CN102008441A - Endoglin antibody coupled liposome as well as preparation method and application thereof - Google Patents
Endoglin antibody coupled liposome as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an Endoglin antibody coupled liposome as well as a preparation method and an application thereof, and relates to an Endoglin antibody coupled liposome. The Endoglin antibody coupled liposome comprises nano liposome particles, Endoglin antibody molecules, a coupling agent and a plasmid DNA (deoxyribonucleic acid) /antineoplastic drug/imaging material, wherein the coupling agent is used for coupling the Endoglin antibody onto the nano liposome particles, and the plasmid DNA/antineoplastic drug/imaging material is carried by the nano liposome particles. The preparation method comprises the following steps: preparing a PEG (polyethylene glycol)-modified phospholipid lamina, filling the plasmid/drug/imaging material to prepare the nano liposome particles, homogenizing and purifying the nano liposome particles, thiolating the Endoglin antibody, and coupling the Endoglin antibody molecules on the PEG-modified nano liposome. The Endoglin antibody coupled liposome can be used for preparing drugs for diagnosing and treating solid tumors.
Description
Technical field
The present invention relates to a kind of liposome of Endoglin antibody coupling, especially relate to liposome of a kind of Endoglin antibody coupling and its production and application.
Background technology
In diagnosis, Drug therapy and the gene therapy of tumor, seek ideal tumor target spot and targeting vector is the research focus always.
Endoglin is a film surface homodimer glycoprotein molecule (molecular weight be about 180kDa) relevant with tumor neogenetic blood vessels of finding the eighties in 20th century, be transforming growth factor-beta 1 (transforming growth factor-β 1, TGF-β 1) and cell surface receptor in conjunction with the time accessory molecule.After Endoglin and TGF-β 1 combination, the signal transduction path in downstream after scalable TGF-β 1 and its receptors bind, thus promote tumor neogenetic blood vessels to generate.Now existing many studies show that: Endoglin mainly is distributed in the endotheliocyte of new vessels and the cell surface of tumor surrounding tissue, and other normal structure is not almost all expressed.Therefore, Endoglin is closely related with tumor-blood-vessel growth, is one of significant molecule of tumor-blood-vessel growth.Because the tumor neogenetic blood vessels of different tissue sources all originates from endotheliocyte, the new vessels of various tumors has homogeneity, expressed specific antigen material has general character, therefore so just can avoid different tumors owing to the heterogeneity of the tumor antigen that the different tissues that hereditary material is caused in continuous sudden change originates from might be applied to various tumors at tumor neovasculature treatment means.As seen, Endoglin is the desirable target spot of anti-angiogenic treatment of tumor and targeted therapy, has tempting potential applicability in clinical practice.
Pegylated immunoliposomes (PILs) through Polyethylene Glycol (PEG) modify, particle diameter is controlled at the nano immune liposome about 100 nanometers, is a kind of new non-viruse gene transferring vector system.In existing research, people with multiple antibodies on liposome, prepare the immunoliposome material of various different purposes.For example: the Chinese invention patent application 200910103657.X that submitted on April 21st, 2009 discloses a kind of immunoliposome based on endostatin gene and its production and application, this liposome combines anti-VIII factor related antigen monoclonal antibody, but specific effect suppresses angiogenesis and pulmonary fibrosis and forms in pulmonary vascular endothelial cell.And for example: the Chinese invention patent application of submitting on July 21st, 2,009 200910100763.2 discloses a kind of preparation and purposes of CD19 monoclonal antibody immunity liposome, this liposome can specific effect in malignant B and bone-marrow-derived lymphocyte leukemic stem cells.
Yet, for the bonded antibody of immunoliposome material institute that is used for oncotherapy of prior art for preparing, its specific antigen all only is present in the tumor cell of particular types, so this lipoid plastid material all can only have targeting at the tumor cell of particular types, therefore its scope of application also be subjected to very big restriction.If foregoing Endoglin antibody and liposome can be carried out coupling, can develop a kind of novel immunoliposome material, this material can be a targeting with the new vessels endotheliocyte that extensively exists in all kinds of tumor tissues, thereby all kinds of tumors are all had good targeting.
Summary of the invention
The purpose of this invention is to provide a kind of can specific effect in the liposome of the Endoglin antibody coupling of the new vessels endotheliocyte of tumor tissues.
Another object of the present invention provides the preparation method of the liposome of described Endoglin antibody coupling.
Another object of the present invention provides the application of the liposome of described Endoglin antibody coupling.
The liposome of Endoglin antibody coupling of the present invention comprises nanometer liposome microgranule, Endoglin antibody molecule, with coupling agent and the nanometer liposome microgranule plasmid DNA/antitumor drug/image forming material that load of Endoglin antibody coupling on the nanometer liposome microgranule; In the liposome of described Endoglin antibody coupling, the mass ratio of each component is: the nanometer liposome microgranule: the Endoglin antibody molecule: the material of coupling agent/loading=(8～12): (0.8～1.2): (0.8～1.2): (0.1～0.3).
The particle diameter of described nanometer liposome microgranule can be 50～250nm.
Described Endoglin antibody molecule can be monoclonal antibody (monoclonal antibody, mAb) or single-chain antibody (single-chain antibody, ScAb), molecular weight can be 20～180k dalton.
The material that described nanometer liposome microgranule loads can be selected from least a etc. in the plasmid, protein or the polypeptide that carry the antitumor genes of interest, anti-tumor medicine, the tumor tissues image forming material, its envelop rate can be 3%～70%, and drug loading can be 0.7%～3%.
Described coupling agent can be polyethyleneglycol derivative etc., described polyethyleneglycol derivative can be selected from DSPE-Polyethylene Glycol (DSPE-PEG) or DSPE-Polyethylene Glycol-maleimide PEG derivants such as (DSPE-PEG-maleimide), and the molecular weight of the PEG of coupling agent can be 1000～4000.
The preparation method of the liposome of Endoglin antibody coupling of the present invention may further comprise the steps:
1) the phospholipid thin layer of preparation PEG modification;
2) plasmid/medicine/image forming material of packing into, preparation nanometer liposome granule;
3) particulate homogenization of nanometer liposome and purification;
4) sulfhydrylation Endoglin antibody;
5) the Endoglin antibody molecule is coupled on the nanometer liposome of PEG modification.
In step 1), the concrete grammar of the phospholipid thin layer that described preparation PEG modifies comprises: with chloroform palmityl oleoyl phosphatidylcholine, didodecyldimethylammbromide bromide, DSPE-Macrogol 2000 and DSPE-Macrogol 2000-maleimide are mixed with the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL respectively, and are mixed in the sample bottle by the amount of 18.6 μ mol, 0.6 μ mol, 0.6 μ mol and 0.2 μ mol; Fully behind the mixing, logical nitrogen current (time should be no less than 10min) is removed organic solvent, and continuous rotary sample bottle, until forming the phospholipid thin layer; And be placed on vacuum evaporation 2h in the Rotary Evaporators.
In step 2) in, described plasmid/the medicine of packing into/image forming material, preparation nanometer liposome particulate concrete grammar comprises: the TRIS-HCL buffer 0.2mL that adds 50mM, pH=7.0 in the described sample bottle of step 1), after slight rotation and the concussion, put the water-bath ultrasonoscope and handle 3min, abundant resuspended mixture of phospholipids should be avoided the generation of bubble simultaneously as far as possible; Dropwise add material 350 μ g, final concentration to be loaded successively and be 40%～80% ethanol and 200mM CaCl
2Solution, and make alcoholic acid final concentration reach 35% (V/V), CaCl
2Final concentration reach 4mM; With bottle cap screwing and sealing, carry out 5 circulation multigelations, obtain the plasmid liposome, wherein, each freeze-thaw cycle comprises that 37 ℃ of water-baths of 5min liquid nitrogen-2min-4min room temperature leaves standstill; Material described to be loaded can be selected from least a in the plasmid that carries the antitumor genes of interest, anti-tumor medicine, tumor tissues image forming material etc.
In step 3), the concrete grammar of particulate homogenization of described nanometer liposome and purification comprises: with step 2) the plasmid liposome that obtains is successively by 400nm, 100nm in the film extruder and the double-deck microporous filter membrane of 50nm each 20 times, 20 times and 21 times; Dialysis 2h reclaims the plasmid liposome obtain in the HEPES buffer, during change dialysis solution once.
In step 4), the concrete grammar of described sulfhydrylation Endoglin antibody comprises; At lucifuge room temperature mercaptolation 1h, ultrafiltration subsequently 3 times when removing Traut ' s Reagent, is replaced as the HEPES buffer with buffer with Traut ' the s Reagent of Endoglin antibody 1.6mg and 200 μ L 10mM; Described Endoglin antibody can be monoclonal antibody or single-chain antibody, and molecular weight can be 20～180k dalton.
In step 5); the described concrete grammar that the Endoglin antibody molecule is coupled on the nanometer liposome that PEG modifies comprises: the sulfhydrylation Endoglin antibody that step 4) is obtained adds rapidly in the liposome particles that step 3) obtains; in the nitrogen protection environment, rock coupling reaction and spend the night under the room temperature.
The liposome of described Endoglin antibody coupling has good targeting to the new vessels endotheliocyte, and therefore the liposome of described Endoglin antibody coupling can be used in preparation diagnosis and treatment entity tumor medicine.This complex liposome can be mounted with purpose DNA (deoxyribonucleic acid) (DNA), ribonucleic acid (RNA), oligonucleotide material, protein, polypeptide, drug molecule, image forming material.When this complex liposome specific effect the time in the new vessels endotheliocyte of tumor tissues, the specific purpose gene of its loading can access expression, biomolecule, drug molecule, the image forming material that loads can obtain discharging, thus the purpose that realization is diagnosed and/or treated tumor.Owing to forming conjugate, DNA, the RNA of its loading, protein and polypeptide drug be counted as a kind of novel drug-loading system, therefore can comprise two or more plasmid/protein and polypeptide/medicine/image forming material, thereby reach the purpose of diagnosis by the biological action of image forming material, discharge the effect that realizes targeted therapy by the expression of genes of interest and the orientation of protein and polypeptide/drug molecule.
Description of drawings
Fig. 1 is the structural representation of the liposome of Endoglin antibody coupling of the present invention.
Fig. 2 is the preparation method flow chart of the liposome of Endoglin antibody coupling of the present invention.
Fig. 3 is the plasmid DNA liposome images of transmissive electron microscope (X10000) of the Endoglin antibody coupling in the embodiment of the invention.In Fig. 2, scale is 0.2 μ m, and the circular granular of arrow indication all is the link coupled liposome of Endoglin monoclonal antibody.
The result of Fig. 4 for using agarose gel electrophoresis and dna content method for measuring that the release efficiency of the plasmid DNA liposome of the Endoglin antibody coupling in the embodiment of the invention is measured.In Fig. 4,1:DNA marker; The 2:EGFP plasmid; 3-4: liposome packing EGFP plasmid; 5-6:5%Triton handles the back liposome and discharges EGFP plasmid (among the figure shown in the arrow).
The result of Fig. 5 for using agarose gel electrophoresis and dna content method for measuring that the stability of the plasmid DNA liposome of the Endoglin antibody coupling in the embodiment of the invention is measured.In Fig. 5,5-1: the liposome that is loaded with the EGFP plasmid is at human serum, 5-2: do not contain 20% hyclone and two anti-DMEM culture medium, 5-3: contain the stability in 20% hyclone and the two anti-DMEM culture medium; 1:marker; The 2:EGFP plasmid; 3: the liposome of packaging plasmid; 4～9: the 10min in human serum of the plasmid vector after the packing, 0.5h, 1.5h, 4h, 6h, the stability behind the 8h; Arrow A is shown in the EGFP plasmid band that discharges in the human serum, and arrow B is shown in and contains the EGFP plasmid band that discharges in 20% hyclone and the two DMEM culture medium that resists.
Fig. 6 is the metabolism situation of carrying LSS670 dyestuff liposome 1h in the mice body of the Endoglin antibody coupling in the embodiment of the invention.
Fig. 7 is the metabolism situation of carrying LSS670 dyestuff liposome 2.5h in the mice body of the Endoglin antibody coupling in the embodiment of the invention.
Fig. 8 is the metabolism situation of carrying LSS670 dyestuff liposome 4h in the mice body of the Endoglin antibody coupling in the embodiment of the invention.
In Fig. 6～8,6-1,7-1 and 8-1 are the matched group of injection free dye, and 6-2,7-2 and 8-2 are the experimental group that carries LSS670 dyestuff liposome of injection Endoglin antibody coupling.
Fig. 9 is the metabolism situation of carrying LSS670 dyestuff liposome 8h in the tumor-bearing mice body of the Endoglin antibody coupling in the embodiment of the invention.In Fig. 9, be followed successively by from left to right: do not inject the LSS670 dyestuff, the link coupled liposome of Endoglin MAb of LSS670 labelling is only injected dyestuff, and tumor is at oxter, right side (shown in the arrow); The signal magnitude mice that differs with whether in bladder position urinates relevantly, and the signal of lower limb and tail is due to the urine that is excluded is soaked.
The specific embodiment
The invention will be further described below in conjunction with drawings and Examples, and these drawings and Examples are appreciated that it is illustrative, but not limitation of the present invention.
Figure 1 shows that the structural representation of the liposome of described Endoglin antibody coupling.
1-1 is the nanometer liposome microgranule, and its particle size range is 50～250nm, has following special nature:
1) can improve the particulate water solublity of nanometer liposome and avoid that the non-specific of reticuloendothelial system engulfs in the body, and prolong nanometer liposome granule circulation time and reaching in vivo and improve target tissue and target cell purpose the nano-particle picked-up;
2) increase the dissolubility and the stability of this type of bio-pharmaceutical, weaken or eliminate immunogenicity, antigenicity and toxicity, improve the therapeutic index of medicine, enlarge the clinical application scope;
3) improve pharmacokinetics character in the body of medicine, prolong drug half-life in vivo etc.
1-2 is the material that described nanometer liposome microgranule loads, and comprising: carry at least a etc. in plasmid, protein or polypeptide, anti-tumor medicine, the tumor tissues image forming material of antitumor genes of interest, its envelop rate is 3%～70%.
1-3 is a coupling agent, be specially polyethyleneglycol derivative, comprise: DSPE-Polyethylene Glycol (DSPE-PEG) or DSPE-Polyethylene Glycol-maleimide PEG derivants such as (DSPE-PEG-maleimide), the molecular weight that is used as the PEG of coupling agent of the present invention is 1000～4000.
1-4 is the Endoglin antibody molecule, this antibody be monoclonal antibody (monoclonal antibody, mAb) or single-chain antibody (single-chain antibody, ScAb), molecular weight is about 20～180k dalton.
Figure 2 shows that the preparation method flow chart of described Endoglin antibody coupling liposome.
The step of the phospholipid thin layer that 2-1 modifies for preparation PEG specifically comprises: with chloroform with palmityl oleoyl phosphatidylcholine (POPC), didodecyldimethylammbromide bromide (DDAB), DSPE-Macrogol 2000 (DSPE-PEG
2000) and DSPE-Macrogol 2000-maleimide (DSPE-PEG
2000-maleimide) be mixed with the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL respectively, and be mixed in the sample bottle by the amount of 18.6 μ mol, 0.6 μ mol, 0.6 μ mol and 0.2 μ mol; Fully behind the mixing, logical nitrogen current (time should be no less than 10min) is removed organic solvent, and continuous rotary sample bottle, until forming the phospholipid thin layer; And be placed on vacuum evaporation 2h in the Rotary Evaporators.
2-2 is the plasmid/medicine of packing into/image forming material, the particulate step of preparation nanometer liposome, specifically comprise: in the described sample bottle of step 2-1, add 0.2mL TRIS-HCL buffer (50mM, pH 7.0), after slight rotation and the concussion, put the water-bath ultrasonoscope and handle 3～5min, abundant resuspended mixture of phospholipids should be avoided the generation of bubble simultaneously as far as possible; Dropwise add successively material 350～500 μ g (purpose DNA (deoxyribonucleic acid) (DNA), ribonucleic acid (RNA), oligonucleotide material, protein, polypeptide and/or drug molecule and/or image forming material) to be loaded, 40%～80% ethanol (final concentration 35%, V/V) and 200mMCaCl
2Solution (final concentration 4mM); With bottle cap screwing and sealing, carry out the multigelation of 5～10 circulations (each circulation comprises: 5～8min liquid nitrogen-37 ℃ of water-baths of 2～3min-4～5min room temperature leaves standstill), obtain the plasmid liposome.
2-3 is the homogenization and the purification step of liposome particles, specifically comprise: the plasmid liposome that step 2-2 is obtained is successively by 400nm (15～25 times), 100nm (15～25 times) and the double-deck microporous filter membrane of 50nm (15～25 times) in the film extruder, to finish the homogenization of liposome; Dialysis 2～4h reclaims the plasmid liposome that obtains in the HEPES buffer (pH 7.0 for 25mM HEPES, 140mM NaCl), during change dialysis solution once, to finish the purification of liposome.
2-4 is the step of sulfhydrylation Endoglin antibody, specifically comprises; (2 Iminothiolane hydrochloride is through the 50mM sodium tetraborate with Traut ' the s Reagent of Endoglin antibody 1.6～2mg and 200～250 μ L10mM, pH 9.4 is formulated), lucifuge room temperature mercaptolation 1～2h, ultrafiltration subsequently 2～4 times, when removing Traut ' s Reagent, buffer is replaced as the HEPES buffer.
2-5 is the step on the nanometer liposome that the Endoglin antibody molecule is coupled at the PEG modification; specifically comprise: the sulfhydrylation Endoglin antibody that step 2-4 is obtained adds rapidly in the liposome particles that step 2-3 obtains; in the nitrogen protection environment, rock coupling reaction and spend the night (10～18h) under the room temperature.
Nanometer liposome microgranule in the liposome of described Endoglin antibody coupling can load at least a in the following material:
1) plasmid DNA, carrying can be at the genes of interest of the cell inner expression of tumor linked groups, and when loading these genes of interest, the liposome of described Endoglin antibody coupling can be further used for preparing the tumor-targeting gene therapy medicament;
2) drug molecule (comprising bio-pharmaceutical molecules such as protein and polypeptide), can act on tumor linked groups and carry out tumor treatment, when loading these medicines, the liposome of described Endoglin antibody coupling can be further used for preparing tumor-targeting sustained release medicine;
3) image forming material can be taken in to carry out the diagnosis of tumor by tumor linked groups, and when loading these image forming materials, the liposome of described Endoglin antibody coupling can be further used for preparing diagnosing tumor medicine/reagent.
Embodiment 1: the preparation particle diameter is the plasmid DNA liposome of the Endoglin antibody coupling of 100nm, and concrete preparation process is as follows:
1) with chloroform with POPC, DDAB, DSPE-PEG
2000And DSPE-PEG
2000-maleimide is mixed with the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL respectively, and is mixed in the Agilent chromatographic sample bottle by the amount of 18.6 μ mol, 0.6 μ mol, 0.6 μ mol and 0.2 μ mol; Fully behind the mixing, logical nitrogen current 15min removes organic solvent, and continuous rotary sample bottle, until forming the phospholipid thin layer; And be placed on vacuum evaporation 2h in the Rotary Evaporators.
2) take out sample bottle, add 0.2mL TRIS-HCL buffer (50mM, pH 7.0), after slightly rotating and shaking, put the water-bath ultrasonoscope and handle 3min, abundant resuspended mixture of phospholipids should be avoided the generation of bubble simultaneously as far as possible; Dropwise add successively enhanced green fluorescence protein (EGFP) plasmid DNA (350 μ g), 60% ethanol (final concentration 35%, V/V), 200mM CaCl
2Solution (final concentration 4mM); With bottle cap screwing and sealing, carry out the multigelation of 5 circulations (each circulation comprises: 37 ℃ of water-baths of 5min liquid nitrogen-2min-4min room temperature leaves standstill), obtain the plasmid liposome.
3) the plasmid liposome that step is obtained by 400nm (20 times), 100nm (20 times) and the double-deck microporous filter membrane of 50nm (21 times) in the film extruder, is finished the homogenization of liposome successively; Dialysis 2h reclaims the plasmid liposome that obtains in the HEPES buffer (pH 7.0 for 25mM HEPES, 140mM NaCl), during change dialysis solution once, finish the purification of liposome.
4) (2 Iminothiolane hydrochloride is through the 50mM sodium tetraborate with Traut ' the s Reagent of Endoglin antibody 1.6mg and 200 μ L10mM, pH 9.4 is formulated), lucifuge room temperature mercaptolation 1h, adopt Microcon Ultracel YM-30 ultra-filtration centrifuge tube ultrafiltration 3 times subsequently, when removing Traut ' s Reagent, buffer is replaced as the HEPES buffer
5) the sulfhydrylation Endoglin antibody that step 2-4 is obtained adds rapidly in the liposome particles that step 2-3 obtains, and in the nitrogen protection environment, rocks coupling reaction and spends the night under the room temperature.
Plasmid DNA liposome to prepared Endoglin antibody coupling has carried out electron microscopic observation, particle size determination, release efficiency mensuration, stability mensuration.
Figure 3 shows that the images of transmissive electron microscope of the plasmid DNA liposome of prepared Endoglin antibody coupling, can see that the liposome particles particle diameter is about 100nm, and particle size distribution is even.
Particle size determination adopts dynamic light scattering method to carry out, and the particle diameter that records the plasmid DNA liposome of prepared Endoglin antibody coupling is 100.78 ± 1.94nm, and is consistent with transmission electron microscope observing result among Fig. 3.
Figure 4 shows that and use agarose gel electrophoresis and dna content method for measuring that the release efficiency of the plasmid DNA liposome of prepared Endoglin antibody coupling is measured, wherein: 1 is DNA marker, 2 is the EGFP plasmid, 3～4 is EGFP plasmid DNA liposome, and 5～6 are 5%Triton processing back liposome release EGFP plasmid.Can see that after 5%Triton handled, the plasmid that is loaded with was released.
In Detection of Stability, in the time of 37 ℃, the plasmid DNA liposome of prepared Endoglin antibody coupling is almost plasmid-free release (seeing Table 1) of 10min, 0.5h, 1.5h, 4h, 6h, 8h in the DMEM basal medium, human serum and contain 20% hyclone and two anti-DMEM culture medium in then all keeps low release, release rate all is lower than 20% after measuring 8h after the processing such as ultrafiltration precipitation, shows stability preferably.
The plasmid DNA liposome of table 1:Endoglin antibody coupling is at the release rate of different time
Detection of Stability also utilizes agarose gel electrophoresis and dna content method for measuring to verify, Figure 5 shows that the checking result, wherein: 5-1 is the stability of prepared plasmid DNA liposome in human serum, 5-2 is the stability of prepared plasmid DNA liposome in containing 20% hyclone and two DMEM culture medium that resists, and 5-3 is the stability of prepared plasmid DNA liposome in not containing 20% hyclone and pair anti-DMEM culture medium; In each width of cloth of Fig. 5,1 is DNA marker, and 2 is the EGFP plasmid, and 3 is the liposome of packaging plasmid, and 4-9 is the plasmid vector stability behind 10min, 0.5h, 1.5h, 4h, 6h and the 8h in human serum after packing.This checking result conforms to testing result shown in the table 1, proves that the plasmid DNA liposome of prepared Endoglin antibody coupling possesses stability preferably.
Embodiment 2: the preparation particle diameter be 100nm the Endoglin antibody coupling carry LSS670 dyestuff (can available from Kodak) liposome, concrete preparation process is as follows:
1) with chloroform with POPC, DDAB, DSPE-PEG
2000And DSPE-PEG
2000-maleimide is mixed with the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL respectively, and is mixed in the Agilent chromatographic sample bottle by the amount of 18.6 μ mol, 0.6 μ mol, 0.6 μ mol and 0.2 μ mol; Fully behind the mixing, logical nitrogen current 15min removes organic solvent, and continuous rotary sample bottle, until forming the phospholipid thin layer; And be placed on vacuum evaporation 2h in the Rotary Evaporators.
2) take out sample bottle, add 0.2mL TRIS-HCL buffer (50mM, pH 7.0), after slightly rotating and shaking, put the water-bath ultrasonoscope and handle 3min, abundant resuspended mixture of phospholipids should be avoided the generation of bubble simultaneously as far as possible; Dropwise add successively LSS670 dyestuff (350 μ g), 60% ethanol (final concentration 35%, V/V), 200mM CaCl
2Solution (final concentration 4mM); With bottle cap screwing and sealing, carry out the multigelation of 5 circulations (each circulation comprises: 37 ℃ of water-baths of 5min liquid nitrogen-2min-4min room temperature leaves standstill), obtain the plasmid liposome.
3) the plasmid liposome that step is obtained by 400nm (20 times), 100nm (20 times) and the double-deck microporous filter membrane of 50nm (21 times) in the film extruder, is finished the homogenization of liposome successively; Dialysis 2h reclaims the plasmid liposome that obtains in the HEPES buffer (pH 7.0 for 25mM HEPES, 140mM NaCl), during change dialysis solution once, finish the purification of liposome.
4) (2 Iminothiolane hydrochloride is through the 50mM sodium tetraborate with Traut ' the s Reagent of Endoglin antibody 1.6mg and 200 μ L 10mM, pH 9.4 is formulated), lucifuge room temperature mercaptolation 1h, adopt Microcon Ultracel YM-30 ultra-filtration centrifuge tube ultrafiltration 3 times subsequently, when removing Traut ' s Reagent, buffer is replaced as the HEPES buffer.
5) the sulfhydrylation Endoglin antibody that step 2-4 is obtained adds rapidly in the liposome particles that step 2-3 obtains, and in the nitrogen protection environment, rocks coupling reaction and spends the night under the room temperature.
The LSS670 dyestuff liposome that carries to prepared Endoglin antibody coupling has carried out testing in the lotus human glioma nude mouse, to measure the endotheliocyte targeting of this year of LSS670 dyestuff liposome.Experiment uses Kunming mouse to carry out, medicine feeds by tail vein injection, be respectively the internal metabolism situation of 1h, 2.5h and 4h shown in Fig. 6～8, wherein, 6-1,7-1 and 8-1 are the matched group of injection free dye, and 6-2,7-2 and 8-2 are the experimental group that carries LSS670 dyestuff liposome of injection Endoglin antibody coupling.Fig. 9 is the metabolism situation of carrying LSS670 dyestuff liposome 8h in lotus human glioma nude mouse of the Endoglin antibody coupling in the embodiment of the invention.
Because free LSS670 has metabolism very fast, can see that from the result of 1h the matched group accretion rate is obviously greater than experimental group.Arrived the data of 2.5h and 4h, can obviously see, near the signal the only remaining bladder of matched group, and experimental group is at whole health, especially internal organs all also have tangible signal.Arrived the metabolite data of 8h, can see obviously that still there is clear signal in the tumor locus of experimental group.The result shows that the liposome of Endoglin antibody coupling of the present invention has good targeting to the new vessels endotheliocyte.
It should be noted that, above embodiment is only in order to describe technical scheme of the present invention, and it is unrestricted, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment to technical scheme of the present invention under the situation that does not deviate from the spirit and scope of the present invention or be equal to replacement, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (10)
1.Endoglin the liposome of antibody coupling is characterized in that comprising nanometer liposome microgranule, Endoglin antibody molecule, with coupling agent and the nanometer liposome microgranule plasmid DNA/antitumor drug/image forming material that load of Endoglin antibody coupling on the nanometer liposome microgranule; In the liposome of described Endoglin antibody coupling, the mass ratio of each component is: the nanometer liposome microgranule: the Endoglin antibody molecule: the material of coupling agent/loading=(8～12): (0.8～1.2): (0.8～1.2): (0.1～0.3).
2. the liposome of Endoglin antibody coupling as claimed in claim 1, the particle diameter that it is characterized in that described nanometer liposome microgranule is 50～250nm;
Described Endoglin antibody molecule is preferably monoclonal antibody or single-chain antibody, and molecular weight is 20～180k dalton.
3. the liposome of Endoglin antibody coupling as claimed in claim 1, it is characterized in that material that described nanometer liposome microgranule loads is selected from least a in the plasmid, protein or the polypeptide that carry the antitumor genes of interest, anti-tumor medicine, the tumor tissues image forming material, its envelop rate is 3%～70%, and drug loading is 0.7%～3%;
Described coupling agent is preferably polyethyleneglycol derivative, described polyethyleneglycol derivative is selected from DSPE-Polyethylene Glycol or DSPE-Polyethylene Glycol-maleimide PEG derivant, and the molecular weight of the PEG of coupling agent is 1000～4000.
4. the preparation method of the liposome of Endoglin antibody coupling as claimed in claim 1 is characterized in that may further comprise the steps:
1) the phospholipid thin layer of preparation PEG modification;
2) plasmid/medicine/image forming material of packing into, preparation nanometer liposome granule;
3) particulate homogenization of nanometer liposome and purification;
4) sulfhydrylation Endoglin antibody;
5) the Endoglin antibody molecule is coupled on the nanometer liposome of PEG modification.
5. the preparation method of the liposome of Endoglin antibody coupling as claimed in claim 4, it is characterized in that in step 1), the concrete grammar of the phospholipid thin layer that described preparation PEG modifies comprises: with chloroform with palmityl oleoyl phosphatidylcholine, didodecyldimethylammbromide bromide, DSPE-Macrogol 2000 and DSPE-Macrogol 2000-maleimide is mixed with 20mg/mL respectively, 5mg/mL, the respective concentration of 20mg/mL and 10mg/mL, and by 18.6 μ mol, 0.6 μ mol, 0.6 the amount of μ mol and 0.2 μ mol is mixed in the sample bottle; Fully behind the mixing, logical nitrogen current (time should be no less than 10min) is removed organic solvent, and continuous rotary sample bottle, until forming the phospholipid thin layer; And be placed on vacuum evaporation 2h in the Rotary Evaporators.
6. the preparation method of the liposome of Endoglin antibody coupling as claimed in claim 4, it is characterized in that in step 2) in, described plasmid/the medicine of packing into/image forming material, preparation nanometer liposome particulate concrete grammar comprises: the TRIS-HCL buffer 0.2mL that adds 50mM, pH=7.0 in the described sample bottle of step 1), after slight rotation and the concussion, put the water-bath ultrasonoscope and handle 3min, abundant resuspended mixture of phospholipids should be avoided the generation of bubble simultaneously as far as possible; Dropwise add material 350 μ g, final concentration to be loaded successively and be 40%～80% ethanol and 200mM CaCl
2Solution, and make alcoholic acid final concentration reach 35% (V/V), CaCl
2Final concentration reach 4mM; With bottle cap screwing and sealing, carry out 5 circulation multigelations, obtain the plasmid liposome, wherein, each freeze-thaw cycle comprises that 37 ℃ of water-baths of 5min liquid nitrogen-2min-4min room temperature leaves standstill; Material described to be loaded can be selected from least a in the plasmid that carries the antitumor genes of interest, anti-tumor medicine, tumor tissues image forming material etc.
7. the preparation method of the liposome of Endoglin antibody coupling as claimed in claim 4, it is characterized in that in step 3) the concrete grammar of particulate homogenization of described nanometer liposome and purification comprises: with step 2) the plasmid liposome that obtains is successively by 400nm, 100nm in the film extruder and the double-deck microporous filter membrane of 50nm each 20 times, 20 times and 21 times; Dialysis 2h reclaims the plasmid liposome obtain in the HEPES buffer, during change dialysis solution once.
8. the preparation method of the liposome of Endoglin antibody coupling as claimed in claim 4 is characterized in that in step 4), and the concrete grammar of described sulfhydrylation Endoglin antibody comprises; At lucifuge room temperature mercaptolation 1h, ultrafiltration subsequently 3 times when removing Traut ' s Reagent, is replaced as the HEPES buffer with buffer with Traut ' the s Reagent of Endoglin antibody 1.6mg and 200 μ L 10mM; Described Endoglin antibody can be monoclonal antibody or single-chain antibody, and molecular weight can be 20～180k dalton.
9. the preparation method of the liposome of Endoglin antibody coupling as claimed in claim 4; it is characterized in that in step 5); the described concrete grammar that the Endoglin antibody molecule is coupled on the nanometer liposome that PEG modifies comprises: the sulfhydrylation Endoglin antibody that step 4) is obtained adds rapidly in the liposome particles that step 3) obtains; in the nitrogen protection environment, rock coupling reaction and spend the night under the room temperature.
10. use in preparation diagnosis and treatment entity tumor medicine as the liposome of Endoglin antibody coupling as described in any claim in the claim 1～3.
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