CN107875123A

CN107875123A - Functionalization vinblastine liposome and its application

Info

Publication number: CN107875123A
Application number: CN201610854962.2A
Authority: CN
Inventors: 吕万良; 沐黎敏; 卜英子; 刘磊; 吴佳栓; 居瑞军
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2016-09-27
Filing date: 2016-09-27
Publication date: 2018-04-06
Anticipated expiration: 2036-09-27
Also published as: CN107875123B

Abstract

The invention discloses functionalization vinblastine liposome and its application.The invention provides a kind of vinblastine liposome, for with functionalization material TfR binding peptide polyethylene glycol DSPE and the poly arginine modified liposome of stearyl eight, liposome after being modified, vinblastine is contained after the modification in liposome again, obtains functionalization vinblastine liposome.The experiment proves that the present invention has synthesized a kind of functionalization material TfR binding peptide polyethylene glycol DSPE (NHS PEG₂₀₀₀DSPE), and with it with targetting the poly arginine of material stearyl eight (stearyl R₈) modification vinblastine liposome, obtain a kind of to cross over blood-brain barrier and killing glioma and its functionalization vinblastine liposome of stem cell.

Description

Functionalization vinblastine liposome and its application

Technical field

The present invention relates to biological technical field, more particularly to a kind of functionalization vinblastine liposome and its application.

Background technology

Vinblastine (vinblastine, VLB) by Canadian University of Western Ontario scientist Robert Noble and Charles Thomas extracted isolated from apocynaceae plant catharanthus roseus first in 1958, and it is a kind of double indoles types Alkaloid, there is antitumor activity.The structural formula of vinblastine is as shown in figure 1, molecular formula is C₄₆H₅₈N₄O₉, molecular weight is 810.97, normal vitriolization salt is not soluble in water, for white or off-white color crystalline powder.

TfR binding peptide TfR-T₁₂It is the polypeptide that sequence is THRPPMWSPVWP, includes 12 amino acid (Thr-His-Arg-Pro-Pro-Met-Trp-Ser-Pro-Val-Trp-Pro), soluble in water and methanol, molecular weight are 1490.8。TfR-T₁₂Polypeptide can be specifically bound with human TfR (transferrin receptor, TfR), and Competion experiment shows that its binding site is different with the binding site of transferrins (transferrin, Tf), and this causes TfR- T₁₂It will not be disturbed when being combined in vivo with TfR by transferrins.Due to high on blood-brain barrier and tumour cell Express TfR, therefore TfR-T₁₂There is potential using value in brain targeted drug delivery system.

The poly arginine of stearyl eight (stearylated octaarginine, Stearyl-R₈) be by eight arginine and Functional material obtained by stearyl (stearyl) synthesis connection, is synthesized to obtain first by Shiroh Futaki etc..Eight gather smart ammonia Acid can help DNA to be transfected into cell, be dramatically increased being combined rear transfection efficiency with stearyl.Stearyl-R₈Energy Help material to enter cell and be primarily due to arginine with abundant cationic species, and (especially tumour is thin for most cells Born of the same parents) and organelle surface be in elecrtonegativity, therefore the function with targets neoplastic cells and subcellular organelle, simultaneously because stearyl In the presence of can be plugged on the phospholipid bilayer of liposome, help liposome target tumor position.

DSPE-PEG (DSPE-PEG₂₀₀₀) it is that one kind is used for long circulating liposome system Standby material, 1992 by Maruyama K etc. reported the material modification big unilamellar liposome circulated in hematological system when Between be extended.By the DSPE-PEG of NHS group activations₂₀₀₀For DSPE-PEG-N- Succinimide (NHS-PEG₂₀₀₀- DSPE), easily reacted with the amino on polypeptide, slough N- hydroxysuccinimidyl acyls during the course of the reaction Imines, amido link is generated, it is easily operated.

Glioma is a kind of great brain diseases, and operative treatment difficulty is big, and radiotherapy side effect is more, and operation is controlled Still can a small amount of brain glioblastoma cell of remaining and its stem cell after treatment or radiotherapy.In conventional chemotherapy, chemotherapeutics It is difficult to cross over blood-brain barrier, and a small amount of medicine that can cross over blood-brain barrier is because human brain glioma stem cells have drug resistance, it is also difficult It is clean to be removed, cause the recurrence of glioma.Therefore, how to help chemotherapeutics across blood-brain barrier, in killing brain It is the important scientific issues to remain unsolved that the glioma of remaining and its stem cell are removed while glioma.

The content of the invention

It is an object of the present invention to provide a kind of liposome or vinblastine liposome.

Liposome provided by the invention is through TfR binding peptide-PEG2000-DSPE Modified with the poly arginine of stearyl eight；

Vinblastine liposome provided by the invention is by the liposome and contains the catharanthus roseus in the liposome Alkali forms；

TfR binding peptide-the PEG2000-DSPE is by TfR-T₁₂Polypeptide and NHS-PEG₂₀₀₀The product that-DSPE is obtained by acid amides key connection.

TfR-T₁₂(Thr-His-Arg-Pro-Pro-Met-Trp-Ser-Pro-Val-Trp-Pro) purchased from the peptide life of Hefei state Thing Science and Technology Ltd..

Another object of the present invention is to provide a kind of method for preparing above-mentioned vinblastine liposome.

Method provided by the invention, for TfR binding peptide-PEG2000-DSPE With the poly arginine (stearyl-R of stearyl eight₈) modified liposome, and vinblastine is contained and grown in the liposome Spring flower alkali liposome.

The above method comprises the following steps：

1) TfR binding peptide-PEG2000-DSPE is prepared；

The preparation method comprises the following steps：By TfR-T₁₂Polypeptide and NHS-PEG₂₀₀₀Effects of-the DSPE in catalyst Lower carry out acylation reaction, obtains TfR binding peptide-PEG2000-DSPE；

It is above-mentioned prepare TfR binding peptide-PEG2000-DSPE method it is also specific Comprise the following steps：After the acylation reaction step, reaction system is transferred in bag filter, is placed in deionized water thoroughly Analysis 24 hours, solvent and unreacting material are removed, obtains TfR binding peptide-polyethylene glycol-distearoylphosphatidyl Monoethanolamine；

The molecular cut off of the bag filter is specially 3000Da；The time of the dialysis is 24 hours.

2) with lecithin, cholesterol, TfR binding peptide-polyethylene glycol-distearoylphosphatidyl ethanol Amine (TfR-T₁₂-PEG₂₀₀₀- DSPE) and the poly arginine (stearyl-R of stearyl eight₈) carry out film formation reaction, fat after being modified Plastid, liposome after being modified；

The method of liposome specifically comprises the following steps after above-mentioned preparation modification：By lecithin, cholesterol and TfR-T₁₂- PEG₂₀₀₀- DSPE is dissolved in organic solvent, and organic solvent is removed under reduced pressure, and forms the system containing film；Ammonium sulfate is added to enter Water-filling obtains thick liposome；Again by thick lipide supersonic, product after ultrasound is obtained, is by aperture by product after ultrasound 200nm poly- carbon ester film, filtrate are dialysed by bag filter, obtain liposome；

3) vinblastine is contained after the modification in liposome, obtains vinblastine liposome.

Above-mentioned contain vinblastine in the liposome comprises the following steps：The vinblastine methanol is molten Solution, lysate is obtained, then liposome and the lysate are mixed, reaction, obtain vinblastine liposome.

In the above method, in step 1), the TfR-T₁₂Polypeptide and NHS-PEG₂₀₀₀- DSPE molar ratio is 1:1- 3:1；

Or, the catalyst is N-methylmorpholine；

Or, the catalyst and the TfR-T₁₂The charge ratio of polypeptide is 200 μ L：8μmol；

Or, the temperature of the acylation reaction is normal temperature, the time of acylation reaction is 48h；

Or, the acylation reaction is carried out in a solvent；

Or, the solvent is specially DMF.

In the above method, in step 2), the lecithin, cholesterol, institute's TfR binding peptide-polyethylene glycol- DSPE and the stearyl-R₈Molar ratio be 60:30:3:5.

In the above method, in step 3), the mass ratio that feeds intake of liposome is 1 after the vinblastine and the modification:20.

TfR binding peptide-polyethylene glycol-distearoylphosphatidyl the second being prepared in the above method Hydramine is also the scope of protection of the invention；

Or liposome is also the scope of protection of the invention after the modification being prepared in the above method.

Above-mentioned TfR binding peptide-PEG2000-DSPE prepare liposome or The application for preparing vinblastine liposome or modified liposome or modifying in vinblastine liposome is also the model of the invention protected Enclose；

Or above-mentioned vinblastine liposome or above-mentioned TfR binding peptide-polyethylene glycol-distearyl phosphorus Acyl monoethanolamine is being prepared with following 1) -9) application in the product of at least one function is also the scope of protection of the invention：

1) brain glioblastoma cell or its stem cell are killed；

2) brain glioblastoma cell or its stem cells hyperplasia are suppressed；

3) blood-brain barrier is crossed over；

4) brain glioblastoma cell or its stem cell apoptosis are promoted；

5) brain glioblastoma cell or the injury of mitochondria of its stem cell are promoted；

6) brain glioblastoma cell or its Stem Cell Activity oxygen level are improved；

7) brain glioblastoma cell or the micro-duct injury of its stem cell are promoted；

8) improve and promote apoptosis-related protein gene expression in brain glioblastoma cell or its stem cell；

9) prevent and/or treat glioma.

3rd purpose of the invention, which is to provide, a kind of has following 1) -9) product of at least one function.

Product provided by the invention, its active component are above-mentioned vinblastine liposome；

1) brain glioblastoma cell or its stem cell are killed；

2) brain glioblastoma cell or its stem cells hyperplasia are suppressed；

3) blood-brain barrier is crossed over；

4) brain glioblastoma cell or its stem cell apoptosis are promoted；

9) prevent and/or treat glioma.

In above-mentioned, the rush apoptosis-related protein is caspase-3/7, caspase-8, caspase-9, Bax, cell color Plain C, Mcl-1, LC3B or FoxO1；

Or the product is medicine or kit.

The experiment proves that the present invention has synthesized a kind of functionalization material TfR binding peptide-poly- second two Alcohol-DSPE (TfR-T₁₂-PEG₂₀₀₀- DSPE), and gather smart ammonia with itself and cationic materials stearyl eight Acid (stearyl-R₈) modification vinblastine liposome, blood-brain barrier can be crossed over and kill glioma and its do by obtaining one kind The functionalization vinblastine liposome of cell.The functionalization vinblastine liposome has across blood-brain barrier and specific killing Human brain glioma stem cells act on, and are a kind of new drug-loading systems, there is important academic significance and potential clinical value.

Brief description of the drawings

Fig. 1 is vinblastine structural formula schematic diagram.

Fig. 2 is synthetic product TfR-T₁₂-PEG₂₀₀₀- DSPE MALDI-TOF-MS collection of illustrative plates.

Fig. 3 is functionalization vinblastine liposome structure schematic diagram.

Fig. 4 is the standard curve that HPLC methods determine vinblastine concentration-peak area.

Fig. 5 is the characteristic spectrum that HPLC methods determine vinblastine.

Fig. 6 is the test limit that HPLC methods determine vinblastine.

Fig. 7 is that functionalization vinblastine liposome AFM (AFM) observes result.

Fig. 8 is that functionalization vinblastine liposome transmission electron microscope (TEM) observes result.

Fig. 9 is vinblastine from the release rate in each group liposome.

Figure 10 is expression of the TfR in brain glioblastoma cell (A) and its stem cell (B).

Figure 11 is the expression feelings of cells and characteristic of stem mark TfR in brain glioblastoma cell and its stem cell Condition.

Figure 12 is each preparation group to brain glioblastoma cell (A₁) and its stem cell (A₂) lethal effect

Figure 13 is expression of the TfR in brain microvessel endothelial cells in vitro (BMVECs).

Figure 14 is that each preparation group is crossed over after blood-brain barrier co-culture model to the lethal effect of brain glioblastoma cell.

Figure 15 is apoptosis induction effect of each preparation group to brain glioblastoma cell.

Figure 16 is apoptosis induction effect of each preparation group to human brain glioma stem cells.

Figure 17 is nucleus degree of impairment of each preparation group to brain glioblastoma cell.

Figure 18 is injury of mitochondria situation of each preparation group to brain glioblastoma cell.

Figure 19 is the facilitation that each preparation group discharges active oxygen to brain glioblastoma cell.

Figure 20 is Microtubule disruption situation of each preparation group to brain glioblastoma cell.

Figure 21 is activation of each preparation group to apoptosis enzyme caspase-3/7 in brain glioblastoma cell.

Figure 22 is activation of each preparation group to apoptosis enzyme caspase-8 in brain glioblastoma cell.

Figure 23 is activation of each preparation group to apoptosis enzyme caspase-9 in brain glioblastoma cell.

Figure 24 is expression facilitation of each preparation group to pro apoptotic protein Bax in brain glioblastoma cell.

Figure 25 be each preparation group to cytochrome c from the release facilitation in brain glioblastoma cell mitochondria.

Figure 26 is that each preparation group is acted on the expression inhibiting of anti-apoptotic proteins Mcl-1 in brain glioblastoma cell.

Figure 27 is that up-regulation of each preparation group to autophagy proteins LC3B in brain glioblastoma cell acts on.

Figure 28 is that up-regulation of each preparation group to autophagy proteins FoxO1 in brain glioblastoma cell acts on.

Figure 29 is normal nude mice brain tissue slice.

Figure 30 is lotus glioma nude mice brain tissue slice.

Figure 31 is real-time living imaging of the medicine in lotus glioma nude mice.

Figure 32 is in vitro imaging of the medicine in each organ of lotus glioma nude mice after administration 48h.

Figure 33 is lotus glioma nude mice Kapp Lan-Mei Ye survivorship curves after treatment.

Figure 34 is anti-human brain glioma stem cells effect of the medicine in lotus glioma nude mice.

Figure 35 is each organ hematoxylin-eosin (HE) dyeing of lotus glioma nude mice.

Embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.

Portion of material and instrument used are as follows in following embodiments 1：

NHS-PEG₂₀₀₀-DSPE(N-hydroxysuccinimidyl-polyethylene glycol distearoyl Phosphatidyl ethanolamine) NOF Corp (Japanese NOF companies) is purchased from, Japan；TfR-T₁₂(Thr- His-Arg-Pro-Pro-Met-Trp-Ser-Pro-Val-Trp-Pro Hefei Guo Tai bio tech ltd) is purchased from, in State；stearyl-R₈(stearyl-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-NH₂) limited purchased from Shanghai gill biochemistry Company, China；Lecithin (egg yolk phosphatidylcholine, EPC), (Japanese NOF is public for NOF Corp Department), Japan；Cholesterol (cholesterol), Haidian District, Beijing City microbiological culture media products factory (train by Beijing bispin microorganism Support based articles factory), China；PEG2000-DSPE (polyethylene glycol- Distearoylphosphatidyl ethanolamine, DSPE-PEG₂₀₀₀), (Japanese NOF is public for NOF Corp Department), Japan；Vinblastine Sulfate (vinblastine), Nanjing Duolun Chemical Co., Ltd., China；DMF (dimethylformamide, DMF, super dry solvent), Beijing lark prestige company, China；Methanol (methanol, HPLC level), write from memory Gram company, Germany；Acetonitrile (acetonitrile, HPLC level), Merck ＆ Co., Inc., Germany；Hepes (4- hydroxyethyl piperazineethanesulfonic acids, 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid), Shanghai life work bioengineering share is limited Company, China；Hyclone (Fetal bovine serum, FBS), PAN biotechnologies company, Germany；Phosphate buffer Pulvis (phosphate buffer saline, PBS), Beijing prosperity Bioisystech Co., Ltd of ancient cooking vessel state, China；Used in remaining Reagent is the pure rank of analysis, and Beijing traditional Chinese medicines Reagent Company is Chinese.

Bag filter (molecular cut off 3000Da, 8000-14000Da), the prosperous great achievement bio tech ltd in Beijing, in State；The a ten thousandth gram precision electronic balances of BT-25S ten, Sartorius companies, Germany；Six magnetic force heating/constant temperature of HJ-6 types Agitator, Ke Xi Instrument Ltd. of Community of Jin Tan County city, China；MALDI TOFMS (MALDI-TOF-MS, matrix-assisted desorption/ionization time of flight mass), Shimadzu companies, Japan；RE52CS Rotary Evaporators, Shanghai Yarong Biochemical Instrument Plant, China；SB-5200 ultrasonic machines, Ningbo Xin Zhi bio tech ltd instrument, China；JY92-IID type ultrasonic cell disruptors, the new sesame biotechnology in Ningbo are limited Company, China；ZWY-103B constant temperature culture oscillators, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd., China；Tecnar G2 20ST types transmission electron microscope (transmission electron microscope, TEM), FEI Co., the U.S.；Laser Scatter particle size determination instrument (Malvern Zetasizer 3000HS), Malvern instruments companies, Britain； SPI3800N series SPA-400 types AFMs (AFM), NSK Co., Ltds, Japan；Shimadzu chromatograph (LC- 10AT), Shimadzu (Shimadzu) Co., Ltd, Japan；Reverse-phase chromatographic column (C18column, 5 μm, 250x4.6mm), Tuo Lunsi Phenomenex companies, the U.S..

All data are represented with Mean ± SD.Using one-way analysis of variance (ANOVA), then by Bonferroni schools Test, P<0.05 thinks there is significant difference.

Portion of material and instrument used are as follows in following embodiments 2：

Glioma U87-MG cell lines are purchased from Tumour Inst., Chinese Medical Academy；Serum-free replenishers, Gibco companies, the U.S.；LIF ELISA (LIF) is purchased from Wuxi Yao Kemei bio tech ltd, China；Tire ox blood Clearly, PAN companies, Germany；MEM, DMEM-F12 culture medium, insulin, EGF (EGF), basic fibroblast life The long factor (bFGF), nonessential amino acid, pancreatin are purchased from Beijing Mai Chen Science and Technology Ltd.s, China；Anti-Human Transferrin Receptor (CD71) antibody-FITC, Sino Biological Inc., China； Anti-Human Nestin antibody-FITC, R＆D companies, the U.S.；Anti-Human ABCG2(CD338)antibody- FITC, Anti-Human CXCR4antibody-PE, and corresponding Isotype antibody are purchased from Biolegend companies, the U.S.；Phosphorus Phthalate buffer (PBS), purchased from Beijing Ding Guo Bioisystech Co., Ltd；Sulforhodamine B (SRB) is purchased from Sigma companies, beautiful State；Triton X-100 are purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge (ZSGB-BIO), China；4% paraformaldehyde is purchased From Mai Chen Science and Technology Ltd.s, China；Trichloroacetic acid (TCA) is purchased from Beijing Xing Jin chemical plant, China；Disposable cell culture Experimental article (including Tissue Culture Plate blake bottle, culture dish, sterile centrifugation tube etc.) is purchased from healthy and free from worry (Corning) company, the U.S.. Cell culture incubator, Thermo companies, the U.S.；AirTECH cell super-clean benches, the safe and sound companies of Su Jing, China；TDZ4-WS low speed platforms Formula centrifuge, Xiang Yi centrifuges Instrument Ltd., China；Tecan infinite F50 ELIASAs, Tecan companies, Switzerland； TS-8 oscillator plates, its woods Bel's instrument manufacturing Co., Ltd of Haimen City, China；Inverted microscope, Chongqing optics electricity instrument Device company, China；DK-S24 electric-heated thermostatic water baths, the upper grand experimental facilities Co., Ltd of Nereid, China；Flow cytometer (BD FACS Calibur), Becton Dickinson companies, the U.S..

The portion of material and instrument of following embodiments 3 are as follows：

Glioma U87-MG cell lines are purchased from Tumour Inst., Chinese Medical Academy；Mouse brain capillary endothelial cell (brain microvascular endothelial cells, BMVECs), purchased from clinical research institute of China-Japan Friendship Hospital；It is double Anti- (penicillin, streptomysin) is purchased from Beijing Suo Laibao Science and Technology Ltd, China；Hyclone, PAN companies, Germany；MEM、 DMEM culture mediums, heparin, endothelial growth factor (ECGF), nonessential amino acid, pancreatin are limited purchased from Beijing morning science and technology advanced in years Company, China；Anti-Mouse Transferrin Receptor (CD71) antibody-FITC, eBioscience companies, The U.S.；Phosphate buffer (PBS), purchased from Beijing Ding Guo Bioisystech Co., Ltd；Sulforhodamine B (SRB) is purchased from Sigma Company, the U.S.；Gelatin is purchased from Beijing Baeyer enlightening biotech company；Trichloroacetic acid (TCA) is purchased from Beijing Xing Jin chemical plant, in State；Disposable cell culture experiments articles for use (including Tissue Culture Plate, Transwell polyester films inserter, blake bottle, culture dish, Sterile centrifugation tube etc.) it is purchased from healthy and free from worry (Corning) company, the U.S..

The a ten thousandth gram precision electronic balances of BT-25S ten, Sartorius companies, Germany；Cell culture incubator, Thermo are public Department, the U.S.；AirTECH cell super-clean benches, the safe and sound companies of Su Jing, China；TDZ4-WS low speed desk centrifuges, Hunan instrument centrifuge Instrument Ltd., China；Tecan infinite F50 ELIASAs, Tecan companies, Switzerland；TS-8 oscillator plates, Haimen Its woods Bel's instrument manufacturing Co., Ltd of city, China；ERS-2 cell resistance instrument, Millipore companies, the U.S..

The portion of material and instrument of following embodiments 4 are as follows：

Glioma U87-MG cell lines are purchased from Tumour Inst., Chinese Medical Academy；Human brain glioma stem cells GSCs is This research is voluntarily obtained by Fiber differentiation；Serum-free replenishers, Gibco companies, the U.S.；LIF ELISA (LIF) is purchased From Wuxi Yao Kemei bio tech ltd, China；Hyclone, PAN companies, Germany；Lowlenthal serum is purchased from Zhejiang day Hangzhoupro Biotech inc, China；MEM, DMEM, DMEM-F12 culture medium, insulin, EGF (EGF), alkali Property fibroblast growth factor (bFGF), nonessential amino acid, pancreatin (being free of EDTA) step the morning limited public affairs of science and technology purchased from Beijing Department, China；Mitochondria red fluorescence probe (Mitotracker Deep Red FM), Hoechst 33342 are purchased from Invitrogen companies, the U.S.；Micro-pipe red fluorescence probe (Tubulin-Tracker Red) is purchased from the green skies in Beijing (Beyotime) company, China；Apoptosis kit (7-AAD and Annexin V-KeyFluor 647) is purchased from the triumphant base in Shanghai Biological (KeyGEN Biotech), China；Active oxygen detection kit (DCFH-DA) is purchased from Beijing Puli's Lay (APPLYGEN), China；anti-caspase 3/7antibody、anti-caspase 8antibody、anti-caspase 9antibody、 anti-Bax antibody、anti-cytochrome c antibody、anti-MCL-1antibody、anti-Forkhead Box protein O1 (FoxO1), antibody and anti-LC3B antibody are purchased from the green skies in Beijing (Beyotime) Company, China；Alexa488-conjugated goat anti-rabbit antibody、Alexa 488-conjugated goat anti-mouse antibody, Triton X-100 are purchased from Beijing Zhong Shan Golden Bridge biotechnology Company (ZSGB-BIO), China；4% paraformaldehyde, which is purchased from, steps morning scientific and technological (Beijing) Co., Ltd, China；Glycine is purchased from traditional Chinese medicines Group's chemical reagent Beijing Co., Ltd, China；Phosphate buffer (PBS), purchased from Beijing Ding Guo Bioisystech Co., Ltd； Disposable cell culture experiments articles for use (including Tissue Culture Plate, blake bottle, sterile centrifugation tube, etc.) be purchased from healthy and free from worry (Corning) Company, the U.S..The a ten thousandth gram precision electronic balances of BT-25S ten, Sartorius companies, Germany；Cell culture incubator, Thermo Company, the U.S.；AirTECH cell super-clean benches, the safe and sound companies of Su Jing, China；TDZ4-WS low speed desk centrifuges, the centrifugation of Hunan instrument Machine Instrument Ltd., China；Inverted microscope, Chongqing optics electro-kinetic instrument company, China；LEICA TCS-SP8 laser is total to Focusing microscope (Confocal laser scanning fluorescent microscope), LEICA companies, Germany；Stream Formula cell instrument (BD FACS Calibur), Becton Dickinson companies, the U.S.；The high intension analysis systems of Operetta, Perkin Elmer companies, the U.S..

The portion of material and instrument of following embodiments 5 are as follows：

BALB/c male nude mouses (18-20g) are purchased from Peking University's Experimental Animal Center, China；Glioma U87-MG is thin Born of the same parents' strain is purchased from Tumour Inst., Chinese Medical Academy；Hyclone, PAN companies, Germany；MEM culture mediums, non-essential amino Acid, pancreatin are purchased from Beijing Mai Chen Science and Technology Ltd.s, China；Lowlenthal serum is purchased from the limited public affairs of Zhejiang day Hangzhoupro biotechnology share Department, China；Hoechst 33342 is purchased from Invitrogen companies, the U.S.；Disposable syringe (1mL, 2mL and 5mL) is purchased from Becton Dickinson (BD) company, the U.S.；DiR fluorescent dyes, purchased from Sigma companies, the U.S.；anti-nestin Antibody is purchased from AbCam companies, Britain；Alexa488-conjugated goat anti-rabbit Antibody, Triton X-100 are purchased from Beijing biotech company of Zhong Shan Golden Bridge (ZSGB-BIO), China；4% paraformaldehyde Purchased from morning advanced in years scientific and technological (Beijing) Co., Ltd, China；Phosphate buffer (PBS), purchased from the limited public affairs of Beijing ancient cooking vessel state biotechnology Department；Disposable cell culture experiments articles for use (including Tissue Culture Plate, blake bottle, sterile centrifugation tube, etc.) purchased from healthy and free from worry (Corning) company, the U.S..

The a ten thousandth gram precision electronic balances of BT-25S ten, Sartorius companies, Germany；Cell culture incubator, Thermo are public Department, the U.S.；AirTECH cell super-clean benches, the safe and sound companies of Su Jing, China；TDZ4-WS low speed desk centrifuges, Hunan instrument centrifuge Instrument Ltd., China；Inverted microscope, Chongqing optics electro-kinetic instrument company, China；LEICA TCS-SP8 laser is copolymerized Focusing microscope (Confocal laser scanning fluorescent microscope), LEICA companies, Germany；Brain is stood Body position indicator, Shenzhen Rui Wode Life Sciences Co., Ltd, China；Caikon XDS-300C systems, Shanghai Cai Kang optical instruments Co., Ltd, China；IVIS Spectrum living imaging systems, Perkin Elmer companies, the U.S..

All data are represented with Mean ± SD.Using one-way analysis of variance (ANOVA), then by Bonferroni schools Test, P<0.05 thinks there is significant difference.Survival rate data are shown with kaplan-Meier survival curve, and are examined with log-rank Test and do statistical procedures, P<0.05 thinks there is significant difference.

The structure and sign of embodiment 1, functionalization vinblastine liposome

First, the structure of functionalization vinblastine liposome

1、TfR-T₁₂-PEG₂₀₀₀- DSPE synthesis

TfR-T₁₂-PEG₂₀₀₀- DSPE is under the conditions of existing for catalyst, by TfR-T₁₂Polypeptide and NHS-PEG₂₀₀₀- DSPE carries out acylation reaction and obtained；Wherein TfR-T₁₂Polypeptide and NHS-PEG₂₀₀₀- DSPE molar ratio is 1:1.

Specific synthetic method is as follows：

8 μm of ol TfR-T are added into reaction bulb₁₂Polypeptide (THRPPMWSPVWP) and 8 μm of ol NHS-PEG₂₀₀₀- DSPE, Dissolved with 2mL DMF, and add 200 μ L catalyst ns-methyl morpholine.It is transferred under normal temperature after stirring reaction 48h in bag filter (molecular cut off 3000Da), it is placed in deionized water and dialyses 24 hours, removes solvent and unreacting material.It is afterwards that sample is cold It is lyophilized dry, it is placed in -20 DEG C of preservations.

Product is verified using MALDI TOFMS (MALDI-TOF-MS), uses 2,5- bis- Hydroxybenzoic acid (DHB) is as detection matrix.

As a result as shown in Fig. 2 the MALDI-TOF-MS collection of illustrative plates of product, the average molecular mass that product is shown in figure is 4441.77Da with reactant PEG₂₀₀₀Difference between-DSPE molecular weight (3000.00Da) is 1441.77Da, this and TfR- T₁₂Molecular weight (1490.80Da) coincide, illustrate target product TfR-T₁₂-PEG₂₀₀₀- DSPE is synthesized successfully.

2nd, the preparation of functionalization vinblastine liposome

1) common vinblastine liposome

In molar ratio 60:30:3 precisions weigh lecithin (EPC), cholesterol (CHOL), PEG₂₀₀₀- DSPE is in eggplant-shape bottle In, by volume 3:1 adds dichloromethane and methanol, has been removed under reduced pressure with Rotary Evaporators under 40 DEG C of water-baths, 90rpm rotating speeds Solvent, obtain one layer of uniform film and be affixed on eggplant-shape bottle bottom.Add 50mL ammonium sulfate (250mM) aqueous solution and carry out aquation：First Water bath sonicator 5min, completely falls off to film, and system is in the thick liposome of uniform milky；Thick liposome is shifted afterwards Into EP pipes, with JY92-IID type ultrasonic cell disruptors further ultrasonic 10min (the setting ultrasound works time is 1s, Intermittent time is 1s, and protection temperature is 30 DEG C, power 200W).After ultrasound terminates, obtain with the translucent of faint blue-opalescent Liquid, the liquid is extruded and by the poly- carbon ester film that aperture is 200nm, obtained liquid is transferred in bag filter (retention point Son amount 8000-14000Da), the dialysis 24h in HBS cushioning liquid (151mM NaCl, 25.2mM Hepes, pH 7.4), per 6h Change liquid once.After dialysis terminates, blank liposomes liquid solution is obtained.

By medicine fat mass ratio 1:20 (w/w) precision weighing vinblastines are added in 25mL eggplant-shape bottles, are added methanol dissolving, are used Methanol is removed under reduced pressure under 40 DEG C of water-baths, 90rpm rotating speeds in Rotary Evaporators, obtains dispersed vinblastine.Utilize sulfuric acid Ammonium gradient carries medicine method, and the blank liposomes liquid solution of above-mentioned acquisition is added in the eggplant-shape bottle, 20min is shaken in 60 DEG C of water-baths, Obtain common vinblastine liposome.

2)TfR-T₁₂The vinblastine liposome of modification

It is identical with common vinblastine method for preparing lipidosome, but by PEG in film forming procedure₂₀₀₀- DSPE replaces with above-mentioned 1) TfR-T prepared₁₂-PEG₂₀₀₀- DSPE, obtain TfR-T₁₂The vinblastine liposome of modification.

3)Stearyl-R₈The vinblastine liposome of modification

It is identical with common vinblastine method for preparing lipidosome, but in molar ratio 60 in film forming procedure:30:3:5 accurate titles Take lecithin (EPC), cholesterol (CHOL), PEG₂₀₀₀-DSPE、stearyl-R₈In eggplant-shape bottle, Stearyl-R is obtained₈Modification Vinblastine liposome.

4) functionalization vinblastine liposome

It is identical with common vinblastine method for preparing lipidosome, but in molar ratio 60 in film forming procedure:30:3:5 accurate titles Take lecithin (EPC), cholesterol (CHOL), TfR-T₁₂-PEG₂₀₀₀-DSPE、stearyl-R₈In eggplant-shape bottle, functionalization is obtained Vinblastine liposome.

Fig. 3 is the schematic diagram of functionalization vinblastine liposome, and lecithin constitutes the bilayer of liposome, has stream Dynamic property, while insert the rigidity of cholesterol increase liposome.Functionalization material TfR-T₁₂-PEG₂₀₀₀- DSPE and stearyl-R₈ Modification actively enters the interior aqueous phase of liposome in surface of liposome, water-soluble vinblastine under ammonium sulphate gradient effect.

2nd, characterize

1st, vinblastine content assaying method

1) chromatographic condition

Chromatographic column：Luna C18 posts (Phenomenex, C18column, 5 μm, 250x4.6mm)；Mobile phase：Methanol- 0.02M sodium dihydrogen phosphates (70:30, v/v)；Detection wavelength：267nm；Flow velocity：1.0mL/min；Sample size：20 μ L, measure Temperature：25℃.

2) standard curve

Precision weighing vinblastine, add proper amount of methanol dissolve and be diluted to concentration be respectively 0.78,1.56,3.12, 6.25th, 12.50,25.00,50.00,100.00 μ g/mL standard liquid.According to the chromatogram of above-mentioned chromatographic condition measure each sample Peak area A values.Using vinblastine concentration C as independent variable, linear regression is carried out to its corresponding chromatographic peak area A value, tries to achieve length The standard curve (Fig. 4) of spring flower alkali.Standard curve is y=19476x+1348.2, R²=1.

Vinblastine such as Fig. 5 of the chromatogram under this liquid-phase condition, its retention time are good in 7min or so, peak shape.3) Return

Yield

Appropriate blank liposome is taken, the methanol for adding nonuploid product is destroyed.Then accurately weighed catharanthus roseus is added Alkali reference substance is appropriate, adds flowing phased soln, is formulated as the solution of the μ g/mL containing vinblastine 12.50,25.00,50.00, mistake successively Filter, peak area is determined with above-mentioned chromatographic condition respectively, calculate the rate of recovery.

As a result as shown in table 1, display, the rate of recovery is in the range of 96.49%-98.56%.

Table 1 is that HPLC-UV is determined in liposome to the rate of recovery of vinblastine

Notes:Data are presented as mean ± standard deviation (SD, n=5)

4) precision

Appropriate blank liposome is taken, the methanol for adding nonuploid product destroys.Then accurately weighed vinblastine pair is added It is appropriate according to product, add flowing phased soln, the solution of the μ g/mL containing vinblastine 12.50,25.00,50.00 is formulated as successively, on 5th Peak area inside is determined with above-mentioned chromatographic condition respectively, calculates in a few days and in the daytime relative standard deviation.

As a result as shown in Table 2, the in a few days and in the daytime relative deviation RSD of the method measure vinblastine<2%, the measure side Method precision is good.

Table 2 is the measure of HPLC-UV method precisions

Notes:Data are presented as mean ± standard deviation (SD, n=5)

5) test limit

It is appropriate that precision weighs vinblastine reference substance, adds methanol to dissolve and dilute, it is molten to prepare standard successively by doubling dilution Liquid, according to the chromatographic peak area of above-mentioned chromatographic condition determination sample.When signal to noise ratio is about 3:Detectable concentration when 1 is this method Minimum detection limit.

As a result such as Fig. 6 is shown, the detection of this method measure vinblastine is limited to 0.024 μ g/mL.

2nd, the measure of envelop rate

The μ L of functionalization blank liposome 500 are taken by Sephadex G-50 sephadex columns, with HBS cushioning liquid (25mM HEPES, 150mM NaCl, pH 7.4) is mobile phase presaturation sephadex column.Take drug-loaded liposome (general afterwards Logical vinblastine liposome, TfR-T₁₂Vinblastine liposome, the stearyl-R of modification₈Vinblastine liposome, the work(of modification Vinblastine liposome can be changed) 500 μ L are by Sephadex G-50 sephadex columns, using HBS cushioning liquid as mobile phase point From the free vinblastine not wrapped into liposome, the liposome after separation is collected, nonuploid product methanol is added and destroys, and with flowing After phase dilution, it is measured with above-mentioned high performance liquid chromatography.The liposome stoste for not crossing gel column is taken, adds nonuploid product first Alcohol destroys, and with after the identical multiple of flowing phase dilution, is measured with above-mentioned high performance liquid chromatography.

The envelop rate of vinblastine in each liposome is calculated using equation below：

Envelop rate %=crosses amount × 100% of the amount of gel column liposome drug containing/do not cross gel column liposome drug containing.

Drug concentration Standard reference (comparing one point method) calculates, and produces.

Envelop rate of the vinblastine in each group liposome is all higher than 97.0%, is shown in Table 3.

Table 3 is the physicochemical characterization of liposome

Notes:Data are presented as mean ± standard deviation (SD, n=3)

3rd, the measure of particle diameter and Zeta potential

1) measure of particle diameter

Common vinblastine liposome, TfR-T are taken respectively₁₂Vinblastine liposome, the stearyl-R of modification₈Modification Vinblastine liposome, functionalization vinblastine liposome, 20 times are diluted with PBS, takes 1mL to add sample cell, is dissipated using laser The particle diameter (angle of wavelength 633nm, incident light and scattered beam is 90 °) of radion footpath analyzer measure liposome, each sample Equilibration time is 120s, determines 20 circulation times, and measurement temperature is set as 25 DEG C.The last measurement result of each sample is 20 The average value of secondary measurement result.The uniformity coefficient of polydispersity expression particle size, the smaller then particle of polydispersity are more uniform.

As a result as shown in table 3, the average grain diameter of each liposome in the range of 90-120nm, polydispersity coefficient (PDI, Polydisperisty indexes) between 0.176-0.253.

2) measure of Zeta potential

After having determined particle diameter, by common vinblastine liposome, TfR-T₁₂Vinblastine liposome, the stearyl- of modification R₈Vinblastine liposome, the functionalization vinblastine liposome of modification are transferred in current potential cup that (solution will flood conductive copper Piece), it is equilibration time 120s to set Zeta potential condition determination, and at least 20 circulation times of measure, measurement temperature are set every time For 25 DEG C.The last measurement result of each sample is the average value of 20 measurement results.

As a result as shown in table 3, stearyl-R₈The liposome Zeta potential of modification is just, other groups of liposomes shows slightly negative Electricity.

4th, morphological observation

1) AFM (AFM) is observed

Using deionized water as decentralized medium, by unmodified common vinblastine liposome and functionalization catharanthus roseus Alkali liposome is diluted to 100 μ g/mL (fat material concentration) and with 0.2 μm of filtering with microporous membrane.10 μ L dilutions are taken to be applied to silicon chip table Face, it is stored at room temperature after drying, is detected and recorded with AFM tapping-mode.

As a result as shown in fig. 7, A1 is common vinblastine liposome, A2 is functionalization vinblastine liposome, atomic force Under microscope, two kinds of liposomes are rendered as surface rounding, smooth form, particle diameter 100nm or so, with particle size results phase Symbol.

2) transmission electron microscope (TEM) is observed

Using deionized water as decentralized medium, by unmodified common vinblastine liposome and functionalization catharanthus roseus Alkali liposome is diluted to 1 μ g/mL (fat material concentration) and with 0.2 μm of filtering with microporous membrane.Then, the copper mesh for being covered with carbon film is placed on On liposome solutions, taken out after 1min, after being blotted with filter paper, it is double that the copper mesh drift that capture there are liposome particles is placed on 1% acetic acid On the oxygen uranium aqueous solution, take out after 1min and blotted with filter paper.After left at room temperature over night dries, using transmission electron microscope instrument in 120kV Under be observed and record.

As a result such as Fig. 8 shows that A1 is common vinblastine liposome, and A2 is functionalization vinblastine liposome, transmission electricity Under mirror, two kinds of liposomes are rendered as the spherical of the smooth of the edge rounding, particle diameter 100nm or so, are consistent with particle size results.

5th, drug release rate

Extracorporeal releasing experiment is carried out in the dissolution medium (PBS for containing 10% hyclone) containing haemocyanin. The common vinblastine liposomes of 1mL, TfR-T are taken respectively₁₂Vinblastine liposome, the stearyl-R of modification₈The catharanthus roseus of modification Alkali liposome, functionalization vinblastine liposome add in bag filter (8000-14000Da), and add 1mL dissolution mediums, use Suture is placed in after tightening in the cillin bottle equipped with 20mL dissolution mediums, is put into shaking table, starts to shake under the conditions of 37 DEG C, 100rpm Swing.0.2mL dissolution mediums are taken out respectively at 1,2,4,8,12,24h, and fill into the new release of equal volume after every sub-sampling immediately Medium.

Each part sample methanol (sample taken out:Methanol=2:8, v/v) destruction dilution is carried out, separates out albumen, at a high speed Centrifugation, then film is crossed with filter membrane, high molecular weight protein is removed, peak area corresponding to vinblastine is measured in each sample with HPLC, Calculate concentration using the equation of linear regression of gained, so as to calculate each sample in burst size at different moments, obtain its The cumulative release amount at a certain moment.

Release in vitro rate is calculated with following formula：

Release in vitro rate %=(amount of medicine in the i-th time point release liquid/with dialysis volume identical dialysis proliposome The amount of drug in solution) × 100%.

Each group liposome such as Fig. 9 of the releasing result in dissolution medium, as a result shows, length of each group liposome in 24h Spring flower alkali release rate is below 12%.

The above results show that functionalization vinblastine liposome is by functionalization material TfR-T₁₂-PEG₂₀₀₀- DSPE and stearyl-R₈Modify on liposome and wrapped into using ammonium sulphate gradient by vinblastine obtained by liposome.

Embodiment 2, functionalization vinblastine liposome are trained to glioma and its stem cell target lethal effect cell Support

First, cell culture

Brain glioblastoma cell U87-MG cells are cultivated in the MEM culture mediums containing 10%FBS and 1% nonessential amino acid, Condition of culture is 37 DEG C, containing 5%CO₂。

The induction of human brain glioma stem cells (glioma stem cells, GSCs) and culture (Yu SC；Ping YF；Yi L；Zhou ZH；Chen JH；Yao XH；Gao L；Wang JM；Bian XW.Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87[J].Cancer Letters,2008,265(1):124-34)：With every mL 2 × 10⁴The concentration of individual U87-MG cells is containing 2%Serum-free Replenishers, 10ng/mL LIF, 20ng/mL EGF, 20ng/mL bFGF, the DMEM-F12 culture mediums of 181.6ng/mL insulin The middle culture that suspends, condition of culture is 37 DEG C, containing 5%CO₂.Identified after culture four weeks.

Mouse brain capillary endothelial cell BMVECs containing 20%FBS, 100U/mL penicillin, 100 μ g/mL streptomysins, 40U/mL heparin, 100ug/mL ECGF DMEM culture mediums in cultivate, condition of culture be 37 DEG C, containing 5%CO₂.Blake bottle shifts to an earlier date 30min is handled with 2% gelatin solution.The compound method of 2% gelatin solution：2g gelatin is weighed, with 100ml Hank ' s solution (80 DEG C) scattered, swelling, filtration sterilization, 4 DEG C of preservations.

2nd, glioma and its stem cell TfR mark

BSA buffer solutions：500mg BSA and 74.45mg EDTA are added in per 100mL PBSs.

Respectively collect GSCs (B), U87-MG cells (A), add BSA buffer solutions cell is resuspended, by cell dispense to In 1.5mL EP pipes, 1 × 10 is often added in pipe⁷Individual cell.5 μ L Anti- are added into GSCs and U87-MG experimental group Human Transferrin Receptor (CD71) antibody-FITC, while add 5 μ L Isotype controls into control group and resist Body, it is incubated at room temperature 20min.Cell is washed twice with PBS afterwards, is resuspended with 300 μ L PBS and is crossed 400 mesh sieves into streaming pipe, on Lucifuge ice bath preserves before machine.

Determined using flow cytometer FACScan flow cytometer, come using forward angle light scatter and side scatter Exclude double cell masses.The excitation wavelength for detecting FITC is 488nm, launch wavelength 530nm.

As a result such as Figure 10 is shown, expression feelings of the TfR in brain glioblastoma cell (A) and its stem cell (B) Condition, showing, TfR has expression on two kinds of cells, wherein, the expression quantity in U87-MG is 94.65% (A).

3rd, the identification of human brain glioma stem cells

Human brain glioma stem cells GSCs is induced and characteristic indication thing is identified after cultivating four weeks, including nestin (nestin)、ABCG2、CXCR4.Equivalent U87-MG cells are taken to be compareed simultaneously.

The cell ball for the culture that suspends is centrifuged, is single cell suspension with the digestion of 0.05% pancreatin, dispenses to 1.5mL EP and manage It is interior, 1 × 10 is often added in pipe⁷Individual cell.

For nestin mark：With the paraformaldehyde of 4% ice in 4 DEG C of fixed 10min, then with 0.2%Triton X- 100 solution are incubated 10min to Cell-transmission model at room temperature, and 10 μ L Anti- are added into the experimental group of GSCs and U87-MG cells Human Nestin antibody-FITC, while 10 μ L isotype control Abs are added into control group, fully piping and druming mixes, room The lower lucifuge of temperature is incubated 30min.Cell is washed twice with PBS afterwards, is resuspended with 300 μ L PBS and is crossed 400 mesh sieves into streaming pipe, on Lucifuge ice bath preserves before machine.

For ABCG2 mark：With the paraformaldehyde of 4% ice in 4 DEG C of fixed 10min, to GSCs and U87-MG cells Experimental group in add 5 μ L Anti-Human ABCG2 (CD338) antibody-FITC, while 5 μ L are added into control group Isotype control Ab, fully piping and druming mix, and lucifuge is incubated 30min at room temperature.Cell is washed twice with PBS afterwards, with 300 μ L PBS It is resuspended and crosses 400 mesh sieves into streaming pipe, lucifuge ice bath preserves before upper machine.

For CXCR4 mark：Use Anti-Human CXCR4 antibody-PE and corresponding Isotype antibody, step Same ABCG2.

Determined using flow cytometer FACScan flow cytometer, come using forward angle light scatter and side scatter Exclude double cell masses.The excitation wavelength for detecting FITC is 488nm, launch wavelength 530nm；Detection FITC excitation wavelength be 488nm, launch wavelength 564nm.

As a result as shown in figure 11, ABCG2 is in brain glioblastoma cell (A₁) and its stem cell (A₂) in expression；CXCR4 is in brain Glioma cell (B₁) and its stem cell (B₂) in expression；Nestin is in brain glioblastoma cell (C₁) and its stem cell (C₂) in Expression；Figure 11 B₁With Figure 11 B₂It has been shown that, expression quantity of the ABCG2 in U87-MG and GSCs is respectively 5.76% and 23.93%； Figure 11 B₁With Figure 11 B₂It has been shown that, expression quantity of the CXCR4 in U87-MG and GSCs is respectively 22.80% and 98.60%；Figure 11 C₁ With Figure 11 C₂It has been shown that, expression quantity of the nestin in U87-MG and GSCs is respectively 39.36% and 97.77%.As a result show, three Expression of the kind characteristic indication thing in GSCs increases than U87-MG, shows to induce stem cell success.

4th, to brain glioblastoma cell and its lethal effect of stem cell

1. cell is inoculated with

U87-MG the and GSCs cells of digestion exponential phase are collected, respectively with each hole 4 × 10³(180 μ L are trained individual cell Support base) it is inoculated on 96 orifice plates, if six multiple holes, at 37 DEG C, containing 5%CO₂Incubator in be incubated 24h.

2. dosage regimen

Free vinblastine, common vinblastine liposome, the TfR-T of series concentration are prepared with serum free medium₁₂Repair Vinblastine liposome, the stearyl-R of decorations₈Vinblastine liposome, the functionalization vinblastine liposome of modification.Add per hole Enter 20 μ L, make final concentration of 0-50 μM of vinblastine.96 well culture plates after dosing, it is placed in 37 DEG C, containing 5%CO₂Culture It is incubated in case, continues to be incubated 48h.

3. measure and calculating

After incubation terminates, 96 well culture plates are taken out, discard culture medium, the trichloroacetic acid (TCA) 200 of 10% ice is added per hole μ L, placed under the conditions of 4 DEG C and fix cell more than 1h.Then with 5 times culture plates of water washing are distilled, to remove trichloroacetic acid. After being dried overnight in air, to every μ L 0.4% of Kong Zhongjia 100 SRB (being prepared with 1% glacial acetic acid), 15min is placed at room temperature After discard liquid, washed 5 times with 1% acetic acid, to remove uncombined dyestuff, 200 μ L added per hole after air drying 10mmol/L Tris base (pH 10.5) dissolve, and vibrate 30min on oscillator plate (decolorization swinging table), whole to dyestuff Dissolving.Put in ELIASA and determined under 540nm wavelength per hole OD value (OD).Using after the dosing culture cell deposit Percentage (Cell survival rate, %) live to evaluate the toxic action of each preparation on cells.Cell survival percentage is pressed Calculated according to equation below：

As a result as shown in figure 12, one group of block diagram under each concentration is followed successively by：Free vinblastine, vinblastine lipid Body, TfR-T₁₂The vinblastine liposome of modification, stearyl-R₈The vinblastine liposome of modification, functionalization vinblastine fat Plastid, functionalization blank liposome；Figure 12 A1 and Figure 12 A₂Represent each preparation group to external U87-MG and GSCs cells respectively Depression effect.As a result show, functionalization vinblastine liposome has most strong Proliferation Ability to make for U87-MG and GSCs With.Proliferation Ability ability sequence consensus of each preparation group to U87-MG and GSCs：Functionalization vinblastine liposome>stearyl- R₈The vinblastine liposome of modification>TfR-T₁₂The vinblastine liposome of modification>Common vinblastine liposome>Free Changchun Flower alkali>Functionalization blank liposome.Functionalization vinblastine liposome suppresses U87-MG and GSCs cell proliferations.

Embodiment 3, functionalization vinblastine liposome are in the effect across blood-brain barrier

First, cell culture

2nd, BMVECs surfaces TfR marks

BSA buffer solutions：500mg BSA and 74.45mg EDTA are added in per 100mL PBSs.

BMVECs cells are collected, adding BSA buffer solutions is resuspended cell, and cell is dispensed into 1.5mL EP pipes, often managed It is middle to add 1 × 10⁷Individual cell.5 μ L Anti-Mouse Transferrin Receptor are added into BMVECs experimental group (CD71) antibody-FITC, while 5 μ L isotype control Abs are added into control group, it is incubated at room temperature 20min.Cell afterwards Washed twice with PBS, be resuspended with 300 μ L PBS and cross 400 mesh sieves into streaming pipe, lucifuge ice bath preserves before upper machine.

As a result if Figure 13, expression quantity of the TfR in BMVECs are 17.68%.

2nd, the foundation and evaluation of blood-brain barrier model

With the advance coated cell inserter 30min of 2% gelatin, by BMVECs with every hole 1 × 10⁴The density of individual cell adds Into the cell inserter being coated with (per hole 1mL culture mediums), and add in lower room the culture medium of 1mL U87-MG cells.Often Change a subculture within two days, co-culture 6 days.

The close connection that BMVECs is formed is observed under the microscope, carries out preliminary assessment；It is thin using the detection of cell resistance instrument Born of the same parents' inserter and the transepithelial electrical resistance (TEER) in lower room.It was found that the blood-brain barrier model TEER values established are all higher than 300 Ω·cm².Therefore, in vitro blood-brain barrier model is successfully established.

3rd, the foundation of BMVECs/U87-MG co-culture models

With every hole 2 × 10³Density add U87-MG cells (1mL culture mediums) in 12 orifice plates, and will before be covered with BMVECs cell inserter is transferred to above 12 orifice plates, at 37 DEG C, containing 5%CO₂Incubator in co-incubation 24h, produce To BMVECs/U87-MG co-culture models.

4th, across killing glioma effect after blood-brain barrier

Each group preparation is separately added into the cell inserter of the BMVECs/U87-MG co-culture models of foundation, to prescription Case is as follows：Free vinblastine, common vinblastine liposome, TfR-T₁₂Vinblastine liposome, the stearyl-R of modification₈ Vinblastine liposome, the functionalization vinblastine liposome of modification, to add the holes of blank cultures as blank control group, Hole not to be covered with BMVECs is used as positive controls, the final concentration of 50nM of vinblastine.At 37 DEG C, containing 5%CO₂Culture After being incubated 48h in case, liquid is discarded, the trichloroacetic acids of 2mL 10% are added per hole, 1h is placed in 4 DEG C of refrigerators and fixes cell.Training Support each hole of plate to be washed with deionized 5 times, to remove TCA.After drying in atmosphere, add 1mL 0.4%SRB per hole, at room temperature 15min is placed, discards in each hole and is washed 5 times with 1% glacial acetic acid-aqueous solution after liquid, uncombined dyestuff is removed, is done in air It is dry after with pH 10.5,10mmol/L Tris alkali 2mL dissolving, 30min is vibrated on oscillator plate, put in ELIASA in Determined under 540nm wavelength per hole trap OD values, calculate survival rate.Survival rate=(A_540nmPreparation group/A_540nmControl group) x 100%, A_540nmIt is the absorbance under 540nm.

As a result as shown in figure 14,1, dissociate vinblastine；2, vinblastine liposome；3, TfR-T₁₂The vinblastine of modification Liposome；4, stearyl-R₈The vinblastine liposome of modification；5, functionalization vinblastine liposome；Represent each preparation group across More depression effect of the in vitro blood-brain barrier to external U87-MG cells.As a result show, functionalization vinblastine liposome has most Strong has most strong inhibited proliferation across in vitro blood-brain barrier ability and for U87-MG.Each group survival rate order be： Blank control group>Free vinblastine>Common vinblastine liposome>stearyl-R₈The vinblastine liposome of modification> TfR-T₁₂The vinblastine liposome of modification>Functionalization vinblastine liposome>Positive controls.Wherein, in positive controls, BMVECs is not laid in cell inserter, the false positive results that are likely to occur in experimentation are excluded with this.

The liposome-induced glioma apoptosis effect of embodiment 4, functionalization vinblastine

First, the liposome-induced glioma apoptosis effect of functionalization vinblastine

1. cell is inoculated with

By brain glioblastoma cell U87-MG and human brain glioma stem cells GSCs with every hole 5 × 10⁵The density difference of individual cell It is inoculated in 6 orifice plates, and adds 2mL culture mediums, at 37 DEG C, containing 5%CO₂Incubator in be incubated 24h.

2. dosage regimen

Culture medium is discarded, is separately added into free vinblastine, common vinblastine fat that 2mL is prepared with serum free medium Plastid, TfR-T₁₂Vinblastine liposome, the stearyl-R of modification₈Vinblastine liposome, the functionalization vinblastine of modification Liposome solutions, it is 30nM to make vinblastine concentration；Blank group adds serum free medium, is placed in 37 DEG C, containing 5%CO₂Training Support and be incubated in case.After being incubated 12h, with PBS cell 2 times, cell dissociation is got off and centrifuged using the pancreatin without EDTA Cell is collected, is scattered in 300 μ l Binding buffer and (is provided in kit), 400 mesh sieves is crossed into streaming pipe, adds 10 μ L Annexin V-KeyFluor 647, room temperature lucifuge are incubated 15min, and 5min adds 10 μ L7-AAD before upper machine.Use 2mM Dioxygen water process cell 2h individually marks as positive control, and with Annexin V-kFluor647 and 7-AAD.

3. measure and calculating

Determined using flow cytometer FACScan flow cytometer, come using forward angle light scatter and side scatter Exclude double cell masses.The excitation wavelength for detecting KeyFluor 647 is 651nm, launch wavelength 667nm；Detect 7-AAD's Excitation wavelength is 546nm, launch wavelength 647nm.Rational cross door is being set using single standard specimen notebook data, and is carrying out data Processing.

Apoptosis rate=experimental group apoptosis rate-blank group apoptosis rate

Apoptosis induction effect such as Figure 15 (a, blank control of the preparation to brain glioblastoma cell and its stem cell；B, length of dissociating Spring flower alkali；C, vinblastine liposome；D, TfR-T₁₂The vinblastine liposome of modification；E, stearyl-R₈The catharanthus roseus of modification Alkali liposome；F, functionalization vinblastine liposome) and Figure 16 (a, blank control；B, dissociate vinblastine；C, vinblastine fat Plastid；D, TfR-T₁₂The vinblastine liposome of modification；E, stearyl-R₈The vinblastine liposome of modification；F, functionalization length Spring flower alkali liposome) shown in.Cross door determines that each result has been divided into four parts by it according to feminine gender group and two single sun groups, Lower-left (Lower Left, LL) represents normal cell, and bottom right (Lower Right, LR) represents apoptosis early stage cell, upper right (Upper Right, UR) represents apoptosis late cell or non-viable non-apoptotic cell.

As a result show, functionalization vinblastine liposome is respectively provided with most strong apoptosis to brain glioblastoma cell and its stem cell Inductive effect, and apoptosis early stage (being early apoptosis in cross door lower right area) is mainly reflected in, and apoptosis late period is without obvious Change.

For brain glioblastoma cell, the order of each preparation induction early apoptosis is：Functionalization vinblastine liposome (40.98%)>stearyl-R₈The vinblastine liposome (28.90%) of modification>TfR-T₁₂The vinblastine liposome of modification (24.01%)>Free vinblastine (23.79%)>Vinblastine liposome (17.82%).

For human brain glioma stem cells, the order of each preparation induction early apoptosis is：Functionalization vinblastine liposome (15.36%)>stearyl-R₈The vinblastine liposome (10.90%) of modification>TfR-T₁₂The vinblastine liposome of modification (9.70%)>Free vinblastine (7.23%)>Vinblastine liposome (7.29%).

2nd, to the damage effect of brain glioblastoma cell core and mitochondria

By brain glioblastoma cell U87-MG with every hole 5 × 10³It is thin that the density of individual cell (180uL culture mediums) is inoculated in 96 holes In born of the same parents' culture plate, at 37 DEG C, containing 5%CO₂Incubator in be incubated 24h.Free vinblastine, common is added into each group respectively Vinblastine liposome, TfR-T₁₂Vinblastine liposome, the stearyl-R of modification₈The vinblastine liposome of modification, function Change vinblastine liposome solutions, it is 30nM to make vinblastine concentration；Blank group adds serum free medium.Every group of preparation is set 6 multiple holes, in 37 DEG C, 5%CO₂It is incubated in incubator.After 12h, culture medium is discarded, PBS is washed 2 times, and it is red to add mitochondria Fluorescence probe (Mitotracker Deep Red FM, 200nM) and Hoechst 33342 (5 μ g/mL) are incubated at 37 DEG C 30min, then, add 200 μ L serum free mediums with PBS twice.

Picture is gathered to every hole under 10 times of eyepieces using Operetta High content screenings system and determines fluorescence intensity.

Picture is gathered to each group using Operetta high intensions, gathered picture is analyzed with Columbus system The degree of variation of middle nucleus, and quantified.Cell nuclear damage ratio=experimental group nucleus Variation factor/blank group Nucleus Variation factor.

As a result as Figure 17 (1, blank control；2, dissociate vinblastine；3, vinblastine liposome；4, TfR-T₁₂Modification Vinblastine liposome；5, stearyl-R₈The vinblastine liposome of modification；6, functionalization vinblastine liposome.) shown in, Cell nuclear damage ratio order be：Functionalization vinblastine liposome (1.64 ± 0.12)>stearyl-R₈The catharanthus roseus of modification Alkali liposome (1.43 ± 0.08)>TfR-T₁₂The vinblastine liposome (1.44 ± 0.08) of modification>Vinblastine liposome (1.21±0.09)>Free vinblastine (1.15 ± 0.05).

Picture is gathered to each group using Operetta high intensions, gathered picture is analyzed with Columbus system The degree of variation of middle nucleus, and quantified.Injury of mitochondria ratio=experimental group mitochondria Variation factor/blank group Mitochondria Variation factor.As a result as Figure 18 (1, blank control；2, dissociate vinblastine；3, vinblastine liposome；4, TfR-T₁₂The vinblastine liposome of modification；5, stearyl-R₈The vinblastine liposome of modification；6, functionalization vinblastine Liposome) shown in, injury of mitochondria ratio order is：Functionalization vinblastine liposome (1.281 ± 0.013)>stearyl- R₈The vinblastine liposome (1.226 ± 0.022) of modification>TfR-T₁₂The vinblastine liposome (1.215 ± 0.023) of modification >Free vinblastine (1.141 ± 0.027)>Vinblastine liposome (1.137 ± 0.035).

3rd, to the regulation and control of brain glioblastoma cell reactive oxygen species

By brain glioblastoma cell U87-MG with every hole 5 × 10⁵The density of individual cell is inoculated in 6 orifice plates, 37 DEG C, contain 5%CO₂Incubator in be incubated 24h.

Culture medium is discarded, is separately added into free vinblastine, common vinblastine fat that 2mL is prepared with serum free medium Plastid, TfR-T₁₂Vinblastine liposome, the stearyl-R of modification₈Vinblastine liposome, the functionalization vinblastine of modification Liposome solutions, it is 30nM to make vinblastine concentration；Blank group adds serum free medium, is placed in 37 DEG C, containing 5%CO₂Training Support and be incubated in case.After being incubated 12h, 500 μ L DCFH-DA (1 μM) are added into each hole, at 37 DEG C with PBS cell twice After lower incubation 0.5h, cell is collected by centrifugation, cell is resuspended with 300 μ L PBS and crosses 400 mesh sieves into streaming pipe.

Determined using flow cytometer FACScan flow cytometer, come using forward angle light scatter and side scatter Exclude double cell masses.The excitation wavelength of detection is 485nm, launch wavelength 525nm.

Each group cell be induced produce active oxygen horizontal result such as Figure 19 (1, blank control；2, dissociate vinblastine；3, Vinblastine liposome；4, TfR-T₁₂The vinblastine liposome of modification；5, stearyl-R₈The vinblastine liposome of modification； 6, functionalization vinblastine liposome) shown in, each group reactive oxygen species order is：Functionalization vinblastine liposome> stearyl-R₈The vinblastine liposome of modification>TfR-T₁₂The vinblastine liposome of modification>Vinblastine liposome>It is free Vinblastine.

4th, to the damage effect of destruction brain glioblastoma cell micro-pipe

By brain glioblastoma cell U87-MG with every hole 2 × 10⁵The density of individual cell is inoculated in Glass bottom culture dish, 37 DEG C, containing 5%CO₂Incubator in be incubated 24h.

After 24h, culture medium is discarded, is separately added into free vinblastine, common Changchun that 1mL is prepared with serum free medium Flower alkali liposome, TfR-T₁₂Vinblastine liposome, the stearyl-R of modification₈The vinblastine liposome of modification, functionalization length Spring flower alkali liposome solutions, it is 30nM to make vinblastine concentration；Blank group adds serum free medium.It is placed in 37 DEG C, containing 5% CO₂Incubator in be incubated.After being incubated 3h, add micro-pipe red fluorescence probe (Tubulin- with PBS cell twice Tracker Red) 0.5h is incubated, add Hoechst 33342 (5 μ g/mL) and be incubated 10min, then, add with PBS twice Enter 1mL serum free mediums, IMAQ is carried out using laser confocal microscope.

Each preparation is as shown in figure 20 to the destruction result of micro-pipe in brain glioblastoma cell, a, blank control；B, Changchun of dissociating Flower alkali；C, vinblastine liposome；D, TfR-T₁₂The vinblastine liposome of modification；E, stearyl-R₈The vinblastine of modification Liposome；F, functionalization vinblastine liposome；In blank group (Figure 20 a), it may be observed that clearly thread tubulin, micro-pipe Prop up whole cell and be in strip fusiform, be the representative configuration of glioma.And in functionalization vinblastine liposome group (figure In 20f), it is impossible to which, it was observed that thread tubulin, whole cell also loses original shape and is rounded.Each preparation group is to brain glue Microtubule disruption ability order is in matter oncocyte：Functionalization vinblastine liposome>stearyl-R₈The vinblastine fat of modification Plastid (Figure 20 e)>TfR-T₁₂The vinblastine liposome (Figure 20 d) of modification>Vinblastine liposome (Figure 20 c)>Free Changchun Flower alkali (Figure 20 b).

5th, the mechanism of brain glioblastoma cell apoptosis is induced

By brain glioblastoma cell U87-MG with every hole 5 × 10³It is thin that the density of individual cell (180uL culture mediums) is inoculated in 96 holes In born of the same parents' culture plate, at 37 DEG C, containing 5%CO₂Incubator in be incubated 24h.Free vinblastine, common is added into each group respectively Vinblastine liposome, TfR-T₁₂Vinblastine liposome, the stearyl-R of modification₈The vinblastine liposome of modification, function Change vinblastine liposome solutions, it is 30nM to make vinblastine concentration；Blank group adds serum free medium.Every group of preparation is set 6 multiple holes, in 37 DEG C, 5%CO₂It is incubated in incubator.After 6h, culture medium is discarded, PBS is washed 2 times, adds 4% paraformaldehyde Fixed 15min, cold PBS are washed 2 times；Add the PBS punchings 15min containing 0.5%Triton X-100 and 0.3M glycine Afterwards, 2h is closed using the PBS room temperatures containing 5% sheep blood serum.It is separately added into and dilutes corresponding times according to specification with antibody diluent Several anti-caspase 3/7antibody, anti-caspase 8antibody, anti-caspase 9antibody, anti-Bax antibody、anti-cytochrome c antibody、anti-MCL-1antibody、anti-Forkhead Box protein O1 (FoxO1) antibody and anti-LC3B antibody, 4 DEG C of overnight incubations, then (are contained with PBST 0.05%Tween20 PBS solution) embathe 3 times, each 2min.Corresponding to adding afterwards488 secondary antibodies (1: 500 dilutions) room temperature lucifuge incubation 2h, is embathed 3 times, each 2min with PBS.Add (the 5 μ g/ of core dyestuff Hoechst 33342 Ml 10min) is incubated, is embathed 3 times with PBS, each 2min.Finally, mounting liquid (PBS for containing 90% glycerine) is added, 4 DEG C of lucifuges are protected Deposit.

The fluorescence intensity in each hole is determined under 20 times of eyepieces using Operetta High content screenings system, is used in combination Columbus system carry out data processing.

1. pro apoptotic protein caspase-3/7

Utilize pro apoptotic protein Caspase in the high intension measure each group cells of Operetta (caspase-3/7) expression, with Columbus system calculate fluorescence intensity in each group. Caspase-3/7 expresses multiple=experimental group fluorescence intensity/blank group fluorescence intensity.As a result display (Figure 21,1, blank control； 2, dissociate vinblastine；3, vinblastine liposome；4, TfR-T₁₂The vinblastine liposome of modification；5, stearyl-R₈Modification Vinblastine liposome；6, functionalization vinblastine liposome), caspase-3/7 order of representation is：Functionalization vinblastine Liposome (1.24 ± 0.05)>TfR-T₁₂The vinblastine liposome (1.15 ± 0.02) of modification>stearyl-R₈The length of modification Spring flower alkali liposome (1.13 ± 0.03)>Free vinblastine (1.11 ± 0.01)>Vinblastine liposome (1.03 ± 0.02).

2. pro apoptotic protein caspase-8

Utilize pro apoptotic protein Caspase in the high intension measure each group cells of Operetta (caspase-8) expression, with Columbus system calculate fluorescence intensity in each group.caspase-8 Express multiple=experimental group fluorescence intensity/blank group fluorescence intensity.As a result display (Figure 22,1, blank control；2, dissociate catharanthus roseus Alkali；3, vinblastine liposome；4, TfR-T₁₂The vinblastine liposome of modification；5, stearyl-R₈The vinblastine fat of modification Plastid；6, functionalization vinblastine liposome), caspase-8 order of representation is：Functionalization vinblastine liposome (1.28 ± 0.02)>TfR-T₁₂The vinblastine liposome (1.24 ± 0.04) of modification>stearyl-R₈The vinblastine liposome of modification (1.22±0.02)>Vinblastine liposome (1.17 ± 0.02)>Free vinblastine (1.10 ± 0.05).

3. pro apoptotic protein caspase-9

Utilize pro apoptotic protein Caspase in the high intension measure each group cells of Operetta (caspase-9) expression, with Columbus system calculate fluorescence intensity in each group.caspase-9 Express multiple=experimental group fluorescence intensity/blank group fluorescence intensity.As a result display (Figure 23,1, blank control；2, dissociate catharanthus roseus Alkali；3, vinblastine liposome；4, TfR-T₁₂The vinblastine liposome of modification；5, stearyl-R₈The vinblastine fat of modification Plastid；6, functionalization vinblastine liposome), caspase-9 order of representation is：Functionalization vinblastine liposome (2.20 ± 0.04)>stearyl-R₈The vinblastine liposome (2.09 ± 0.02) of modification>TfR-T₁₂The vinblastine liposome of modification (2.00±0.12)>Vinblastine liposome (1.65 ± 0.05)>Free vinblastine (1.57 ± 0.03).

4. pro apoptotic protein Bax

Using the expression of pro apoptotic protein Bax in the high intension measure each group cells of Operetta, Columbus is used System fluorescence intensity in each group is calculated.Bax expression multiple=experimental group fluorescence intensity/blank group fluorescence is strong Degree.As a result display (Figure 24,1, blank control；2, dissociate vinblastine；3, vinblastine liposome；4, TfR-T₁₂The length of modification Spring flower alkali liposome；5, stearyl-R₈The vinblastine liposome of modification；6, functionalization vinblastine liposome), Bax expression Sequentially it is：Functionalization vinblastine liposome (1.13 ± 0.03)>TfR-T₁₂Modification vinblastine liposome (1.10 ± 0.02)>stearyl-R₈The vinblastine liposome (1.09 ± 0.01) of modification>Vinblastine liposome (1.04 ± 0.03)> Free vinblastine (1.01 ± 0.03).

5. cromoci (Cytochrome C)

Using the yield of cromoci (Cytochrome C) in the high intension measure each group cells of Operetta, use Columbus system fluorescence intensity in each group is calculated.Cytochrome C express multiple=experimental group fluorescence Intensity/blank group fluorescence intensity.As a result display (Figure 25,1, blank control；2, dissociate vinblastine；3, vinblastine liposome； 4, TfR-T₁₂The vinblastine liposome of modification；5, stearyl-R₈The vinblastine liposome of modification；6, functionalization catharanthus roseus Alkali liposome), Cytochrome C order of representation is：Functionalization vinblastine liposome (1.54 ± 0.02)>stearyl-R₈ The vinblastine liposome (1.47 ± 0.04) of modification>TfR-T₁₂The vinblastine liposome (1.40 ± 0.04) of modification>It is free Vinblastine (1.37 ± 0.02)>Vinblastine liposome (1.29 ± 0.04).

6. anti-apoptotic proteins Mcl-1

Using the expression of anti-apoptotic proteins Mcl-1 in the high intension measure each group cells of Operetta, Columbus is used System fluorescence intensity in each group is calculated.Mcl-1 expression multiple=experimental group fluorescence intensity/blank group fluorescence is strong Degree.As a result display (Figure 26,1, blank control；2, dissociate vinblastine；3, vinblastine liposome；4, TfR-T₁₂The length of modification Spring flower alkali liposome；5, stearyl-R₈The vinblastine liposome of modification；6, functionalization vinblastine liposome), Mcl-1 tables Up to being sequentially：Vinblastine liposome (1.03 ± 0.02)>Free vinblastine (0.86 ± 0.03)>TfR-T₁₂The Changchun of modification Flower alkali liposome (0.85 ± 0.03)>stearyl-R₈The vinblastine liposome (0.69 ± 0.02) of modification>Functionalization Changchun Flower alkali liposome (0.54 ± 0.01).

7. autophagy GAP-associated protein GAP LC3B

Using the expression of autophagy GAP-associated protein GAP (LC3B) in the high intension measure each group cells of Operetta, use Columbus system fluorescence intensity in each group is calculated.LC3B expresses multiple=experimental group fluorescence intensity/blank Group fluorescence intensity.As a result display (Figure 27,1, blank control；2, dissociate vinblastine；3, vinblastine liposome；4, TfR-T₁₂ The vinblastine liposome of modification；5, stearyl-R₈The vinblastine liposome of modification；6, functionalization vinblastine lipid Body), LC3B order of representation is：Functionalization vinblastine liposome (1.55 ± 0.05)>stearyl-R₈The vinblastine of modification Liposome (1.48 ± 0.05)>TfR-T₁₂The vinblastine liposome (1.45 ± 0.05) of modification>Vinblastine liposome (1.36 ±0.04)>Free vinblastine (1.34 ± 0.05).

8. autophagy GAP-associated protein GAP FoxO1

Using the expression of autophagy GAP-associated protein GAP (FoxO1) in the high intension measure each group cells of Operetta, use Columbus system fluorescence intensity in each group is calculated.FoxO1 expresses multiple=experimental group fluorescence intensity/sky White group fluorescence intensity.As a result display (Figure 28,1, blank control；2, dissociate vinblastine；3, vinblastine liposome；4, TfR- T₁₂The vinblastine liposome of modification；5, stearyl-R₈The vinblastine liposome of modification；6, functionalization vinblastine lipid Body), FoxO1 order of representation is：Functionalization vinblastine liposome (1.14 ± 0.02)>stearyl-R₈The vinblastine of modification Liposome (1.09 ± 0.01)>TfR-T₁₂The vinblastine liposome (1.08 ± 0.02) of modification>Free vinblastine (1.05 ± 0.01)>Vinblastine liposome (1.03 ± 0.01).

The above results show that (1) functionalization vinblastine liposome is respectively provided with most to brain glioblastoma cell and its stem cell Strong apoptosis induction effect, and apoptosis early stage is mainly reflected in, and apoptosis late period is without significant change；(2) functionalization vinblastine Cell and and injury of mitochondria maximum of the liposome to brain glioblastoma cell；(3) each group reactive oxygen species order is：Functionalization is grown Spring flower alkali liposome>stearyl-R₈The vinblastine liposome of modification>TfR-T₁₂The vinblastine liposome of modification>Catharanthus roseus Alkali liposome>Free vinblastine；(4) functionalization vinblastine liposome has micro-pipe in most strong destruction brain glioblastoma cell Ability；(5) functionalization vinblastine lipid physical efficiency significantly improve pro apoptotic protein caspase-3/7, caspase-8, Caspase-9, Bax and cromoci (Cytochrome C) level, and anti-apoptotic proteins Mcl-1 level is reduced, simultaneously Autophagy GAP-associated protein GAP FoxO1 and LC3B expression increase can be caused.

Embodiment 5, anti-glioma and its stem cell effect in nude mouse

First, the foundation and evaluation of lotus glioma nude mice model

1. animal

Only, starting weight is about 18-20g to BALB/c male nude mouses number.

2. cell prepares

Cell is in exponential phase, with cell is resuspended in serum free medium after digestion, is prepared as every 3 μ L containing 2 × 10⁵ Individual U87-MG brain glioblastoma cells, and cell suspension is placed in 4 DEG C of ice baths, it is stand-by.

3. brain glioblastoma cell inoculation method

Using Naoliqing capsule technology, human brain glioma stem cells are inoculated in the cranium brain of BALB/c nude mices, establish brain glue Matter knurl primary tumor animal model, operating procedure are as follows：

(1) weigh；

(2) anaesthetize, it is fixed：After 4% chloraldurate (1mL/100g) intraperitoneal injection of anesthesia, stereotaxic apparatus is fixed on On；

(3) sterilization, otch and drilling：It is longitudinal backward along endocanthion line midpoint with 75% alcohol disinfecting mouse skin of vertex Cut scalp 1cm, exposure skull.According to Naoliqing capsule collection of illustrative plates：1.0mm after bregma, on the right 2.0mm of sagittal suture skull, with Electric drill drills, and to prevent from puncturing endocranium, only permits drilling through skull depth 1.0-1.5mm；

(4) inoculation human brain glioma stem cells in right caudate nucleus in brain：3.0 μ L gliomas are sucked using 20 μ L micro syringes Stem cell suspension is (containing about 6 × 10⁵Individual cell), vertical inserting needle depth 3.5mm (away from endocranium), 0.5mm is returned to after standing 1min, with 1 μ L/min speed injects 3.0 μ L cell suspensions in caudate nucleus.Let the acupuncture needle remain at a certain point after injection 5min, slowly pulls out pin, bone hole is used immediately Bacteria-free bone wax is closed, and visual area normal saline flushing, meets conjunction otch.

Daily regular check its consciousness, haptoreaction, movement defect, cranial nerve lesion, vision response, observation have unbiased Paralysed, aged and epileptic attack.During Post operation about two weeks, the obvious pathological characters of observable, judge whether to can be used for being administered.

4. model evaluation

After 12 days, 100ml PBS and 100ml 4% is carried out to postanesthetic normal BALB/c male nude mouses and tumor bearing nude mice Paraformaldehyde cardiac perfusion, take out brain tissue, done otch by inoculation point of puncture and done continuous frozen section (4 μm), and carried out Nissl's staining, observed using Caikon XDS-300C systems.

Normal observation is carried out to glioma tumor bearing nude mice, it is found that nude mice 0.5h from after being inoculated with brain glioblastoma cell revives. After operation 5 days, tumor bearing nude mice feed is reduced to be increased with drinking-water, and action is reduced, and body weight gradually mitigates, and bilateral or offside then occurs Reflection infringement, generally become apparent with hind leg, skin tarnishes；(the about the 10th day) during the late stages of developmet, tumor bearing nude mice is shown as Face is unclean, in aged face, lassitude, and slow to stimulate the reaction, body depressed neck part, occasionally there is epileptic attack in shape is rolled up.

Internal Brain Glioma Model is evaluated using pathological examination, is as a result shown, normal cerebral tissue (Figure 29) contaminates through Nissl Visible cell is sparse after color, and each provincial characteristics is obvious；And the brain tissue (Figure 30) for being inoculated with glioma is visible after Nissl's staining Brain glioblastoma cell dense accumulation in groups, in invasive growth.Illustrate that internal Brain Glioma Model is successfully established.

2nd, the living imaging of tumor bearing nude mice

The preparation of 1.DiR liposomes

Precision weighs each group liposome materials and DiR, by DiR：Fat material=1:500 (w/w) are added in eggplant-shape bottle, dissolving In the methanol of proper volume, methanol is removed by 40 DEG C of vacuum drying of rotary evaporation, one is formed in eggplant-shape bottle bottom and inwall The very thin uniform films of layer.Appropriate HEPES buffer solution (10mM, pH7.4,150mM NaCl) is added into eggplant-shape bottle again at 60 DEG C Aquation 30min in water-bath, room temperature is cooled to, excessive DiR is filtered to remove by 0.2 μm of polycarbonate membrane, produces DiR marks Targeted hposome.

2. the living imaging in tumor bearing nude mice body

Brain glioblastoma cell is inoculated with after 12 days, the tumor-bearing mice mould for being successfully established Brain Glioma Model is randomly divided into 5 groups, Every group of 3 difference tail vein injections administration, dosage regimen are as follows：

(1) physiological saline blank control group；

(2) free DiR groups (2.0mg/kg)；

(3)TfR-T₁₂The DiR liposomes group (2.0mg/kg) of modification；

(4)stearyl-R₈The DiR liposomes group (2.0mg/kg) of modification；

(5) functionalization DiR liposomes group (2.0mg/kg)；

1 after administration, 3,6,12,24,48h, utilize IVIS Spectrum Pre-clinical In Vivo Imaging The functionalization liposome of System investigation DiR marks real-time positioning scenarios in Mice Body.And after 48h, every group of mouse is put to death, The heart, liver, spleen, lung, kidney and lotus knurl brain tissue are taken out immediately, and different tissues are imaged.

Targeting effect and medicine real-time distribution of the functionalization liposome of DiR marks in glioma tumor bearing nude mice body are such as Shown in Figure 31, a, physiological saline group；B, dissociate DiR；C, TfR-T₁₂The DiR liposomes of modification；D, stearyl-R₈The DiR of modification Liposome；E, functionalization DiR liposomes.In functionalization DiR liposome groups (Figure 31 e), during 1h, brain fluorescence intensity is maximum, its He is evenly distributed at position, increases over time, and brain fluorescence slightly weakens, and liver region fluorescence is most strong in 3h, Zhi Housui Time decrease, other positions have no significant medicine fluorescence；Fluorescence is had no in physiological saline group (Figure 31 a)；Free DiR groups In (Figure 31 b), fluorescence concentrates on liver region, and fluorescence intensity is increased over time and strengthened, and brain has no fluorescence；TfR-T₁₂ The DiR liposome groups (Figure 31 c) and stearyl-R of modification₈In the DiR liposome groups (Figure 31 d) of modification, brain has certain Fluorescence, liver region fluorescence intensity is maximum, and other positions also have certain fluorescence.

The functionalization liposome of DiR marks drug distribution when glioma tumor bearing nude mice respectively organizes 48h is as shown in figure 32, A, physiological saline group；B, dissociate DiR；C, TfR-T₁₂The DiR liposomes of modification；D, stearyl-R₈The DiR liposomes of modification；E, Functionalization DiR liposomes.In each group, functionalization DiR liposome group (Figure 32 e) deutocerebral region fluorescence is most strong, TfR-T₁₂The DiR of modification Liposome group (Figure 32 c) deutocerebral region also has strong fluorescence, stearyl-R₈DiR liposome group (Figure 32 e) deutocerebral region of modification With week fluorescent, free DiR group (Figure 32 b) deutocerebral region of physiological saline group (Figure 32 a) has no fluorescence.Except physiological saline group, its Remaining each group center, liver, spleen, lung, kidney have certain fluorescence, but mainly concentrate on liver.

3rd, the anti-glioma effect in tumor bearing nude mice body

1. life cycle is investigated

Brain glioblastoma cell is inoculated with after 12 days, the tumor-bearing mice mould for being successfully established Brain Glioma Model is randomly divided into 6 groups, Every group of 9 difference tail vein injections administration, dosage regimen are as follows：

(1) physiological saline blank control group；

(2) free vinblastine group (50 μ g/kg)；

(3) vinblastine liposome group (50 μ g/kg)；

(4)TfR-T₁₂The vinblastine liposome group (50 μ g/kg) of modification；

(5)stearyl-R₈The vinblastine liposome group (50 μ g/kg) of modification；

(6) functionalization vinblastine liposome group (50 μ g/kg)；

Give a medicine within every three days, be administered three times altogether.After administration, every group takes 9 mouse, and tumor bearing nude mice behavior state is entered daily Row observation, records its activity situation, symptom, date of death, investigates survivorship curve.Draw Kaplan-Meier survivorship curve (Kaplan-Meier survival curves), and calculate life span median (the median survival Times, MeST) and life span average value (the mean survival times, MST).Life cycle is calculated as follows Increase percentage (the percentage-increased life span, ILS%)：

t：Represent the nude mice for receiving treatment, U：Represent the nude mice for not receiving treatment.

Figure 33 is that glioma tumor bearing nude mice receives the Kaplan-Meier survivorship curve after each preparation group treatment, and a is raw Manage salt solution group；B, vinblastine group of dissociating；C, vinblastine liposome group；D, TfR-T₁₂The vinblastine liposome group of modification； E, stearyl-R₈The vinblastine liposome group of modification；F, functionalization vinblastine liposome group.As a result show, functionalization length Spring flower alkali liposome group has maximum median survival interval (29 days), and 52.63% is added than physiological saline group (19 days).This Outside, compared with physiological saline group, free vinblastine group (20 days) adds 5.26%, and vinblastine liposome group increases for (21 days) 10.53%, TfR-T is added₁₂The vinblastine liposome group (26 days) of modification adds 36.84%, stearyl-R₈Modification Vinblastine liposome group (27 days) adds 42.11%.As a result show, compared with control formulation, functionalization vinblastine fat Plastid can significantly improve the therapeutic action to glioma tumor bearing nude mice brain tumor.

2. anti-human brain glioma stem cells effect in body

After being inoculated with brain glioblastoma cell and drug treatment according to the method described above, physiological saline blank control group, catharanthus roseus are taken Alkali liposome group and each three of functionalization vinblastine liposome group tumor bearing nude mice, not to be inoculated with the healthy naked of brain glioblastoma cell Mouse is negative control group, through physiological saline and 4% paraformaldehyde cardiac perfusion after anesthesia, take out brain tissue (while take out the heart, Liver, spleen, lung, kidney are standby), sucrose dehydrated overnight.By mouse brain surface seeding point of puncture as otch, continuous frozen section (4 is done μm)。

Section is dipped in 0.2%Triton X-100 solution after 30min progress Cell-transmission model, is transferred to containing 10% goat 5h is closed in the PBS solution of serum, with anti-nestin antibody (1：100 dilutions) it is incubated overnight under the conditions of 4 DEG C, then With anti-rabbit secondary antibody conjugated with Alexafluor-488 (1：500 dilutions) and Hoechst 33342 ((5 μ g/mL)) is incubated 1h.Glioma in observation each group is carried out using laser confocal scanning microscope Stem cell situation.

Immunofluorescence dyeing is carried out using stem cell labeling thing nestin, to evaluate tumor bearing nude mice brain brain glue after administration The quantity of matter knurl stem cell.Immunofluorescence dyeing result is as shown in figure 34, a, normal cerebral tissue；B, the lotus brain of injecting normal saline Glioma brain tissue；C, inject the lotus glioma brain tissue of vinblastine liposome；D, function of injection vinblastine lipid The lotus glioma brain tissue of body, blue-fluorescence is the nucleus dyed by Hoechst 33342, by thin to all brain tissues Born of the same parents' mark sketches the contours of full brain form；Green fluorescence is nestin pigmented sections, can serve to indicate that human brain glioma stem cells.From figure In as can be seen that Normal brain cortical tissue (not being inoculated with brain glioblastoma cell) region, nucleus is evenly distributed and more sparse； And tumor tissue sections, nucleus distribution are fine and close, it is seen that glioma region.The therapeutic effect of each preparation group, Ke Yiyong Nestin expression region accounts for the ratio in full brain area domain as evaluation criterion.As a result show, in functionalization vinblastine liposome group, The fluorescence area marked by nestin is minimum, in Local map, it was observed that tumour cell is less and distribution is sparse；And in physiology salt In water group, the fluorescence area marked by nestin is larger, has accounted for the area of right brain about half, and cell is more in Local map It is intensive；In vinblastine liposome, the fluorescence area marked by nestin is more compared with functionalization vinblastine liposome group, and in brain Portion to form two focuses there occurs transfer, and Local map shows that cell is very intensive.Anti- glioma is dry thin in vivo for each preparation group Born of the same parents' effect is ordered as：Functionalization vinblastine liposome group>Vinblastine liposome group>Physiological saline group.

4th, safety evaluatio

The paraformaldehyde of above-mentioned middle the taken heart, liver, spleen, lung, nephridial tissue 4% is fixed, is prepared as paraffin section (4 μ M), HE (hematoxylin-eosin) dyeing is carried out, and utilizes the observation of Caikon XDS-300C systems.

As a result as shown in figure 35, a, physiological saline group；B, functionalization vinblastine liposome group and physiological saline group it is each Tissue HE coloration results compare, and respectively tissue obvious pathological change to functionalization vinblastine liposome group nude mice does not occur.

In summary, through functional vector material TfR-T₁₂-PEG₂₀₀₀- DSPE and stearyl-R₈The function of modification simultaneously Changing liposome has most obvious tumor region drug accumulation and most strong anti-glioma and human brain glioma stem cells effect, energy Dramatically increase the life span of tumor bearing nude mice.

Claims

1. a kind of liposome or vinblastine liposome,

The liposome is poly- through TfR binding peptide-PEG2000-DSPE and stearyl eight Arginine is modified；

The vinblastine liposome is by the liposome and contains the vinblastine in the liposome and forms；

TfR binding peptide-the PEG2000-DSPE is by TfR-T₁₂Polypeptide and NHS- PEG₂₀₀₀The product that-DSPE is obtained by acid amides key connection.

2. a kind of method for preparing vinblastine liposome described in claim 1, for TfR binding peptide-poly- second two Alcohol-DSPE and the poly arginine modified liposome of stearyl eight, and vinblastine is contained in the fat Vinblastine liposome is obtained in plastid.

3. according to the method for claim 2, it is characterised in that：Methods described comprises the following steps：

1) TfR binding peptide-PEG2000-DSPE is prepared；

The preparation method comprises the following steps：By TfR-T₁₂Polypeptide and NHS-PEG₂₀₀₀- DSPE enters in the presence of catalyst Row acylation reaction, obtain TfR binding peptide-PEG2000-DSPE；

2) with lecithin, cholesterol, TfR binding peptide-PEG2000-DSPE and The poly arginine of stearyl eight carries out film formation reaction, liposome after being modified, liposome after being modified；

4. according to the method for claim 3, it is characterised in that：

In step 1), the TfR-T₁₂Polypeptide and NHS-PEG₂₀₀₀- DSPE molar ratio is 1:1-3:1；

Or, the catalyst is N-methylmorpholine；

Or, the acylation reaction is carried out in a solvent；

Or, the solvent is specially DMF.

5. the method according to claim 3 or 4, it is characterised in that：

In step 2), the lecithin, cholesterol, institute's TfR binding peptide-polyethylene glycol-distearoylphosphatidyl second The molar ratio of hydramine and the poly arginine of the stearyl eight is 60:30:3:5.

6. according to any described method in claim 3-5, it is characterised in that：

In step 3), the mass ratio that feeds intake of liposome is 1 after the vinblastine and the modification:20.

7. the TfR binding peptide-polyethylene glycol-two being prepared in claim 3-6 in any methods described Stearyl phosphatidyl monoethanolamine；

Or liposome after the modification being prepared in claim 3-6 in any methods described.

8. TfR binding peptide-PEG2000-DSPE described in claim 6 is preparing fat Plastid prepares vinblastine liposome or modified liposome or the application modified in vinblastine liposome；

Or the vinblastine liposome described in claim 1 or the TfR binding peptide described in claim 6-poly- second two Alcohol-DSPE is being prepared with following 1) -9) application in the product of at least one function：

1) brain glioblastoma cell or its stem cell are killed；

2) brain glioblastoma cell or its stem cells hyperplasia are suppressed；

3) blood-brain barrier is crossed over；

4) brain glioblastoma cell or its stem cell apoptosis are promoted；

9) prevent and/or treat glioma.

9. one kind has following 1) -9) product of at least one function, its active component is the vinblastine described in claim 1 Liposome；

1) brain glioblastoma cell or its stem cell are killed；

2) brain glioblastoma cell or its stem cells hyperplasia are suppressed；

3) blood-brain barrier is crossed over；

4) brain glioblastoma cell or its stem cell apoptosis are promoted；

9) prevent and/or treat glioma.

10. the product described in application according to claim 8 or claim 9, it is characterised in that：The rush apoptosis is related Albumen is caspase-3/7, caspase-8, caspase-9, Bax, cromoci, Mcl-1, LC3B or FoxO1；

Or the product is medicine or kit.