissue Culture Plate is put and hatched 48 hours in the CO2 gas incubator.Hatch finish after, inhale and to abandon culture fluid, every hole adds 10% trichloroacetic acid, 100 μ L, places the 1h fixed cell then in 4 ℃ of refrigerators.Trichloroacetic acid is removed in deionized water wash 5 times of each hole of culture plate.Behind air drying, every hole adds 0.4%SRB (with the preparation of 1% acetic acid) 100 μ L, places 15min under the room temperature, discards in each hole and washs 5 times with 1% acetic acid behind the liquid, removes not combination dye.Behind the air drying, with the Tris alkali dissolution of 100 μ L pH10.5,10mmol/L, the 10min that vibrates on oscillator plate puts and measures every hole trap OD value (540nm) in the microplate reader.
After the different disposal, the survival rate of cell is seen Fig. 8.The survival rate measurement result shows that in the different preparations of daunorubicin, drug-loaded liposome C1 all shows the effect of the strongest anti-C6 glioma cell propagation under each concentration.
Calculate suppression ratio (suppression ratio=administration group cell absorbance
540nm/ cellular control unit absorbance
540nm), and use SPSS computed in software 50% inhibition concentration (IC50).IC50 result of calculation sees Table 5.
The IC50 value of the different drug-loaded liposomes of table 5
A compares P＜0.05 with free daunorubicin processed group; B, drug-loaded liposome A1 processed group is compared P＜0.05 with drug-loaded liposome D1 processed group; C, drug-loaded liposome C1 processed group is compared P＜0.01 with drug-loaded liposome A1 processed group.
Low 1.39 times of the IC50 value of the IC50 value specific ionization daunorubicin of drug-loaded liposome C1.Between each drug-loaded liposome, the IC50 value of drug-loaded liposome C1 is respectively than low 3.47 times of drug-loaded liposome A1, than low 3.22 times of drug-loaded liposome B1, than low 3.08 times of drug-loaded liposome D1.The daunorubicin liposome that MAN and TF modify shows the effect of the strongest anti-C6 glioma cell propagation.
3, C6 glioma cell picked-up
Mus C6 glioma cell is inoculated in 6 orifice plates (5 * 10
5Individual cells/well) (is divided into five groups, establishes 4 multiple holes for every group); After cultivating 24h, first group adds free daunorubicin (the daunorubicin final concentration is 10 μ g/mL), second group adds drug-loaded liposome B1 (the daunorubicin final concentration is 10 μ g/mL), the 3rd group adds drug-loaded liposome D1 (the daunorubicin final concentration is 10 μ g/mL), the 4th group adds drug-loaded liposome C1 (the daunorubicin final concentration is 10 μ g/mL), and the 5th group adds drug-loaded liposome A1 (the daunorubicin final concentration is 10 μ g/mL); Hatch 2h at 37 ℃.Hatching the back that finishes and giving a baby a bath on the third day after its birth inferiorly with cold PBS, after 0.25% trypsinization, blowing and beating into cell suspension, with cells were tested by flow cytometry and the bonded daunorubicin fluorescence intensity of cell (excitation wavelength of mensuration is 488nm, and the mensuration wavelength is 550nm) with PBS.The used cell number of each analysis is no less than 10
5Individual, the cell number of collection is 10000.Data use FCS Express V3 software to analyze.
The results are shown in Figure 9.Among Fig. 9, A1: drug-loaded liposome A1; A2: drug-loaded liposome B1; A3: drug-loaded liposome D1; A4: drug-loaded liposome C1; The control:C6 glioma cell.Moving to right of peak shows the drug-loaded liposome that application is different, the situation of C6 glioma cell picked-up daunorubicin.With drug-loaded liposome D1, drug-loaded liposome B1, drug-loaded liposome A1 compares, and drug-loaded liposome C1 demonstrates picked-up ability in the strongest C6 glioma cell.
4, C6 cell surface TF receptor competition experiment
Mus C6 glioma cell is inoculated in 6 orifice plates (5 * 10
5Individual/hole) (be divided into four groups, establish 4 multiple holes for every group), behind the cultivation 24h, add free TF (500 μ g) respectively with saturated cell surface TF receptor at the 3rd group and the 4th group; TF added after 30 minutes, added drug-loaded liposome D1 (the daunorubicin final concentration is 10 μ g/mL) respectively at first group and the 3rd group, added drug-loaded liposome C1 (the daunorubicin final concentration is 10 μ g/mL) respectively at second group and the 4th group; Hatch 2h at 37 ℃.Hatching the back that finishes and giving a baby a bath on the third day after its birth time, after 0.25% trypsinization, blowing and beating into cell suspension, with the interior daunorubicin fluorescence intensity (excitation wavelength of mensuration is 488nm, and the mensuration wavelength is 550nm) of cells were tested by flow cytometry cell with PBS with cold PBS.The used cell number of each analysis is no less than 10
5, the cell number of collection is 10000.Data use FCS Express V3 software to analyze.
The results are shown in Figure 10.Among Figure 10, use drug-loaded liposome D1 behind the B1:TF presaturation 30min; B2: directly use drug-loaded liposome D1; Use drug-loaded liposome C1 behind the B3:TF presaturation 30min; B4: directly use drug-loaded liposome C1; The control:C6 glioma cell.Moving to left of peak shows that there is a large amount of TF receptors on the glioma cell surface.Hatch with free TF and glioma cell C6 in advance, the C6 glioma cell is to drug-loaded liposome C1, and the intake significance of drug-loaded liposome D1 reduces.This presentation of results liposome can increase the endocytosis of C6 glioma cell after transferrins is modified.The daunorubicin liposome group that MAN and TF modify can be observed the strongest endocytosis.
5, Laser Scanning Confocal Microscope research
The laser co-focusing method detects cell to free daunorubicin, drug-loaded liposome B1, drug-loaded liposome D1, the picked-up of drug-loaded liposome C1 and drug-loaded liposome A1 and in intracellular distribution behavior.Mus C6 glioma cell is inoculated at the bottom of the glass in the culture dish, and adherent growth to 60% is compiled; Be provided with five groups, add free daunorubicin (the daunorubicin final concentration is 5 μ g/mL), drug-loaded liposome B1 (the daunorubicin final concentration is 5 μ g/mL), drug-loaded liposome D1 (the daunorubicin final concentration is 5 μ g/mL), drug-loaded liposome C1 (the daunorubicin final concentration is 5 μ g/mL), drug-loaded liposome A1 (the daunorubicin final concentration is 5 μ g/mL) respectively; Put in the CO2 gas incubator, cultivated 3 hours for 37 ℃, use cold PBS buffer rinsing three times successively, 4% paraformaldehyde is 10min fixedly, uses Hoechst33258 (excitation wavelength 352nm detects wavelength 461nm) to carry out nucleus dyeing 7 minutes then.Excite daunorubicin (excitation wavelength 488nm detects wavelength 560nm) to carry out graphical analysis with laser confocal microscope (LEICATCS SP2) with the laser beam of 488nm wavelength.
The results are shown in Figure 11.Among Figure 11, C: drug-loaded liposome A1, D: drug-loaded liposome B1, E: drug-loaded liposome D1, F: drug-loaded liposome C1; C1-F1: blue expression is through the nuclear of the painted C6 glioma cell of Hochest33258; C2-F2: the distribution of medicine in the C6 glioma cell behind the red expression application daunorubicin liposome; C3-F3: drug distribution is examined at the C6 glioma cell behind the superimposed image display application daunorubicin liposome.The result shows that medicine mainly is distributed in the nucleus behind C6 glioma cell endocytosis, the daunorubicin abundance is maximum in the drug-loaded liposome C1 group cell.
Three, external dual-target research---BMVEC/C6 co-culture experiments
Six groups of tests (every group of 3 cell inserters) are set.Mus brain endothelial cell kind was gone into cell inserter upper strata after the 4th day, went into Mus C6 glioma cell (10000/hole) in pond, end kind, and culture fluid is two kinds of cell culture fluids each half, cultivates 24h altogether; First group upper storage reservoir culture medium is done blank, contain the free daunorubicin of 5 μ g in second group the upper storage reservoir, contain drug-loaded liposome B1 (containing 5 μ g daunorubicins) in the 3rd group the upper storage reservoir, contain drug-loaded liposome D1 (containing 5 μ g daunorubicins) in the 4th group the upper storage reservoir, contain drug-loaded liposome C1 (containing 5 μ g daunorubicins) in the 5th group the upper storage reservoir, contain drug-loaded liposome A1 (containing 5 μ g daunorubicins) in the 6th group the upper storage reservoir; Continue to cultivate 48h.Hatch finish after, inhale and to abandon culture fluid, every hole adds 10% trichloroacetic acid 2mL, places the 1h fixed cell then in 4 ℃ of refrigerators.Trichloroacetic acid is removed in deionized water wash 5 times of each hole of culture plate.Behind air drying, every hole adds 0.4%SRB (with the preparation of 1% acetic acid) 1mL, places 20min under the room temperature, discards in each hole and washs 5 times with 1% acetic acid behind the liquid, removes not combination dye.With 2ml, pH 10.5,10mmol/L Tris alkali dissolution, the 10min that vibrates on oscillator plate puts and measures every hole trap OD value (540nm) in the microplate reader, calculates suppression ratio (suppression ratio=administration group cell absorbance behind the air drying
540nm/ cellular control unit absorbance
540nm), do statistical analysis.
The results are shown in Figure 12.Preparation is respectively the suppression ratio of C6 glioma cell after striding across blood brain barrier: drug-loaded liposome C1 (64.0 ± 0.7%)＞drug-loaded liposome B1 (58.6 ± 0.7%)＞drug-loaded liposome A1 (46.0 ± 1.0%) 〉=free daunorubicin (45.5 ± 0.7%)＞drug-loaded liposome D1 (41.3 ± 0.7%).The result shows that the daunorubicin liposome that MAN and TF modify demonstrates " secondary targeting " (P＜0.01), promptly strides across blood brain barrier, then targeting C6 glioma cell.
Four, glioma cell ball experiment
Six groups of tests are set, 3 every group multiple holes.
1. use 2% agarose (with the serum-free medium configuration, 80 ℃ are heated 30min down), 40 μ l to wrap, again Mus C6 glioma cell is seeded in (2000 cells/well) in 96 orifice plates, hatched 24 hours for 37 ℃ by 96 orifice plates.
2. respectively with the free daunorubicin of serum-free medium configuration, drug-loaded liposome B1, drug-loaded liposome D1, drug-loaded liposome C1, drug-loaded liposome A1.
3. administration: the drug-loaded liposome (or free daunorubicin) that respectively 2. 20 μ l steps is obtained adds in step 96 orifice plates 1., and the final concentration of daunorubicin is 10 μ g/ holes, is blank with the serum-free medium.
4. whether form, the ball in the 0th, 1,2,3,5 day observation of cell ball under inverted microscope after the administration has necrosis, and measures diameter with eyepiece micrometer.The calculating gross tumor volume (
), tumor sphere volume rate=(volume
My god i/ volume
My god 0) * 100%, volume
My god iBe meant i days C6 glioma cell sphere volumes after the administration, volume
My god 0Be meant the preceding C6 glioma cell sphere volume of administration, the results are shown in Figure 13.The only faint increase of inhibition C6 glioma cell ball on volume of energy in several days of beginning of free daunorubicin.Drug-loaded liposome C1 processed group C6 glioma cell ball produces reducing of significance on size and volume.C6 glioma cell sphere volume rate of change in the time of the 5th day is respectively: free daunorubicin is 74.6 ± 5.1%, drug-loaded liposome A1 is 71.7 ± 5.2%, drug-loaded liposome B1 is 73.5 ± 3.9%, and drug-loaded liposome D1 is 67.7 ± 3.1%, and drug-loaded liposome C1 is 54.7 ± 2.4%.
Targeting evaluation of effect in embodiment 4, the liposome body
Use the male SD rat of 200-250g to test.Adopt the drug-loaded liposome of embodiment 1 preparation to carry out following test.
One, the foundation of cerebral glioma rat model
1, prepares the C6 cell
Get the Mus C6 glioma cell that is in exponential phase, dilution is 5 * 10
5The cell suspension of cell/10 μ l.Cell suspension is put into 37 ℃ of water-baths to be preserved.
2, inoculation is handled
1) weighs
2) anesthesia, fixing with sterilize: rat with 20% urethane (0.6ml/100g) intraperitoneal injection of anesthesia after, the ruff of decaptitating is sent out about 1.0cm * 1.5cm, is fixed on the position finder alcohol disinfecting.
3) otch and bore position: vertically cut rat scalp 1cm backward along endocanthion line mid point, expose skull.According to the rat brain stereotaxic atlas: 0.4mm behind the bregma, with dental burr boring, dental burr is plastic bushing in addition, only allows the dark 1.0-1.5mm of the saturating skull of bit drills (to prevent to puncture cerebral dura mater) on the skull of sagittal suture side 3.5mm.
4) the right brain caudatum inoculation of C6 cell: use the 20uL microsyringe to suck 10 μ L C6 cell suspension, the vertical dark 5mm of inserting needle (apart from cerebral dura mater) injects cell suspension in the caudatum with 1 μ l/min speed.Injection back let the acupuncture needle remain at a certain point the 5min that finishes slowly pulls out pin, and the bone hole is immediately with the bacteria-free bone wax sealing, and art is wild use normal saline flushing, and otch meets with No. 4 lines and closes, and 2 stylus printers are tied, and can obviously reduce tumor growth under the scalp.
5) postoperative lumbar injection penicillin, 40000U/ are only.
Two, targeting evaluation of effect in the liposome body
Rat model is divided into 6 groups at random, 9 every group.Postoperative beginning in the 8th day tail intravenously administrable, every group administration situation is as follows, and dosage refers to the dosage of daunorubicin:
The 1st group (daunorubicin): be administered three times weekly, dosage is 5mg/kg, one week of successive administration;
The 2nd group (drug-loaded liposome D1): be administered three times weekly, dosage is 5mg/kg, one week of successive administration;
The 3rd group (drug-loaded liposome B1): be administered three times weekly, dosage is 5mg/kg, one week of successive administration;
The 4th group (drug-loaded liposome C1): be administered three times weekly, dosage is 5mg/kg, one week of successive administration;
The 5th group (drug-loaded liposome A1): be administered three times weekly, dosage is 5mg/kg, one week of successive administration;
The 6th group (normal saline): use the physiologic saline for substitute administration, in contrast.
Adopt liquid-solid the separating surely of 4%PBS paraformaldehyde to cut tumor,, calculate gross tumor volume (mm with the high diameter of maximum length and width with the maximum aspect diameter of vernier caliper measurement pathological anatomy tumor
3) (
), gross tumor volume * 100% of the gross tumor volume of gross tumor volume suppression ratio=test group/normal saline matched group.
The gross tumor volume suppression ratio is seen Figure 14 after one week of administration.Free daunorubicin is 13.0 ± 2.4%, and drug-loaded liposome A1 is 14.2 ± 6.2%, and drug-loaded liposome B1 is 25.3 ± 3.3%, and drug-loaded liposome D1 is 22.8 ± 4.1%, and drug-loaded liposome C1 is 37.4 ± 1.0%.Compare with other group, the daunorubicin liposome treated animal that MAN and TF modify demonstrates reducing of gross tumor volume significance, and the normal saline group does not have effect to the inhibition of gross tumor volume.
The record death time of animal is drawn kaplan-Meier survival curve, the results are shown in Figure 15.The mean survival time of drug-loaded liposome C1 group rat is 22 days, significance be longer than the normal saline group (13 days, P=0.001), free daunorubicin group is (17 days, P=0.001), drug-loaded liposome A1 group is (18 days, P=0.005), drug-loaded liposome B1 group (19 days, P=0.038) organize with drug-loaded liposome D1 (18 days, P=0.034).
Claims (7)
1. a target liposomes is made up of liposome and its surperficial trim, and it is characterized in that: the trim of described surface of liposome is p-aminophenyl-α-D-manno-pyranoside and transferrins; The surface of described liposome has amino and carboxyl; Described p-aminophenyl-α-D-manno-pyranoside is connected on the amino of described liposome; Described transferrins is connected on the carboxyl of described liposome; The amino of described liposome is by DSPE-Polyethylene Glycol-NH
2Provide; The carboxyl of described liposome is provided by DSPE-Polyethylene Glycol-COOH; Described liposome is with lecithin, cholesterol, Polyethylene Glycol-DSPE, DSPE-Polyethylene Glycol-NH
2And DSPE-Polyethylene Glycol-COOH obtains as feedstock production; Lecithin, cholesterol, Polyethylene Glycol-DSPE, DSPE-Polyethylene Glycol-NH
2And the mass ratio of DSPE-Polyethylene Glycol-COOH is (104.5-105.5): (44.5-45.5): (28-28.5): (3.4-3.6): (3.4-3.6);
In the described target liposomes, the mass ratio of described transferrins and described liposome is (4-5): 15, and the mass ratio of described p-aminophenyl-α-D-manno-pyranoside and described liposome is (1.5-2): 15.
2. the preparation method of a target liposomes comprises the steps:
1) with lecithin, cholesterol, Polyethylene Glycol-DSPE, DSPE-Polyethylene Glycol-NH
2And DSPE-Polyethylene Glycol-COOH has the liposome of amino and carboxyl for the feedstock production surface;
2) p-aminophenyl-α-D-manno-pyranoside is connected with the amino of the liposome of step 1) preparation; The carboxyl of transferrins with the liposome of step 1) preparation is connected;
Obtain the target liposomes liposome.
3. the application of the described target liposomes of claim 1 in the preparation pharmaceutical carrier.
4. pharmaceutical carrier, its active component is the described target liposomes of claim 1.
5. a drug-loaded liposome obtains with the described target liposomes bag of claim 1 medicine carrying thing.
6. medicine for the treatment of the cerebral tumor, its active component is the described drug-loaded liposome of claim 5; The medicine that described drug-loaded liposome bag carries is a daunorubicin.
7. the application of the described drug-loaded liposome of claim 5 in the medicine of the preparation treatment cerebral tumor; The medicine that described drug-loaded liposome bag carries is a daunorubicin.
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| CN104174024B (en) * | 2013-05-21 | 2017-12-01 | 复旦大学 | A kind of Brain targeting polymer micelle delivery system of myristic acid mediation and its preparation method and application |
| CN103405783B (en) * | 2013-08-05 | 2015-08-12 | 中山大学孙逸仙纪念医院 | OX26/CTX-PL/pC27 complex and the application in treatment glioma thereof |
| CN105030681B (en) * | 2015-08-27 | 2018-04-24 | 石河子大学 | A kind of liposome medicament and preparation method thereof |
| CN105055318B (en) * | 2015-08-31 | 2018-08-24 | 石河子大学 | A kind of dual target liposome and its preparation method and application modified with MAN and WGA |
| CN106800650B (en) * | 2015-11-26 | 2019-02-19 | 北京大学 | Function targeting vector material distearoylphosphatidylethanolamine-polyethylene glycol-phenylglucopyranoside and the preparation method and application thereof |
| CN106798923B (en) * | 2015-11-26 | 2020-06-09 | 北京大学 | Function targeting carrier material distearoyl phosphatidyl ethanolamine-polyethylene glycol-polyethylene imine compound and liposome modified by same |
| CN105641711B (en) * | 2016-01-25 | 2019-01-15 | 四川大学 | Using organic amine-modified vitamin C as the dual Brain targeting prodrug of carrier |
| CN105708802B (en) * | 2016-03-24 | 2019-04-02 | 石河子大学 | A kind of casticin pulmonary targeting liposomes and the preparation method and application thereof |
| CN105853360A (en) * | 2016-04-29 | 2016-08-17 | 石河子大学 | Functional lipidosome for treating brain tumor and preparing method and application thereof |
| CN107875123B (en) * | 2016-09-27 | 2019-07-16 | 北京大学 | Functionalization vinblastine liposome and its application |
| CN110522917A (en) * | 2018-05-25 | 2019-12-03 | 成都瑞博克医药科技有限公司 | A kind of targeted nano preparation of mannose-modified |
| CN108743970B (en) * | 2018-06-07 | 2021-09-24 | 上海应用技术大学 | Mitochondrial targeting liposome modified by hyaluronic acid and preparation method thereof |
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