CN108653743A - Double target liposomes of a kind of heart and brain and its preparation method and application - Google Patents

Double target liposomes of a kind of heart and brain and its preparation method and application Download PDF

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CN108653743A
CN108653743A CN201810299429.3A CN201810299429A CN108653743A CN 108653743 A CN108653743 A CN 108653743A CN 201810299429 A CN201810299429 A CN 201810299429A CN 108653743 A CN108653743 A CN 108653743A
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peg
heart
brain
lip
dspe
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CN108653743B (en
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杨宝峰
彭海生
刘肖莹
李明慧
廉明明
唐淑坤
刘云翠
柴彦群
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Harbin Engineering University
Harbin Medical University
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • A—HUMAN NECESSITIES
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/335—Polymers modified by chemical after-treatment with organic compounds containing phosphorus
    • C08G65/3356—Polymers modified by chemical after-treatment with organic compounds containing phosphorus having nitrogen in addition to phosphorus

Abstract

The invention discloses double target liposomes of a kind of heart and brain and its preparation method and application.The double target liposomes of the heart and brain are obtained after the surface for the liposome modified by the acetylation sugar ester polyethylene glycol phosphatidyl-ethanolamine conjugate shown in Formulas I is coupled Anti cTnI protein antibodies or its segment.The present invention in surface of liposome by modifying mannose molecules derivative and Anti cTnI protein antibodies so that the liposome is provided with Brain targeting and the dual-targeting function of heart targeting, can realize that heart and brain are same and control.It proves that the liposome can both treat cardiomyopathies through cell assay in vitro, affinity experiment, internal distribution experiments, at the same time also may span across blood-brain barrier, treat the encephalopathy caused by Cardiac disease.In addition, can change the spatial position of glycan molecule derivative, antibody by changing surface of liposome PEG (Linker) length, to change distribution of the drug in heart and brain tissue, regulating medicine realizes individualized treatment in the heart, brain changes in distribution.

Description

Double target liposomes of a kind of heart and brain and its preparation method and application
Technical field
The liposome and its preparation method and application that the present invention relates to a kind of to use as pharmaceutical carrier, more particularly to it is a kind of The double targetings for having the function of brain and heart can realize heart and brain with the liposome and preparation method thereof controlled.The invention belongs to Pharmaceutical technology field.
Background technology
Cardiovascular and cerebrovascular disease (aase's syndrome, cardiac and cerebral blood supply insufficiency etc.) seriously threatens human health, has higher dead Die rate and incidence.Cardiovascular and cerebrovascular disease along with heart and brain blood supply deficiency situation.Due to myocardial ischemia, heart, which is in, to be lacked The state of weary energy can cause arrhythmia cordis and angina pectoris.When this occurs, acute myocardial infarction AMI is also resulted in, often Caused cardiogenic shock.At the same time, blood supply in brain is because it is also to be reduced by core function abnormality that cardiac output, which declines, sometimes It is cardiogenic cerebral apoplexy to deteriorate.The treatment of cardiogenic cerebral apoplexy relies primarily on the recovery of the normal and blood supply of heart function.
In recent years, researcher has studied many strategies based on nanotechnology, effectively by drug transport to disease Stove, to restore the function of infracted cardiac.In these strategies, ligand and receptor are generally selected, antibody or antigen binding and logical The molecule of transport protein transhipment is crossed to modify the surface of nano-carrier, to improve specificity of the nano particle to Ischemic Heart.To the greatest extent Pipe achieves some improvement, and most of particles still run up to reticuloendothelial system, and reach destination organization in advance.In order to overcome These above-mentioned problems, some seminars devise some double target nano-carriers based on two kinds of different biological phenomenons and mechanism, such as Transport protein, antibody and receptor.
Inventor has proven to anti-cTnI antibody (Anti-cTnI, a species specificity for surface of liposome modification early period Monoclonal antibody) modification can improve the drug concentrations of myocardial sites in surface of liposome.However, cardiac muscle cell absorbs Anti- The speed of cTnI antibody modification liposomes is fast unlike conventional liposome.Glucose transporter (GLUT) can pass through blood brain screen Hinder (BBB) and transports glucose, fructose, galactolipin, mannose and other substances with similar structure.The lipid of mannose-modified Body surface reveals excellent brain targeting, and the liposome of mannose-modified can rapidly enter target cell.These discoveries tell me , if we modify antibody and mannose simultaneously in surface of liposome simultaneously, it will vesica is accelerated to enter impacted cell Internalization speed, save those and be destined to dead cells.
First choice of the polyethylene glycol (PEG) as many researchers with 2000Da chains passes through due to the availability in market Click chemistry is easily used for targeted molecular conjugation to vesicle surface.The different densities and chain of the polyethylene glycol of modified liposome The possibility that length can cause intermolecular interaction and cellular uptake different with accumulation is targeted.However, the various molecules of lipid film surface Steric hindrance may limit the combination of ligand and its receptor, to influence the realization that nano-carrier targets efficiency.With it is common Drug is compared, and target administration has dosage low, the high significant feature of curative effect, and the treatment of Central nervous systemic disease is with important Meaning.Although myocardium targeted therapy has been explored for many years, there are still some side effects and inefficient challenge.In addition, people It always concentrates on the drug concentration in single target organs and/or tissue, and has ignored some internal companies Lock reactor.Cardiogenic cerebral apoplexy may need the treatment of brain and ischemic heart, systematically ensure the function of heart and brain It is not influenced by ischemic state, reduces subsequent disaster.Therefore, how to be realized by shadow based on the steric hindrance of above-mentioned PEG chains The requirement of loud heart and brain realizes that rational drug delivery is an attractive research hotspot.
Invention content
The purpose of the present invention is to provide one kind having brain and heart dual-targeting function, and heart and brain can be realized with the fat controlled Plastid and preparation method thereof.
In order to achieve the above object, present invention employs following technological means：
A kind of double target liposomes (the Anti-cTnI Ab-PEG/Ac of heart and brain of the present invention4MAN-PEG-LIP), it is to pass through Liposome (the Ac of the acetylation sugar ester shown in Formulas I-mPEG2000-DSPE conjugate modification4MAN-PEG-LIP) Surface coupling Anti-cTnI protein antibodies or its segment after obtain；
Wherein, n=10~120, preferably 13~45, it is furthermore preferred that the polyethylene glycol is Macrogol 600, poly- second Glycol 1000 or polyethylene glycol 2000.
Wherein, it is preferred that acetylation sugar ester-mPEG2000-DSPE conjugate shown in Formulas I passes through following step Suddenly it is prepared：In methylene chloride by p- to carboxyl phenyl-α-D- acetylmannosamines (Ac4) and polyethylene glycol (PEG) MAN PEG-Ac is formed by condensation reaction4MAN, then by distearoylphosphatidylethanolamine (DSPE) and PEG-Ac4MAN is carried out Coupling, obtains DSPE-PEG-Ac4MAN, as Formulas I compound represented.
Wherein, it is preferred that acetylation sugar ester-mPEG2000-DSPE conjugate shown in Formulas I passes through following step Suddenly it is prepared：Nitrine-polyethylene glycol-carboxyl is dissolved in tetrahydrofuran, Pd/c and acetic acid are added later, is passed through at room temperature Hydrogen reacts a night；After crude product is filtered, rotary evaporated to dryness is dry, is then dissolved in dichloromethane, and triethylamine and p- pairs is added Carboxyl phenyl-α-D- acetylmannosamines (Ac4MAN), a night is reacted at room temperature, is isolated and purified, and PEG-Ac is obtained4MAN；It will PEG-Ac4MAN is dissolved in dichloromethane, sequentially adds n-hydroxysuccinimide (NHS) and 1- ethyls-(3- dimethylaminos Propyl) carbodiimide hydrochloride (EDCI), it reacts at room temperature overnight, isolates and purifies, obtain succimide polyethylene glycol acetyl Mannoside；Succimide polyethylene glycol acetylated mannan glucosides is dissolved in dichloromethane, TEA is added and is dissolved in chloroform Distearoylphosphatidylethanolamine (DSPE), reacts a night at room temperature, is extracted 2 times with saturation NaCl, isolates and purifies, obtain DSPE-PEG-Ac4MAN, as Formulas I compound represented.
Further, the invention also provides a kind of method preparing the double target liposomes of the heart and brain, including it is following Step：
(1) liposome (Ac of acetylation sugar ester-mPEG2000-DSPE conjugate modification4MAN-PEG-LIP) Preparation
By acetylation sugar ester-polyethylene glycol-phospholipids shown in egg PC (EPC), cholesterol (CHO) and Formulas I Acyl ethanol amine conjugate is dissolved in absolute ethyl alcohol in revolving bottle, and by evaporation under reduced pressure, the mixture forms thin fat Plasma membrane；With the normal saline solution aquation lipid film；So that liposome solutions is disperseed by being ultrasonically treated, and passes through polycarbonate membrane It squeezes out, obtains the liposome of acetylation sugar ester-mPEG2000-DSPE conjugate modification, be named as Ac4MAN-PEG- Liposomal samples are stored in 4 DEG C by LIP, spare；
(2) liposome preparation (Anti-cTnIAb-PEG/Ac of Anti-cTnI antibody modifications4MAN-PEG-LIP)
Synthesize Anti-cTnI Ab-DSPE-PEG solution：By Anti-cTnI antibody and DSPE-PEG- maleimides point It is not dissolved in HEPES solution, is then blended at 4 DEG C overnight, obtains Anti-cTnIAb-DSPE-PEG solution；By Anti- The Ac that cTnI Ab-DSPE-PEG solution is prepared with step (1)4MAN-PEG-LIP liposome solutions incubate 2 hours at 37 DEG C, system It is standby to obtain the Ac of Anti-cTnI antibody modifications4MAN-PEG-LIP is named as Anti-cTnI-PEG/Ac4MAN-PEG-LIP, i.e., For the double target liposomes of heart and brain.
Wherein, it is preferred that acetylation sugar ester-polyethylene glycol-phosphorus acyl ethyl alcohol shown in EPC in step (1), CHO and Formulas I The molar ratio of amine conjugate is (40-49)：(50-55)：(1-5), more preferably 49：50：1.
Wherein, Anti-cTnI Ab-DSPE-PEG and Ac in step (2)4The molar ratio of MAN-PEG-LIP liposomes is (1-100)：(50-1000), more preferably 1：500.
Wherein, it is preferred that the DSPE-PEG- maleimides described in step (2) are DSPE-PEG600- maleimides Amine, DSPE-PEG1000- maleimides or DSPE-PEG2000- maleimides.
Further, the invention also provides double target liposomes the answering in preparing pharmaceutical carrier of the heart and brain With.
Wherein, the double target liposomes of the heart and brain have the function of double targetings of brain and heart, to realize heart and brain With the purpose controlled.
Wherein, it is preferred that the drug is for treating cardiomyopathies and cardiogenic encephalopathy, it is furthermore preferred that described Cardiogenic encephalopathy includes cardiogenic cerebral ischemia syndrome, cardiogenic cerebral insufficiency and cardiogenic cerebral apoplexy.
Compared to the prior art, the beneficial effects of the invention are as follows：
1, the present invention is made by modifying mannose molecules derivative and Anti-cTnI protein antibodies in surface of liposome It obtains the liposome and is provided with Brain targeting and the dual-targeting function of heart targeting, can realize that heart and brain are same and control.It is tried through cell in vitro Test, affinity experiment, internal distribution experiments prove that the liposome can both treat cardiomyopathies, at the same time also may span across blood Brain barrier treats the encephalopathy caused by Cardiac disease.
2, the space bit of glycan molecule derivative, antibody can be changed by changing surface of liposome PEG (Linker) length It sets, to change distribution of the drug in heart and brain tissue, regulating medicine realizes individualized treatment in the heart, brain changes in distribution.
Description of the drawings
Fig. 1 is DSPE-PEG600-Ac4MAN、DSPE-PEG1000-Ac4MAN and DSPE-PEG2000-Ac4The nuclear-magnetism of MAN Resonate hydrogen spectrum and carbon spectrogram；
Fig. 2A is Ac4The schematic diagram of the micelle modified ATO liposomes (by taking PEG1000 as an example) of M；
Fig. 2 B are at Ni (OAC)2Green deposition can be obviously observed when ATO solution being added in solution, show ATO and Ni (OAC)2In Ac4Existence in the liposome of MAN modifications；
Fig. 3 is to determine liposome ATO and Ni (OAC) at 37 DEG C in the PBS that pH is 7.42Cumulative in vitro release Rate curve；
(A) ATO is in ATO-LIP and Ac4Release profiles in M-ATO-LIP (by taking PEG1000 as an example)；(B)Ni(OAC)2 Ni-LIP and Ac4Release profiles in M-Ni-LIP；Data are indicated with mean+SD (n=3)；
Fig. 4 is research of the different liposome to growth of glioma cells inhibiting effect；
Respectively use mtt assay and cell imaging method detection various concentration ATO, ATO-LIP and Ac4M-ATO-LIP (with For PEG1000) and dosing 48h (A, a), the survival rate of 72h (B, b) and 96h (C, c) U87 cells afterwards；ATO concentration is respectively 40.6,81.5,162.5,325,650 and 1300 μM, data are mean+SD (n=3)；(scale, 300nm)；
Fig. 5 is the intake situation using Drug by Flow Cytometry in vitro；
Wherein, Fig. 5 A are that intake is tested：Histogram peak moves to the right, show U87 to Ac4M-ATO-LIP (with For PEG1000) uptake ratio increase；
Fig. 5 B are statistical analysis：The fluorescence intensity of each preparation is indicated with column diagram；Data need to be used in combination flat through normalized Means standard deviation (n=3, * * * p<0.001, be compared with the cellular uptake of Rho-LIP) indicate；
Fig. 6 A are during U87 glioma cells are incubated 0~20min with the Ac4MAN-PEG1000-LIP that Rho is marked The living cells figure of shooting；(RED sector is the liposome marked by Rho；Green is the cell membrane dyed by DIO；Blue is quilt The nucleus that Hoechst 33258 is dyed) (scale, 30 μm)；
Fig. 6 B are during U87 glioma cells are incubated 0~20min with the Ac4MAN-PEG1000-LIP that Rho is marked The statistical analysis of delay living cells imaging experiment；The fluorescence intensity of each preparation makes curve graph with normalization numerical value；Data are aobvious It is shown as mean+SD (n=3)；
Fig. 7 A are to be clapped during U87 glioma cells are incubated 0~20min with the Ac4MAN-PEG600-LIP that Rho is marked The living cells figure taken the photograph；(RED sector is the liposome marked by Rho；Green is the cell membrane dyed by DIO；Blue is quilt The nucleus that Hoechst 33258 is dyed) (scale, 30 μm)；
Fig. 7 B are to prolong during U87 glioma cells are incubated 0~20min with the Ac4MAN-PEG600-LIP that Rho is marked When living cells imaging experiment statistical analysis；The fluorescence intensity of each preparation makes curve graph with normalization numerical value；Data are shown For mean+SD (n=3)；
Fig. 8 A are during U87 glioma cells are incubated 0~20min with the Ac4MAN-PEG2000-LIP that Rho is marked The living cells figure of shooting；(RED sector is the liposome marked by Rho；Green is the cell membrane dyed by DIO；Blue is quilt The nucleus that Hoechst 33258 is dyed) (scale, 30 μm)；
Fig. 8 B are during U87 glioma cells are incubated 0~20min with the Ac4MAN-PEG2000-LIP that Rho is marked The statistical analysis of delay living cells imaging experiment；The fluorescence intensity of each preparation makes curve graph with normalization numerical value；Data are aobvious It is shown as mean+SD (n=3)；
Fig. 9 is antitumor actions of the Ac4MAN-PEG-LIP to glioma tumor-bearing mice；
Wherein, Fig. 9 A are the H.E dyeing for the glioma frozen section for injecting Ac4MAN-PEG1000-LIP；(scale, 50 μm)；
Fig. 9 B are the KaplanMeier survivorship curves for the mouse for injecting Ac4MAN-PEG-LIP；
Wherein, 1) the KaplanMeier survivorship curves (n=7) of the mouse of injection Ac4MAN-PEG1000-LIP；2) it injects The KaplanMeier survivorship curves (n=7) of the mouse of Ac4MAN-PEG600-LIP；3) inject Ac4MAN-PEG2000-LIP's The KaplanMeier survivorship curves (n=7) of mouse；
Figure 10 is the structural characterization of the double target liposomes of heart and brain；
Wherein：(A) schematic diagram of Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP is prepared；(B) it uses The Zeta potential of the Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP of Zetasizer Nano ZS 90 is distributed； (C) Anti-cTnIAb-PEG2000/Ac4MAN-PEG2000 of TEM (engineer's scale, 100) is used.(D) Zetasizer is used The particle diameter distribution of the Anti-cTnIAb-PEG2000/Ac4MAN-PEG2000-LIP of Nano ZS 90；
Figure 11 is double Targeting distributions of the target liposomes in MI rats of heart and brain of various DiR labels；
Wherein：(A) time dependence of rat body fluorescence signal is distributed after the various preparations of intravenous injection：MI rats are distinguished Give Ac4MAN-PEG2000-LIP, DiR of Anti-cTnI Ab-PEG2000-LIP, the DiR labels of isometric DiR labels The Anti-cTnI Ab-PEG2000/ of Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600-LIP, the DiR label of label The Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP of Ac4MAN1000-LIP and DiR labels；(B) upon administration 1,2,3,4,5 and 6 hours, the fluorescence intensity in the in vitro heart tissue from all experimental rats；(C) 1,3 and 6 after being administered Hour, the fluorescence intensity in the heart tissue sections of all experimental rats.
Figure 12 A are the double targets of heart and brain in 1,2,3,4,5 and 6 hour heart tissue in statistics with histogram analysis mouse heart tissue Distribution to liposome is expressed as fluorescence intensities of the DiR in heart.Data indicate average value ± S.D.(n=3).With 1, it 2,3,4,5 is compared with 6 hours Anti-cTnI Ab-PEG2000-LIP, * P<0.05, * * P<0.01 and * * * P<0.001；
Figure 12 B are the double targetings of heart and brain in 1,3 and 6 hour in the heart tissue of statistics with histogram Analyze ＆ separate heart tissue The distribution of liposome is expressed as the fluorescence intensity of DiR.With Anti-cTnI Ab-PEG2000-LIP compared with 1,3 and 6 hour, * P<0.05, * * P<0.01 and * * * P<0.001.
Figure 13 be MicroScale Thermophoresis detect the double target liposomes of heart and brain of various DiR label with The affinity of GLUT1, and show trend；X-axis indicates liposome concentrate, and Y-axis indicates score associated value；Anti-cTnI The binding constant of Ab PEG2000/Ac4MAN-PEG600-LIP is 2746.9 ± 13182nM；Anti-cTnI Ab PEG2000/ The binding constant of Ac4MAN-PEG1000-LIP is 41.8 ± 36.37nM；And Anti-cTnI Ab PEG2000/Ac4MAN- 1.17 ± 5.69nM of binding constant of PEG2000-LIP；
Figure 14 is that the liposome of the Ac4MAN modifications for the connection for having different PEG chains using Flow Cytometry Assay passes through the heart The intake (A and B) of myocyte and C6 cells.The intake of cardiac muscle cell is as shown in Figure 14 A.Figure 14 B show the flat of C6 cells Equal fluorescence intensity.Data are expressed as average value ± S.D. (n=3).*P<0.05, * * * P<0.001, compared with LIP.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be apparent from.But Embodiment is merely to illustrate the present invention, does not constitute any restrictions to protection scope of the present invention.Those skilled in the art should Understand, can modify without departing from the spirit and scope of the invention to the details and form of technical solution of the present invention Or replace, but these modifications and replacement are each fallen in protection scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments Material, reagent etc. commercially obtain unless otherwise specified.
Material used in embodiment is as follows：
Healthy adult male Wistar ratPurchased from Jilin University's Experimental Animal Center.
Dioleoylphosphatidylglycerol (DOPG) is bought (Japan, Osaka) from Japanese Fine Chemical Co., Ltd.
Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE), distearyl acyl group phosphatide Acyl ethanol amine-polyethylene glycol 2000 (DSPE-PEG 2000) and distearoylphosphatidylethanolamine-polyethylene glycol 2000- horses Carry out acid imide (DSPE-PEG2000-MAL) to buy in (China, Shanghai) from advanced carrier technique pharmaceutical Co. Ltd.
PEG 600/1000/2000, glutaric acid n-hydroxysuccinimide (NHS) and 1- ethyls -3- (3- dimethyl-different Propyl) carbodiimide hydrochloride (EDCI).From Aladdin industrial group (China, Shanghai).
P- is to carboxyl phenyl-α-D- acetylmannosamines (Ac4MAN) from BeiJing, China, Innochem is obtained.
Egg PC (EPC) and cholesterol (CHO) are purchased from Bio Life Science＆Technology Co., Ltd (Chinese Shanghai).
Arsenic trioxide (ATO) is that pharmaceutcal corporation, Ltd of Harbin Medical University (China, Harbin) gifts.
Rhodamine B (Rho) and 1,1'- bis- (octadecyl) -3,3,3', 3'- tetramethyls-two Iodine Cyanine of iodate (DiR) From HEDE Bioisystech Co., Ltd (Beijing, China).
Anti- cTnI antibody (Anti-cTnI Ab) is purchased from Immunoway (Beijing, China).
D-MANNOSE is purchased from Innochem (Beijing, China).
MST capillaries and polysorbas20 are bought from Quantum Design (Beijing, China).
1 Brain targeting molecule diisoamyl phosphorus ethanol amine of embodiment-polyethylene glycol-1000-p- is to carboxyl phenyl-α-D- acetyl The synthesis of mannosamine (DSPE-PEG-1000-Ac4MAN)
Formulas I (wherein polyalkylene glycol moiety is PEG-1000)
Nitrine-polyethylene glycol-carboxyl 1000 (1.3g) is dissolved in tetrahydrofuran (20mL), and Pd/c (0.066g) is added later With acetic acid (1mL), it is passed through hydrogen at room temperature, reacts a night.After crude product is filtered, rotary evaporated to dryness is dry, is then dissolved in two Triethylamine (TEA, 0.65mL) and Ac is added in chloromethanes (20mL)4MAN (1.5g), reacts a night at room temperature.Pillar is crossed, is obtained To PEG1000-Ac4MAN (1.18g), yield 62%.PEG 1000-Ac4MAN (1.18g) are dissolved in dichloromethane, according to Secondary addition NHS (0.2g) and EDCI (0.45g), room temperature reaction overnight, cross column, obtain succimide polyethylene glycol acetylated mannan Glucosides (1.0g), yield 78%.Succimide polyethylene glycol acetylated mannan glucosides (0.95g) is dissolved in dichloromethane In (20mL), the DSPE (0.4g) TEA (0.2mL) and be dissolved in chloroform is added, reacts a night at room temperature, with saturation NaCl extractions It takes 2 times, crosses column, obtain DSPE-PEG1000-Ac4MAN (0.86g), yield 86%.
Fig. 1 is 1HNMR and 13CNMR figures, for confirming DSPE-PEG-1000-Ac4The successful synthesis of MAN.Such as Fig. 1 institutes Show, the peak at 3.8ppm is PEG, and at 1.3ppm and 0.8ppm, respectively there are two peak representative-CH3- and-CH2-, this is The characteristic peak of DSPE, and the characteristic peak that the peak for corresponding to 5.5ppm is sugar.
2 Brain targeting molecule diisoamyl phosphorus ethanol amine of embodiment-polyethylene glycol -600-p- is to carboxyl phenyl-α-D- acetyl Mannosamine (DSPE-PEG-600-Ac4MAN synthesis)
Formulas I (wherein polyalkylene glycol moiety is PEG-600)
Nitrine-polyethylene glycol-carboxyl 600 (1.3g) is dissolved in tetrahydrofuran (20mL), and Pd/c (0.078g) is added later With acetic acid (1mL), it is passed through hydrogen at room temperature, reacts a night.After crude product is filtered, rotary evaporated to dryness is dry, is then dissolved in two Chloromethanes (20mL) is added triethylamine (TEA, 0.79mL) and Ac4MAN (1.7g), reacts a night at room temperature.Pillar is crossed, is obtained To PEG600-Ac4MAN (1.08g), yield 62%.PEG 600-Ac4MAN (0.95g) are dissolved in dichloromethane (20mL) In, NHS (0.3g) and EDCI (0.51g) are sequentially added, room temperature reaction overnight, crosses column, obtains succimide polyethylene glycol second Acyl mannoside (0.82g), yield 80%.TEA is added in succimide polyethylene glycol acetylated mannan glucosides (0.82g) (0.3mL) and the DSPE (0.49g) being dissolved in chloroform, reacts a night at room temperature, is extracted 2 times with saturation NaCl, crosses column, obtain DSPE-PEG1000-Ac4MAN (0.76g), yield 76%.
Fig. 1 is 1HNMR and 13CNMR figures, for confirming DSPE-PEG-600-Ac4The successful synthesis of MAN.As shown in Figure 1, Peak at 3.8ppm is PEG, and at 1.3ppm and 0.8ppm, respectively there are two peak representative-CH3- and-CH2-, this is DSPE Characteristic peak, and correspond to 5.5ppm peak be sugar characteristic peak.
3 Brain targeting molecule diisoamyl phosphorus ethanol amine of embodiment-Polyethylene glycol-2000-p- is to carboxyl phenyl-α-D- acetyl Mannosamine (DSPE-PEG-2000-Ac4MAN synthesis)
Formulas I (wherein polyalkylene glycol moiety is PEG-2000)
Nitrine-polyethylene glycol-carboxyl 2000 (1.3g) is dissolved in tetrahydrofuran (20mL), and Pd/c (0.052g) is added later With acetic acid (1mL), it is passed through hydrogen at room temperature, reacts a night.After crude product is filtered, rotary evaporated to dryness is dry, is then dissolved in two Chloromethanes (20mL) is added triethylamine (TEA, 0.54mL) and Ac4MAN (1.34g), reacts a night at room temperature.Pillar is crossed, is obtained To PEG2000-Ac4MAN (1.15g), yield 63%.PEG 2000-Ac4MAN (0.95g) are dissolved in dichloromethane (20mL) In, NHS (0.09g) and EDCI (0.19g) are sequentially added, room temperature reaction overnight, crosses column, obtains succimide polyethylene glycol second Acyl mannoside (0.87g), yield 82%.By succimide polyethylene glycol acetylated mannan glucosides (0.87g), TEA is added (0.15mL) and the DSPE (0.3g) being dissolved in chloroform, reacts a night at room temperature, is extracted 2 times with saturation NaCl, crosses column, obtain DSPE-PEG2000-Ac4MAN (0.75g), yield 84%.
Fig. 1 is 1HNMR and 13CNMR figures, for confirming DSPE-PEG-2000-Ac4The successful synthesis of MAN.Such as Fig. 1 institutes Show, the peak at 3.8ppm is PEG, and at 1.3ppm and 0.8ppm, respectively there are two peak representative-CH3- and-CH2-, this is The characteristic peak of DSPE, and the characteristic peak that the peak for corresponding to 5.5ppm is sugar.
Embodiment 4 carries the preparation of medicine (ATO) brain targeted liposome
(1) preparation of ATO-LIP
Blank liposome is prepared using film hydration method：By dipalmitoylphosphatidylcholine, dioleoylphosphatidylglycerol, courage Sterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in molar ratio 49.4：3.2：43.3：4.1 weigh, and are dissolved in second It in alcohol, is evaporated by rotary evaporation, lipid film can be obtained in round-bottomed flask.Dry lipid film nickel acetate (Ni (OAC)2) Solution (600mm, pH=6.8) is further disperseed with probe sonication (200W) ultrasound 5min, then pass through later through ultrasonic aquation The polycarbonate membrane (220nm) of 220nm squeezes out 3 times.The liposome squeezed crosses SephadexG-50 columns, removes double points of liposome Ni (OAC) outside sublayer2Buffer solution 1 (300mM NaCl+200mM HEPEs, pH 6.8) is added to liposome colloid by solution In solution, a gradient is formed between the inside and outside water phase of liposome, that is, forms Ni (OAC)2Liposome (Ni-LIP).Then, exist 1mLATO solution (33.4mM) is added in 2mL Ni-LIP (8.73mg lipids/mL), it is 7.2 to adjust pH, earthquake shaking table (50 DEG C, 10~15h is incubated in 110rpm), later, Liposomal suspensions cross SephadexG-50 columns, with (the 300mM NaCl+ of buffer solution 2 200mM HEPEs, pH4) remove excessive H3AsO3.Liposome pH value is finally recalled to 7.2, to obtain ATO-LIP.
Fig. 2A is the schematic diagram of liposome preparation.By Ni (OAC)2It is encapsulated in liposome interior, is then actively loaded into ATO In liposome, then with Ni (OAC)2It is complexed.Finally by Ac4M micellas are inserted into surface of liposome.Fig. 2 B show in room temperature Under, Ni (OAC)2Solution can form green precipitate with ATO solution, show ATO and Ni (OAC)2In liposome there are shapes State.When they are combined together, ATO is improved from the quick release in vesica.
(2)DSPE-PEG 1000-Ac4MAN modifies ATO-LIP
The DSPE-PEG 1000-Ac synthesized with embodiment 14MAN modifies ATO-LIP.DSPE-PEG is prepared first 1000-Ac4MAN micellas, wherein containing DSPE-PEG1000-Ac4MAN and DSPE-PEG2000, molar ratio 8.32：3.75. 1ml ATO-LIP (5.82mg fat/mL) and 0.5mlDSPE-PEG 1000-Ac4MAN micellas (8mg lipids/mL) are incubated at room temperature 2h is educated, Ac is can get4M-ATO-LIP(PEG1000)。Ac4M-ATO-LIP (PEG600) and Ac4M-ATO-LIP (PEG2000) is pressed Photograph is added with molar ratio, and preparation method is identical.
(3) release in vitro of encapsulation rate and drug
ATO-LIP and Ac is analyzed using atomic absorption spectrophotometry (AAS)4The drug of M-ATO-LIP (PEG1000) contains Amount.ATO or Ni (OAC) is measured using dialysis2In vitro release rate.Bag filter both ends linear system is tight, by 1mL ATO-LIP or Ac4M-ATO-LIP (PEG1000) is placed in bag filter (8000~14400Da of Cutoff).Then bag filter is put into and is contained In the 500mL centrifuge tubes of 300mL PBS, centrifuge tube is made constantly to be shaken in 37 DEG C, the shaking table of 100rpm.At specific time point (0.5h, 1h, 2h, 4h, 8h, 12h, for 24 hours, 48h, 72h and 96h) acquisition sample, every time collect after be added same volume PBS.
With following formula computational envelope rate (%)：Encapsulation rate (%)=(before crossing drug concentration/mistake G-50 columns after G-50 columns Drug concentration) × 100%.Calculate release rate (%) formula be：Release rate (%)=(Mn/M) × 100%.Mn be ATO or Ni(OAC)2Cumulative release amount at some time point.M is the ATO being loaded into liposome or Ni (OAC)2Total amount.
As a result：ATO is in ATO-LIP and Ac4Encapsulation rate in M-ATO-LIP (PEG1000) is respectively 22.48% ± 3.95 With 25.85% ± 2.64.Due to by modification and without the encapsulation rate no significant difference between the liposome of modification, it is known that repair Adorn ligand DSPE-PEG 1000-Ac4MAN micellas do not influence the encapsulating performance of liposome.ATO-LIP is shown in Fig. 3 A and 3B And Ac4ATO and Ni (OAc) in M-ATO-LIP (PEG1000)2In vitro release rate.After dialysis, ATO and Ni (OAc)2 Release rate be detected with elemental analyser.Experimental result shows, at 2 hours of beginning, ATO and Ni (OAc)2In dialyzate In release rate be respectively smaller than 28.18% ± 6.23 and 27.76% ± 2.56.After 48 hours, the release of ATO and Ni (OAc) 2 Rate respectively may be about 84.82% ± 2.78 and 47.44% ± 3.51.ATO and Ni (OAc)2In ATO-LIP and Ac4M-ATO-LIP (PEG1000) in vitro release rate in is similar.
The vivo efficacy research experiment of 5 brain targeted liposome of embodiment
(1) cell culture
Human glioma cells U87 derives from Cell Bank of Chinese Academy of Sciences (Shanghai).Containing 1% Pen .- Strep and U87 glioma cells are cultivated in the DMEM of 20% fetal calf serum.The cell is in 37 DEG C, 5%CO2Cell incubator in cultivate.
(2) influence of the cytotoxicity to brain glioblastoma cell
The cytotoxic effect of liposome is detected using cell imaging experiment and mtt assay.In 96 well culture plates, with 2.0 ×104The density in a/hole is inoculated with U87 cells, overnight incubation, then use respectively the blank liposome of various concentration, free ATO, ATO-LIP and Ac4M-ATO-LIP (PEG1000) incubated cell is incubated 48h, 72h and 96h respectively.ATO concentration is respectively 40.6,81.5,162.5,325,325,650 and 1300 μM.Later, the former culture medium of drug containing is changed to the 100 new culture mediums of μ L With 10 μ L MTT (5mg/mL), continues at 37 DEG C after being incubated 4h, remove MTT, 100 μ L dimethyl sulfoxide (DMSO)s are added per hole (DMSO).We measure optical density (OD) value at 490nm with enzyme-linked immunosorbent assay instrument (Tecan, Austria).
By U87 cell inoculations, then overnight incubation is incubated with various different liposomes, will after incubation 48h, 72h or 96h Original fluid is changed to new culture solution, is then dyed with Hoechst 33258 (0.2mg/mL), uses phosphate buffer (PBS) it rinses 3 times, immediately begins to cell imaging experiment.We carry out image taking using Cytation 5 and are carried out to data Analysis.
Cytotoxicity of the liposome to U87 glioma cells is shown in Fig. 4.To inquire into ATO to U87 glioma cells Growth whether have effective inhibiting effect, we give U87 glioma cells free ATO, ATO-LIP of various concentration Or Ac4M-ATO-LIP (PEG1000) is used in combination mtt assay and cell imaging method to be observed.It is observed that different liposome pair The growth of U87 glioma cells has apparent inhibiting effect, and the inducing cell death in a manner of dose-dependent.With when Between extension, cytotoxicity enhancing.Each preparation is to the inhibition strengths of U87 cells sequence：Free ATO>Ac4M-ATO-LIP (PEG1000)>ATO-LIP.Compared with liposome, the ATO that dissociates is most strong to the growth inhibition effect of cell, the reason is that small molecule It is in direct contact with cell, cellular uptake speed is fast, and this is that a kind of having anti-personnel natural material.Ac4M-ATO-LIP (PEG1000) ATO-LIP is better than to the inhibiting effect of U87 cells, illustrates the Ac for being present in surface of liposome4M micellas, enhance Inhibiting effect of the ATO-LIP to U87 cell growths.
(3) U87 glioma cells absorb
1) Rho-LIP and Ac4The preparation of M-Rho-LIP
EPC, CHO, DSPE-mPEG2000 are weighed, DSPE-PEG1000-Ac4MAN (DSPE-PEG600-Ac4MAN are added Or DSPE-PEG2000-Ac4MAN) it is dissolved in absolute ethyl alcohol, hair film forming is disperseed once using film, absolute ethyl alcohol is solvent；By The liposome of one-pass film-forming is added absolute ethyl alcohol and redissolves；Rho (6.57mM methanol) is added into the liposome for be dissolved in ethyl alcohol；With rotation Steaming makes it form a film again；Physiological saline ultrasound aquation is added under the conditions of being protected from light；Liposome after aquation is transferred to and is surrounded by tin In the EP pipes of foil paper, carries out Probe Ultrasonic Searching and be crushed power 150W or so.Parameter, which works 10 seconds, suspends operation in 30 seconds 30 times；By lipid Body crosses 0.22 μm of water phase filter membrane 3 times；Liposomal particle size and current potential are measured, dialysis removes the Rho not being packed in, obtains Ac4M- Rho-LIP (being respectively that PEG1000, PEG600 and PEG2000 are linker).
Preparing for Rho-LIP is same as mentioned above, and difference lies in be added without DSPE-PEG-Ac4MAN.
2) flow cytometry
With the cellular uptake situation of Flow cytometry different liposome.U87 glioma cells are inoculated into six by us In orifice plate, and cell culture is made to stay overnight, then uses DMEM/ low sugar to substitute DMEM/ high sugar, continue culture 12 hours, distinguish later Add free Rho, Rho-LIP and Ac4M-Rho-LIP (PEG1000) at 37 DEG C, 5%CO2 (marked incubated cell with Rho by liposome Note).Blank control group is DMEM/ low sugar culture mediums.After being incubated 4 hours, pancreatin digestion is carried out to cell, is centrifuged, then is heavy with PBS It is outstanding, cell is collected, is detected.The fluorescence intensity of the intracellular Rho of flow cytomery.The launch wavelength of Rho is 560nm, With FL2-A optical filter fluorescence intensities, and data are analyzed using 7.6 softwares of FlowJo.
Fig. 5 A and 5B show quantitative analysis of the liposome in U87 glioma cells, disclose tumour cell to vesica Absorb speed.Using the fluorescence signal in Flow cytometry U87 cells, monitoring cell to free Rho, Rho-LIP and Ac4The intake situation of M-Rho-LIP (PEG1000).The experimental results showed that the average fluorescent strength of free Rho is 6162, Rho- The average fluorescent strength of LIP is 7125, Ac4The average fluorescent strength of M-Rho-LIP (PEG1000) is 10827.Experimental data is aobvious Show, in vitro, U87 cells are to Ac4The intake of M-Rho-LIP is compared to Rho-LIP significantly (P<0.05).According to cellular uptake knot Fruit, due to DSPE-PEG-1000-Ac4The presence of MAN, DSPE-PEG-1000-Ac4Liposome micelle modified MAN has brain target Tropism and glioma targeting.
3) living cells is imaged
Use DeltaVision microscopic systems observation liposome cellular uptake ability over time and uptake ratio. U87 glioma cells are inoculated in glass bottom capsule by we, and cell is at 37 DEG C, 5%CO2Under the conditions of overnight incubation.Cell Nucleus and film use Hoechst33258 (0.2mg/mL) and DIO (0.2mg/mL) to dye respectively, are then washed three times, are added with PBS The new culture solutions of 1mL, by Rho-LIP or Ac4M-Rho-LIP (being respectively that PEG1000, PEG600 and PEG2000 are linker) adds Enter into capsule, then start to shoot, and every the image of acquisition in 5 minutes until 20 minutes.Finally use DeltaVisionSoftwx software analysis results.
Fig. 6-8 is delay living cells image, shows the process that different preparations are internalized by by U87 glioma cells.Picture is aobvious That show is Rho-LIP and Ac4M-Rho-LIP (being respectively that PEG1000, PEG600 and PEG2000 are linker) is in 0~20min By the process of cellular uptake.With Rho-LIP or Ac4In the 20min of M-Rho-LIP effects, occur in U87 cells red glimmering Optical signal.Image shows that two kinds of preparations can be by U87 cellular uptakes.And with Rho-LIP be incubated U87 cells in, discovery it is glimmering Optical signal is weaker, at the same time, Ac4M-Rho-LIP is strong in the fluorescence signal ratio Rho-LIP at each time point.Image and data point Analysis shows that Rho-LIP enters the speed ratio Ac of glioma cell4M-Rho-LIP is slow.Ac4The fluorescence signal of M-Rho-LIP is thin There is display in cytoplasm and nucleus, illustrates distribution no significant difference of the vesica in each organelle.
The Anticancer effect in vivo of 6 brain targeted liposome of embodiment
(1) structure of glioma bearing mouse model
5~6 week old male Balb/c nude mices (18~20g) derive from Beijing Life River experimental animal Technology Co., Ltd. (China, Beijing).It is anaesthetized, is fixed on stereotactic apparatus with 5% chloraldurate (15mL/20g), first cut one on scalp and open Mouthful, bregma is found on cranium, one hole of brill the front 0.5mm of bregma, side 2mm at, with the speed of 0.75 μ L/min, By U87 cells (2 × 106The μ LPBS of cell/15) it is slowly injected into intracerebral, depth 2.5mm.Notch is closed with tissue glue, has been performed the operation At weighing daily to model mouse later.
(2) treatment of glioma tumor-bearing mice
After having performed the operation 3 weeks, mouse is divided into four groups, is administered once by tail vein within every 2 days, give respectively physiological saline, Free ATO, ATO-LIP and Ac4M-ATO-LIP (preparation of embodiment 4), dosage are that every gram of weight gives 2 μ g ATO.Daily The physical condition and weight of observation and record mouse.
(3) time-to-live
Every group has 7 mouse for monitoring the time-to-live, on the day of starting within the 1st day to calculate to dead mouse upon administration, and Draw the KaplaneMeier survivorship curves of each group.
KaplaneMeier survivorship curves (Fig. 9 B) are shown, give physiological saline, free ATO, ATO-LIP and Ac4M- The mean survival time of the Glioma Model mouse of ATO-LIP (PEG1000) is 23,25,30 and 32 days.Ac4M-ATO-LIP (PEG1000) time-to-live of group mouse is considerably longer than physiological saline group, free ATO groups and ATO-LIP groups.This demonstrate that DSPE-PEG-1000-Ac4The ATO-LIP of MAN modifications has huge potentiality to treatment glioma.And Ac4MAN-ATO-LIP (PEG600) and Ac4MAN-ATO-LIP (PEG2000) life span is respectively 33 days and 35 days.
(4) H.E. is dyed
The effect of different preparations are to tumor-bearing mice is monitored, before dead mouse, heart filling is carried out with 4% paraformaldehyde solution Note 10 minutes takes out brain and is used for preparing freezing microtome section later (every is sliced 5 μm).Sections stained with hematoxylin and Yihong are dyed, Then in fluorescence microscopy microscopic observation.
Using tumor bearing nude mice as model, the antitumor action of different ATO preparations is had studied.From H.E. coloration results (Fig. 9 A) Tumor tissue section and normal cerebral tissue can be observed directly and be sliced different, giving model mouse ATO-LIP and Ac4M- After ATO-LIP treatments, the tumour cell in mice with tumor brain is reduced.The glioma cell that ATO-LIP is killed is higher than free ATO, and Ac4M-ATO-LIP (PEG1000) can make more glioma cells dead compared with ATO-LIP.These results indicate that lipid Accumulation of the body at glioma position is more than small molecule, is caused by high-permeability and retention effect (EPR).In addition, Ac4M glue Beam can also promote more liposomes to be accumulated at glioma position.
The preparation and representation of the double target liposomes of 7 heart and brain of embodiment
(1) Ac is prepared4MAN-PEG2000-LIP
It is 49 to weigh molar ratio：50：1 egg PC (EPC), cholesterol (CHO) and DSPE-PEG-2000- Ac4MAN prepares liposome according to the method being previously reported by film dispersion.In brief, fat timber-used absolute ethyl alcohol is dissolved In revolving bottle.By evaporation under reduced pressure, the mixture forms thin lipid film.With normal saline solution aquation adipose membrane. So that liposome solutions is disperseed by being ultrasonically treated 3 minutes (operation 10 seconds suspends 6 seconds, 10 times), and passes through polycarbonate membrane (220nm and 150nm, successively 3 times) is squeezed out.Liposomal samples are stored in 4 DEG C with for further study.
(2) Anti-cTnI antibody modifications liposome preparation (Anti-cTnI-PEG2000/Ac4MAN-PEG-LIP)
With method synthesis Anti-cTnI Ab-DSPE-PEG solution [the The use of antibody being previously reported modified liposomes loaded with AMO-1 to deliver oligonucleotides to ischemic myocardium for arrhythmia therapy,MeifangLiu,et.al.Biomaterials,Volume 35, Issue 11,April 2014,Pages 3697-3707].By Anti-cTnI antibody (Ab) (molecular weight 23KD) and DSPE- PEG-2000- maleimides (DSPE-PEG2000-MAL, molecular weight 2900Da) (molar ratio 1:500) it is dissolved in respectively In HEPES solution (10mM, pH7.4), then it is blended at 4 DEG C overnight, it is molten obtains Anti-cTnI Ab-DSPE-PEG2000 Liquid.Liposome solutions prepared by Anti-cTnI Ab-DSPE-PEG2000 solution and step (1) are incubated 2 hours at 37 DEG C, are made It is standby to obtain the Ac of Anti-cTnI antibody modifications4MAN-PEG2000-LIP is named as Anti-cTnI-PEG2000/Ac4MAN- PEG2000-LIP。
Anti-cTnI-PEG2000/Ac is prepared respectively according to the above identical method4MAN-PEG1000-LIP and Anti-cTnI-PEG2000/Ac4MAN-PEG600-LIP。
(3) liposome characterizes
By dynamic light scattering method (Nano ZS 90, Malvern) and transmission electron microscope (Tecnai G2F20, STEM；FEI, Hillsboro).Before measuring a degree of liposome, three parts of each sample are diluted with liposome.
Figure 10 A are the Anti-cTnI antibody and Ac of surface of liposome4The schematic diagram of MAN.Figure 10 B and D show liposome The result of the Zeta potential and grain size of the various PEG coatings of data.Figure 10 C indicate the form of LIP by TEM.The display of table 1 is average Grain size is within the scope of 100-130nm.Ac4MAN-PEG2000-LIP negatively charged (- 1.74mV), and Anti-cTnI Ab- PEG2000/Ac4MAN-PEG600-LIP is -32.0mV.During being incubated 24 hours with 10%FBS, LIP is predominantly smaller than 150nm.With the increase of PEG chain lengths, the Zeta potential of vesica continuously decreases.PEG chains are longer, and the surface charge of vesica is lower, It may be the shielding action due to PEG.
The size distribution and Zeta potential of 1 liposome of table
Note：Data are expressed as mean+SD (S.D.) (n=3)
The internal fluorescent tracing of the double target liposomes of 8 heart and brain of embodiment
1, acute myocardial infarction of rat model foundation
0.3mL chloral hydrate anesthesia rats are injected intraperitoneally per 100g weight, and connect ECG electrode.Rat carries out left side Open chest surgery, and left anterior descending coronary artery is ligatured immediately at about 2-3mm below the tie point between left auricle and lung cone.So Heart is put back into chest immediately afterwards and is kept for electrocardiographic recorder half an hour.Iodophor is used to bruised wounds skin and avoids infection. Successfully mark includes the white surface of heart, and heart rate is reduced to be raised with ST sections.
2, the preparation of the liposome of DiR labels
Weigh EPC, CHO, DSPE-mPEG2000, DSPE-PEG600-Ac4MAN (DSPE-PEG1000-Ac4MAN, DSPE-PEG2000-Ac4MAN it) is dissolved in absolute ethyl alcohol, hair film forming is disperseed once using film, absolute ethyl alcohol is solvent；By first The liposome of secondary film forming is added absolute ethyl alcohol and redissolves；DiR (6.57mM methanol) is added into the liposome for be dissolved in ethyl alcohol；With revolving It is set to form a film again；Physiological saline ultrasound aquation is added under the conditions of being protected from light；Liposome after aquation is transferred to and is surrounded by tinfoil paper In the EP pipes of paper, carries out Probe Ultrasonic Searching and be crushed power 150W or so.Parameter, which works 10 seconds, suspends operation in 30 seconds 30 times；By liposome Cross 0.22 μm of water phase filter membrane 3 times；Measure liposomal particle size and current potential.Anti-cTnI Ab are added and are incubated 2h, or add Nacl To same volume, dialysis removes the DiR not being packed in.The Ac4MAN-PEG2000-LIP, Anti-cTnI of DiR labels are obtained respectively Ab-PEG2000-LIP (is added without DSPE-PEG-Ac4MAN) when preparation, Anti-cTnI Ab-PEG2000/Ac4MAN- PEG600-LIP, Anti-cTnI Ab-PEG2000/Ac4MAN-PEG1000-LIP, Anti-cTnI Ab-PEG2000/ Ac4MAN-PEG2000-LIP。
3, internal fluorescent tracing
For optics at the logical filter of the excitation for being equipped with 720nm using Carestream in vivo FX profession imaging systems The transmitting pass filter of mating plate and 790nm.Photographic schemes：The x-ray bombardment time is each image 2 minutes, when the exposure of fluorescence Between be 30 seconds.MI rats inject the Anti-cTnI-PEG2000-LIP, Ac of DiR labels respectively4MAN-PEG2000-Lip, Anti-cTnI-PEG2000/Ac4MAN-PEG600-LIP, Anti-cTnI-PEG2000/Ac4MAN-PEG1000-LIP and cTnI-PEG2000/Ac4MAN-PEG600-LIP.Rat is put into equipped with 720nm bandpass filters and 790nm long pass filters Imaging system in.The time for exposure of fluorescence is 30 seconds, and X-ray exposure is each image 2 minutes.By what is integrated with imaging table CCD camera shoots image.Data are assessed using Carestream imaging systems
In order to study the Ac of different mol ratio example4The vesicle surface of MAN and Anti-cTnI antibody is to the shadow that is distributed in vivo It rings, we specify Anti-cTnI antibody and Ac herein4MAN (molar ratios 5:12) on the surface of the liposome connected On ratio pass through and connect different PEG molecules such as Anti-cTnI-PEG2000/Ac4MAN-PEG600-LIP, Anti- cTnI-PEG2000/Ac4MAN-PEG1000-LIP and cTnI-PEG2000/Ac4MAN-PEG600-LIP.Use in-vivo imaging system Overall view examines the distribution of the preparation in the ischemic heart and brain of rat in different time points, and the pseudo-colours representative wherein in image includes The fluorescence signal (Figure 11 A) of varying strength in the given tissue of heart and brain.Research before us has confirmed injection DiR marks The fluorescence intensity of Ischemic Heart reaches peak value after the Anti-cTnI antibody PEG2000-LIP of note.Anti-cTnI Ab- PEG2000-LIP and Ac4As a contrast, the liposome of the double targetings of presentation of information heart and brain is glimmering in figure for MAN-PEG2000-LIP groups Optical signal is most strong.
After heart perfusion, distribution of the heart tissue observation liposome in heart tissue is taken out, it was demonstrated that fluorescence signal is certain Carry out self-organizing rather than blood vessel.Before rat is perfused, rat injecting lipid body, and the 6th hour after injection are given by tail vein Take out heart.Fluorescence signal intensity in isolated heart illustrates to select Ac4The shorter PEG chains of MAN, observe lipid in heart Volume poly- more (Figure 11 B).The fluorescence imaging of heart tissue vertical section is consistent with the variation that heart is distributed (Figure 11 C).Histogram Figure indicate heart and isolated heart tissue (1,2,3,4,5 and 6) fluorescence intensity, further confirm, with Ac4MAN connections PEG chain lengths increase, liposome becomes weaker (Figure 12 A and 12B) to the Targeting distribution of heart.With PEG connections Ac4MAN The increase of length chain, due to the free cTnI in Anti-cTnI antibody competition combination ischemic myocardium mesostromas, liposome is in the heart Target distribution in dirty becomes weaker.
9 interaction of molecules of embodiment is studied
Various fat are determined by MicroScale Thermophoresis (MST) (Quantum Design China) The interaction of plastid and GLUT1, the technology can quantitative determine the interaction of biomolecule.EP pipes are numbered, physiological saline is added, Then GLUT1 is mixed with polysorbas20, then the liposome of DiR labels is added in each pipe.It is incubated after ten minutes, capillary Mixture in suction pipe is simultaneously sequentially placed pallet.The binding force of the liposome and GLUT1 of DiR labels, data are detected by MST Processing Manual_MO.Affinity Analysis_V04 softwares.
PEG, Ac are measured using MicroScale Thermophoresis (MST)4The different chain length of MAN connectors and GLUT1 The affinity of degree has studied targeting efficiency of the liposome to brain.Based on these data, we may safely draw the conclusion, liposome with Exist between GLUT1 and combines.The dissociation constant (Kd) of bond strength shows tendency, wherein Anti-cTnI Ab- PEG2000/Ac4The Kd values of MAN-PEG2000-LIP modified liposomes are 1.17 ± 5.69nM；Anti-cTnI Ab- PEG2000/Ac4MAN-PEG1000-LIP is 41.8 ± 36.37nM；Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600- 2746.9 ± 13182nM of LIP.GLUT1 plays important work in the blood glucose transhipment in various tissues, especially blood-brain barrier With.Enhance the Brain targeting ability of vesica using the mannose-modified of surface of liposome.In our current research, modified liposome can be with GLUT 1 is combined, and the binding force of 2000 mannose bonding agent of surface PEG is higher than PEG1000, and the binding force of PEG 600 is minimum (Figure 13).Ac4MAN has significant difference with the ability of the PEG combination GLUT1 albumen of different chain length.
The cellular uptake research of the double target liposomes of 10 heart and brain of embodiment
1, the culture of primary scheming cell
It is put to death after the Wistar rat pups of 3 ages in days are impregnated 1-2min in 75% ethanol solution, takes out heart rapidly, Cleaning, shreds and is ground into fritter.With the doubling dose of the tissue volume of trypsin-EDTA solutions in 37 DEG C of 10mL centrifuge tubes Tissue fragment is digested 1-3 minutes.When solution turbid, the culture medium containing serum is added to terminate digestion.Repetitive process, Until tissue disappears.Fragment of tissue is removed using 200 mesh screens and centrifuges 3 minutes with 2500rpm to collect cell.It then will be thin Dysuria with lower abdominal colic moves on in batch cultur bottle.At 37 DEG C, after being cultivated 2 hours under the conditions of 5%CO2, according to cell adhesive ability removal at Fibrocyte.Cell is incubated 48h at 37 DEG C under the conditions of 5%CO2, follow-up test is carried out in the culture medium more renewed.
2, the preparation of the liposome of Rho labels
For method with the preparation of the liposomes marked of DiR in embodiment 8, difference lies in DiR is replaced with Rho.
3, cellular uptake is studied
It is carried out by flow cytometry (Becton Dickinson FACS Aria I, Mountain view, CA) primary The intake of cardiac muscle cell and the Rho of C6 cells label liposomes.By primary cell and C6 cells with every hole 2 × 105A cell connects Kind is in six orifice plates and cultivates 48 hours.Then include Anti-cTnI Ab-PEG2000-LIP by the Rho LIP marked, Ac4MAN-PEG2000-LIP, Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600-LIP, Anti-cTnI Ab- PEG2000/Ac4MAN-PEG1000-LIP and Anti--cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP and cardiac muscle cell Or C6 cells are incubated altogether, DMEM culture mediums are as blank group.After 4 hours, by 0.25% trypsinization of cell, It collects and is resuspended in PBS and be used for Flow Cytometry Assay.The liposome for measuring Rho labels absorbs, and uses Flow Jo 7.6 Software analysis data.
The liposome of the PEG modifications of the different chain length being connect with Ac4MAN marked using Flow cytometry Rho Cellular uptake.Unmodified LIP as a contrast, and passes through all fluorescent values of normalized.Cardiac muscle cell takes the photograph being averaged for LIP Taken amount is that 1.5476, Anti-cTnI Ab-PEG2000-LIP are 1.8680, Ac4MAN-PEG2000-LIP 2.0820, Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600 are 2.3954, for Anti-cTnI Ab-PEG2000/Ac4MAN- PEG1000-LIP is that 2.435, Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000-LIP are 2.7132.With control group phase Than the intake of the cardiac muscle cell of the liposome of the PEG modifications of different chain length obviously increases (* P<0.05 and * * * P<0.001).With It the PEG chain lengths connecting with Ac4MAN to increase, cardiac muscle cell becomes stronger (Figure 14 A) intake of liposome.C6 cells are put down Equal fluorescence intensity is that LIP is that 3.9440, Anti-cTnI Ab-PEG2000-LIP are for 4.6351, Ac4MAN-PEG2000-LIP 6.5682, it is 6.1121, Anti-cTnI Ab- for Anti-cTnI Ab-PEG2000/Ac4MAN-PEG600-LIP fluorescent values PEG2000/Ac4MAN-PEG1000-LIP is 6.8042, for Anti-cTnI Ab-PEG2000/Ac4MAN-PEG2000- LIP is 8.2738.Compared with the control group, C6 cells increase (* P significant to the intake of various liposomes<0.05, * * * P< 0.001).As the PEG chain lengths being connect with Ac4MAN increase, the intake of brain increases (Figure 14 B).

Claims (10)

1. a kind of double target liposomes of heart and brain, which is characterized in that the double target liposomes of the heart and brain are by shown in Formulas I The surface of the liposome of acetylation sugar ester-mPEG2000-DSPE conjugate modification is coupled Anti-cTnI protein antibodies Or obtained after its segment；
Wherein, n=10~120, preferably 13~45, it is furthermore preferred that the polyethylene glycol is Macrogol 600, polyethylene glycol 1000 or polyethylene glycol 2000.
2. the double target liposomes of heart and brain as described in claim 1, which is characterized in that acetylation sugar ester shown in Formulas I-poly- second two Alcohol-phosphatidyl-ethanolamine conjugate is prepared by following steps：In methylene chloride by p- to carboxyl phenyl-α-D- acetyl Mannosamine (Ac4MAN) and polyethylene glycol (PEG) forms PEG-Ac by condensation reaction4MAN, then by distearyl acyl group phosphatide Acyl ethanol amine (DSPE) and PEG-Ac4MAN is coupled, and DSPE-PEG-Ac is obtained4MAN, as Formulas I compound represented.
3. the double target liposomes of heart and brain as claimed in claim 2, which is characterized in that acetylation sugar ester shown in Formulas I-poly- second two Alcohol-phosphatidyl-ethanolamine conjugate is prepared by following steps：Nitrine-polyethylene glycol-carboxyl is dissolved in tetrahydrofuran, Pd/c and acetic acid are added later, is passed through hydrogen at room temperature, reacts a night；After crude product is filtered, rotary evaporated to dryness is dry, then It is dissolved in dichloromethane, triethylamine and p- is added to carboxyl phenyl-α-D- acetylmannosamines (Ac4MAN), react at room temperature It at one night, isolates and purifies, obtains PEG-Ac4MAN；PEG-Ac4MAN is dissolved in dichloromethane, N- hydroxysuccinimidyl acyls are sequentially added Imines (NHS) and 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride (EDCI), room temperature reaction overnight, detach Purifying, obtains succimide polyethylene glycol acetylated mannan glucosides；Succimide polyethylene glycol acetylated mannan glucosides is dissolved in In dichloromethane, the distearoylphosphatidylethanolamine (DSPE) that TEA is added and is dissolved in chloroform reacts one at room temperature Night is extracted 2 times with saturation NaCl, isolates and purifies, obtain DSPE-PEG-Ac4MAN, as Formulas I compound represented.
4. a kind of method preparing the double target liposomes of claim 1-3 any one of them heart and brain, which is characterized in that including with Lower step：
(1) preparation of the liposome of acetylation sugar ester-mPEG2000-DSPE conjugate modification
By acetylation sugar ester-polyethylene glycol-phosphorus acyl second shown in egg PC (EPC), cholesterol (CHO) and Formulas I Hydramine conjugate is dissolved in absolute ethyl alcohol in revolving bottle, and by evaporation under reduced pressure, the mixture forms thin lipid Film；With the normal saline solution aquation lipid film；So that liposome solutions is disperseed by being ultrasonically treated, and is squeezed by polycarbonate membrane Go out, obtains the liposome of acetylation sugar ester-mPEG2000-DSPE conjugate modification, be named as Ac4MAN-PEG- Liposomal samples are stored in 4 DEG C by LIP, spare；
(2) liposome preparation of Anti-cTnI antibody modifications
Synthesize Anti-cTnI Ab-DSPE-PEG solution：Anti-cTnI antibody and DSPE-PEG- maleimides difference is molten It in HEPES solution, then mixes, at 4 DEG C overnight, Anti-cTnI Ab-DSPE-PEG solution is obtained, by Anti-cTnI The Ac that Ab-DSPE-PEG solution is prepared with step (1)4MAN-PEG-LIP liposome solutions incubate 2 hours at 37 DEG C, are prepared into To the Ac of Anti-cTnI antibody modifications4MAN-PEG-LIP is named as Anti-cTnI-PEG/Ac4MAN-PEG-LIP, the as heart The double target liposomes of brain.
5. method as claimed in claim 4, which is characterized in that acetylation sugar ester-shown in EPC in step (1), CHO and Formulas I The molar ratio of mPEG2000-DSPE conjugate is (40-49)：(50-55)：(1-5), preferably 49：50：1.
6. method as claimed in claim 4, which is characterized in that Anti-cTnI Ab-DSPE-PEG and Ac in step (2)4MAN- The molar ratio of PEG-LIP liposomes is (1-100)：(50-1000), preferably 1：500.
7. method as claimed in claim 4, which is characterized in that the DSPE-PEG- maleimides described in step (2) are DSPE-PEG600- maleimides, DSPE-PEG1000- maleimides or DSPE-PEG2000- maleimides.
8. double applications of the target liposomes in preparing pharmaceutical carrier of claim 1-3 any one of them heart and brain.
9. application as claimed in claim 8, which is characterized in that the double target liposomes of the heart and brain have brain and heart The effect of double targetings, to realize heart and brain with the purpose controlled.
10. application as claimed in claim 8, which is characterized in that the drug is for treating cardiomyopathies and cardiogenic Encephalopathy, it is preferred that the cardiogenic encephalopathy includes cardiogenic cerebral ischemia syndrome, cardiogenic cerebral insufficiency and cardiogenic Cerebral apoplexy.
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