CN102008441B - 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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- CN102008441B CN102008441B CN201010579418.4A CN201010579418A CN102008441B CN 102008441 B CN102008441 B CN 102008441B CN 201010579418 A CN201010579418 A CN 201010579418A CN 102008441 B CN102008441 B CN 102008441B
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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 preparation method and application.
Background technology
In diagnosis, Drug therapy and the gene therapy of tumor, find desirable tumor target spot and targeting vector is study hotspot 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, transforming growth factor-beta 1 (transforming growth factor-β 1, TGF-β 1) and cell surface receptor in conjunction with 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 angiogenesis.Now existing much research shows: Endoglin is mainly 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, so just can avoid different tumors due to the heterogeneity of hereditary material at the tumor antigen of the different tissues origin that constantly sudden change causes, therefore for tumor neovasculature treatment means, likely be applied to various tumors.Visible, Endoglin is the desirable target spot of the anti-angiogenic treatment of tumor and targeted therapy, has tempting potential applicability in clinical practice.
Pegylated immunoliposomes (PILs), through the nano immune liposome that Polyethylene Glycol (PEG) is modified, particle diameter is controlled at 100 nanometer left and right, is a kind of new non-viruse gene transferring vector system.In existing research, people are combined in Multiple Antibodies on liposome, prepare the immunoliposome material of various different purposes.For example: the Chinese invention patent application 200910103657.X that on April 21st, 2009 submits to discloses a kind of immunoliposome based on endostatin gene and its preparation method and application, this liposome combines anti-VIII factor Ⅷ related antigen monoclonal antibody, can specific effect in pulmonary vascular endothelial cell, suppress angiogenesis and pulmonary fibrosis and form.And for example: the Chinese invention patent application 200910100763.2 of submitting on July 21st, 2009 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 antibody of the immunoliposome material institute combination for oncotherapy of preparing for prior art, its specific antigen is all only present in the tumor cell of particular types, therefore this lipoid plastid material all can only have targeting for the tumor cell of particular types, therefore its scope of application be also subject to very big restriction.If foregoing Endoglin antibody and liposome can be carried out to coupling, can develop a kind of novel immunoliposome material, it is targeting that this material can be take the new vessels endotheliocyte extensively existing in all kinds of tumor tissues, thereby all kinds of tumors are all had to good targeting.
Summary of the invention
The object 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 is to provide the preparation method of the liposome of described Endoglin antibody coupling.
Another object of the present invention is to provide the application of the liposome of described Endoglin antibody coupling.
The liposome of Endoglin antibody coupling of the present invention comprises plasmid DNA/antitumor drug/image forming material that nanometer liposome microgranule, Endoglin antibody molecule, the coupling agent by Endoglin antibody coupling on nanometer liposome microgranule and nanometer liposome microgranule load; In the liposome of described Endoglin antibody coupling, the mass ratio of each component is: nanometer liposome microgranule: 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), and molecular weight can be 20～180k dalton.
The material that described nanometer liposome microgranule loads can be selected from and carry plasmid, protein or polypeptide, the anti-tumor medicine of antitumor genes of interest, at least one in tumor tissues image forming material etc., 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 the PEG derivants such as PEG-DSPE (DSPE-PEG) or PEG-DSPE-maleimide (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 comprises the following steps:
1) prepare the phospholipid thin layer that PEG modifies;
2) pack plasmid/medicine/image forming material into, prepare nanometer liposome granule;
3) homogenization of nanometer liposome granule and purification;
4) sulfhydrylation Endoglin antibody;
5) Endoglin antibody molecule is coupled on the nanometer liposome of PEG modification.
In step 1) in, the concrete grammar of the phospholipid thin layer that described preparation PEG modifies comprises: with chloroform, POPC, didodecyldimethylammbromide bromide, PEG-DSPE 2000 and PEG-DSPE 2000-maleimide are mixed with respectively to the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL, and are mixed in sample bottle by the amount of 18.6 μ mol, 0.6 μ mol, 0.6 μ mol and 0.2 μ mol; After fully mixing, logical nitrogen current (time should be no less than 10min) is removed organic solvent, and continuous rotary sample bottle, until form phospholipid thin layer; And be placed on vacuum evaporation 2h in Rotary Evaporators.
In step 2) in, described plasmid/medicine/the image forming material that packs into, the concrete grammar of preparing nanometer liposome granule comprises: toward step 1) add the TRIS-HCL buffer 0.2mL of 50mM, pH=7.0 in described sample bottle, after slight rotation and concussion, put water-bath ultrasonoscope and process 3min, abundant resuspended mixture of phospholipids should be avoided the generation of bubble simultaneously as far as possible; Dropwise add successively ethanol and 200mM CaCl that material to be loaded 350 μ g, final concentration are 40%～80%
2solution, and make the final concentration of ethanol reach 35% (V/V), CaCl
2final concentration reach 4mM; By bottle cap screwing sealing, the multigelation that carries out 5 circulations, obtains plasmid liposome, and wherein, each freeze-thaw cycle comprises that 37 ℃ of water-bath-4min room temperatures of 5min liquid nitrogen-2min are standing; Material described to be loaded can be selected from least one in the plasmid, anti-tumor medicine, tumor tissues image forming material etc. that carries antitumor genes of interest.
In step 3) in, the homogenization of described nanometer liposome granule and the concrete grammar of purification comprise: by step 2) the plasmid liposome that obtains is successively by 400nm, 100nm in film extruder and the double-deck microporous filter membrane of 50nm each 20 times, 20 times and 21 times; The 2h that dialyses in HEPES buffer reclaims the plasmid liposome obtain, during change dialysis solution once.
In step 4) in, the concrete grammar of described sulfhydrylation Endoglin antibody comprises; By Traut ' the s Reagent of Endoglin antibody 1.6mg and 200 μ L 10mM, at lucifuge room temperature mercaptolation 1h, ultrafiltration subsequently 3 times, when removing Traut ' s Reagent, is replaced as HEPES buffer by buffer; Described Endoglin antibody can be monoclonal antibody or single-chain antibody, and molecular weight can be 20～180k dalton.
In step 5) in; the described concrete grammar that Endoglin antibody molecule is coupled on the nanometer liposome that PEG modifies comprises: by step 4) the sulfhydrylation Endoglin antibody that obtains adds rapidly step 3) in the liposome particles that obtains; under room temperature, in nitrogen protection environment, rock coupling reaction and spend the night.
The liposome of described Endoglin antibody coupling has good targeting to new vessels endotheliocyte, and therefore the liposome of described Endoglin antibody coupling can be applied in preparation diagnosis and treatment entity tumor medicine.This complex liposome can be mounted with object DNA (deoxyribonucleic acid) (DNA), ribonucleic acid (RNA), oligonucleotide material, protein, polypeptide, drug molecule, image forming material.When this complex liposome specific effect is in the new vessels endotheliocyte of tumor tissues time, the specific purpose gene of its loading can access expression, the biomolecule, drug molecule, the image forming material that load can be discharged, thereby realize the object that tumor is diagnosed and/or treated.Because forming conjugate, DNA, the RNA of its loading, proteins and peptides class medicine be counted as a kind of novel drug-loading system, therefore can comprise two or more plasmid/proteins and peptides/medicine/image forming materials, thereby by the biological action of image forming material, reach the object of diagnosis, by the expression of genes of interest and the targeted release of proteins and peptides/drug molecule, realize the effect of targeted therapy.
Accompanying drawing explanation
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 present invention.In Fig. 2, scale is 0.2 μ m, and the circular granular of arrow indication is all the liposome of Endoglin monoclonal antibody coupling.
The result of Fig. 4 for using agarose gel electrophoresis and DNA content method for measuring to measure the release efficiency of the plasmid DNA liposome of the Endoglin antibody coupling in the embodiment of the present invention.In Fig. 4,1:DNA marker; 2:EGFP plasmid; 3-4: liposome packing EGFP plasmid; After 5-6:5%Triton processes, liposome discharges EGFP plasmid (in figure shown in arrow).
The result of Fig. 5 for using agarose gel electrophoresis and DNA content method for measuring to measure the stability of the plasmid DNA liposome of the Endoglin antibody coupling in the embodiment of the present invention.In Fig. 5,5-1: be loaded with the liposome of EGFP plasmid at human serum, 5-2: containing 20% hyclone and dual anti-DMEM culture medium, 5-3: containing the stability in 20% hyclone and dual anti-DMEM culture medium; 1:marker; 2:EGFP plasmid; 3: the liposome of packaging plasmid; 4～9: the 10min in human serum of the plasmid vector after packing, 0.5h, 1.5h, 4h, 6h, the stability after 8h; Arrow A is shown in the EGFP plasmid band discharging in human serum, and arrow B is shown in the EGFP plasmid band discharging containing in 20% hyclone and dual anti-DMEM culture medium.
Fig. 6 is the metabolism situation of carrying LSS670 dyestuff liposome 1h in Mice Body of the Endoglin antibody coupling in the embodiment of the present invention.
Fig. 7 is the metabolism situation of carrying LSS670 dyestuff liposome 2.5h in Mice Body of the Endoglin antibody coupling in the embodiment of the present invention.
Fig. 8 is the metabolism situation of carrying LSS670 dyestuff liposome 4h in Mice Body of the Endoglin antibody coupling in the embodiment of the present 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 tumor-bearing mice body of the Endoglin antibody coupling in the embodiment of the present invention.In Fig. 9, be followed successively by from left to right: do not inject LSS670 dyestuff, the liposome of the Endoglin MAb coupling of LSS670 labelling, only injects dyestuff, and tumor is at oxter, right side (shown in 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
Below in conjunction with drawings and Examples, the invention will be further described, 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 nanometer liposome microgranule, and its particle size range is 50～250nm, has following special nature:
1) can improve the water solublity of nanometer liposome granule and avoid the non-specific of reticuloendothelial system in body to engulf, and extending circulation time in vivo of nanometer liposome granule and reach and improve target tissue and the object of target cell to nano-particle picked-up;
2) increase dissolubility and the stability of this type of bio-pharmaceutical, weaken or eliminate immunogenicity, antigenicity and toxicity, improve the therapeutic index of medicine, expand clinical application scope;
3) improve the interior pharmacokinetics character of body of medicine, prolong drug half-life in vivo etc.
1-2 is the material that described nanometer liposome microgranule loads, and comprising: carry plasmid, protein or polypeptide, the anti-tumor medicine of antitumor genes of interest, at least one in tumor tissues image forming material etc., its envelop rate is 3%～70%.
1-3 is coupling agent, be specially polyethyleneglycol derivative, comprise: the PEG derivants such as PEG-DSPE (DSPE-PEG) or PEG-DSPE-maleimide (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 Endoglin antibody molecule, and this antibody is monoclonal antibody (monoclonal antibody, mAb) or single-chain antibody (single-chain antibody, ScAb), and 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 by POPC (POPC), didodecyldimethylammbromide bromide (DDAB), PEG-DSPE 2000 (DSPE-PEG
2000) and PEG-DSPE 2000-maleimide (DSPE-PEG
2000-maleimide) be mixed with respectively the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL, and be mixed in sample bottle by the amount of 18.6 μ mol, 0.6 μ mol, 0.6 μ mol and 0.2 μ mol; After fully mixing, logical nitrogen current (time should be no less than 10min) is removed organic solvent, and continuous rotary sample bottle, until form phospholipid thin layer; And be placed on vacuum evaporation 2h in Rotary Evaporators.
2-2 is for packing plasmid/medicine/image forming material into, the step of preparing nanometer liposome granule, specifically comprise: in sample bottle described in step 2-1, add 0.2mL TRIS-HCL buffer (50mM, pH 7.0), after slight rotation and concussion, put water-bath ultrasonoscope and process 3～5min, abundant resuspended mixture of phospholipids should be avoided the generation of bubble simultaneously as far as possible; Dropwise add successively material to be loaded 350～500 μ g (object DNA (deoxyribonucleic acid) (DNA), ribonucleic acid (RNA), oligonucleotide material, protein, polypeptide and/or drug molecule and/or image forming material), 40%～80% ethanol (final concentration 35%, V/V) and 200mMCaCl
2solution (final concentration 4mM); By bottle cap screwing sealing, carry out 5～10 circulations multigelation of (each circulation comprises: 37 ℃ of water-bath-4～5min room temperatures of 5～8min liquid nitrogen-2～3min are standing), obtain plasmid liposome.
2-3 is 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 film extruder, to complete the homogenization of liposome; Dialysis 2～4h reclaims the plasmid liposome obtaining in the HEPES buffer (pH 7.0 for 25mM HEPES, 140mM NaCl), during change dialysis solution once, to complete the purification of liposome.
2-4 is the step of sulfhydrylation Endoglin antibody, specifically comprises; By Traut ' the s Reagent of Endoglin antibody 1.6～2mg and 200～250 μ L10mM, (2 Iminothiolane hydrochloride is through 50mM sodium tetraborate, 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 to HEPES buffer.
2-5 is the step on the nanometer liposome that Endoglin antibody molecule is coupled to PEG and modifies; specifically comprise: the sulfhydrylation Endoglin antibody that step 2-4 is obtained adds rapidly in the liposome particles that step 2-3 obtains; under room temperature in nitrogen protection environment, rock coupling reaction spend the night (10～18h).
Nanometer liposome microgranule in the liposome of described Endoglin antibody coupling can load at least one in following material:
1) plasmid DNA, carrying can be at the genes of interest of the cell inner expression of Tumor-assaciated tissue, and when loading these genes of interest, the liposome of described Endoglin antibody coupling can be further used for preparing tumor-targeting gene therapy medicament;
2) drug molecule (comprising the bio-pharmaceutical molecules such as proteins and peptides), can act on the treatment that Tumor-assaciated tissue carries out tumor, when loading these medicines, the liposome of described Endoglin antibody coupling can be further used for preparing tumor-targeting controlled release drug;
3) image forming material, can be taken in to carry out the diagnosis of tumor by Tumor-assaciated tissue, 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: preparing particle diameter is the plasmid DNA liposome of the Endoglin antibody coupling of 100nm, and concrete preparation process is as follows:
1) with chloroform by POPC, DDAB, DSPE-PEG
2000and DSPE-PEG
2000-maleimide is mixed with respectively the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL, and is mixed in 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; After fully mixing, logical nitrogen current 15min removes organic solvent, and continuous rotary sample bottle, until form phospholipid thin layer; And be placed on vacuum evaporation 2h in Rotary Evaporators.
2) take out sample bottle, add 0.2mL TRIS-HCL buffer (50mM, pH 7.0), after slightly rotating and shaking, put water-bath ultrasonoscope and process 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); By bottle cap screwing sealing, carry out 5 circulations multigelation of (each circulation comprises: 37 ℃ of water-bath-4min room temperatures of 5min liquid nitrogen-2min are standing), obtain plasmid liposome.
3) plasmid liposome step being obtained by 400nm (20 times), 100nm (20 times) and the double-deck microporous filter membrane of 50nm (21 times) in film extruder, completes the homogenization of liposome successively; Dialysis 2h reclaims the plasmid liposome obtaining in the HEPES buffer (pH 7.0 for 25mM HEPES, 140mM NaCl), during change dialysis solution once, complete the purification of liposome.
4) by Traut ' the s Reagent of Endoglin antibody 1.6mg and 200 μ L10mM, (2 Iminothiolane hydrochloride is through 50mM sodium tetraborate, pH 9.4 is formulated), lucifuge room temperature mercaptolation 1h, adopt subsequently Microcon Ultracel YM-30 ultra-filtration centrifuge tube ultrafiltration 3 times, when removing Traut ' s Reagent, buffer is replaced as to HEPES buffer
5) sulfhydrylation Endoglin antibody step 2-4 being obtained adds rapidly in the liposome particles that step 2-3 obtains, and under room temperature, in nitrogen protection environment, rocks coupling reaction and spends the night.
The plasmid DNA liposome of prepared Endoglin antibody coupling has been carried out to electron microscopic observation, particle size determination, release efficiency mensuration, Stability Determination.
Figure 3 shows that the images of transmissive electron microscope of the plasmid DNA liposome of prepared Endoglin antibody coupling, can see that 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, consistent with transmission electron microscope observing result in Fig. 3.
Figure 4 shows that and use agarose gel electrophoresis and DNA content method for measuring to measure the release efficiency of the plasmid DNA liposome of prepared Endoglin antibody coupling, wherein: 1 is DNA marker, 2 is EGFP plasmid, 3～4 is EGFP plasmid DNA liposome, and 5～6 discharge EGFP plasmid for 5%Triton processes rear liposome.Can see, after 5%Triton processes, the plasmid being loaded with is released.
In Detection of Stability, in the time of 37 ℃, the plasmid DNA liposome of prepared Endoglin antibody coupling is almost plasmid-free release (in Table 1) of 10min, 0.5h, 1.5h, 4h, 6h, 8h in DMEM basal medium, at human serum and in containing 20% hyclone and dual anti-DMEM culture medium, all keep low release, measure 8h after the processing such as ultrafiltration precipitation after, release rate, all lower than 20%, shows good stability.
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 result, wherein: 5-1 is the stability of prepared plasmid DNA liposome in human serum, 5-2 is prepared plasmid DNA liposome in the stability containing in 20% hyclone and dual anti-DMEM culture medium, and 5-3 is prepared plasmid DNA liposome not containing the stability in 20% hyclone and dual anti-DMEM culture medium; In each width of Fig. 5,1 is DNA marker, and 2 is EGFP plasmid, 3 liposomees that are packaging plasmid, plasmid vector in human serum in stability 10min, 0.5h, 1.5h, 4h, 6h and 8h after of 4-9 after for packing.This result conforms to testing result shown in table 1, proves that the plasmid DNA liposome of prepared Endoglin antibody coupling possesses good stability.
Embodiment 2: prepare particle diameter and be 100nm Endoglin antibody coupling carry LSS670 dyestuff (can purchased from Kodak) liposome, concrete preparation process is as follows:
1) with chloroform by POPC, DDAB, DSPE-PEG
2000and DSPE-PEG
2000-maleimide is mixed with respectively the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL, and is mixed in 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; After fully mixing, logical nitrogen current 15min removes organic solvent, and continuous rotary sample bottle, until form phospholipid thin layer; And be placed on vacuum evaporation 2h in Rotary Evaporators.
2) take out sample bottle, add 0.2mL TRIS-HCL buffer (50mM, pH 7.0), after slightly rotating and shaking, put water-bath ultrasonoscope and process 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); By bottle cap screwing sealing, carry out 5 circulations multigelation of (each circulation comprises: 37 ℃ of water-bath-4min room temperatures of 5min liquid nitrogen-2min are standing), obtain plasmid liposome.
3) plasmid liposome step being obtained by 400nm (20 times), 100nm (20 times) and the double-deck microporous filter membrane of 50nm (21 times) in film extruder, completes the homogenization of liposome successively; Dialysis 2h reclaims the plasmid liposome obtaining in the HEPES buffer (pH 7.0 for 25mM HEPES, 140mM NaCl), during change dialysis solution once, complete the purification of liposome.
4) by Traut ' the s Reagent of Endoglin antibody 1.6mg and 200 μ L 10mM, (2 Iminothiolane hydrochloride is through 50mM sodium tetraborate, pH 9.4 is formulated), lucifuge room temperature mercaptolation 1h, adopt subsequently Microcon Ultracel YM-30 ultra-filtration centrifuge tube ultrafiltration 3 times, when removing Traut ' s Reagent, buffer is replaced as to HEPES buffer.
5) sulfhydrylation Endoglin antibody step 2-4 being obtained adds rapidly in the liposome particles that step 2-3 obtains, and under room temperature, in nitrogen protection environment, rocks coupling reaction and spends the night.
The LSS670 dyestuff liposome that carries to prepared Endoglin antibody coupling has carried out testing in lotus human glioma nude mouse, to measure the endotheliocyte targeting of this year LSS670 dyestuff liposome.Experiment is used Kunming mouse to carry out, medicine feeds by tail vein injection, shown in Fig. 6～8, be respectively the internal metabolism situation of 1h, 2.5h and 4h, 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 present invention.
Because free LSS670 has metabolism very fast, from the result of 1h, can see, matched group accretion rate is obviously greater than experimental group.Arrived the data of 2.5h and 4h, can obviously see, matched group is near the signal remaining bladder only, and experimental group is at whole health, and especially internal organs all also have obvious signal.Arrived the metabolite data of 8h, can obviously see that the tumor locus of experimental group still exists clear signal.Result shows, the liposome of Endoglin antibody coupling of the present invention has good targeting to 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 in the situation that not deviating from the spirit and scope of the present invention, to technical scheme of the present invention, modify or be equal to replacement, it all should be encompassed in the middle of claim scope of the present invention.
Claims (3)
- The liposome of 1.Endoglin antibody coupling, is characterized in that the plasmid DNA/antitumor drug/image forming material that comprises that nanometer liposome microgranule, Endoglin antibody molecule, the coupling agent by Endoglin antibody coupling on nanometer liposome microgranule and nanometer liposome microgranule load; In the liposome of described Endoglin antibody coupling, the mass ratio of each component is: nanometer liposome microgranule: 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 is 50～250nm;The material that described nanometer liposome microgranule loads is selected from and carries plasmid, protein or polypeptide, the anti-tumor medicine of antitumor genes of interest, at least one in tumor tissues image forming material, and its envelop rate is 3%～70%, and drug loading is 0.7%～3%;Described Endoglin antibody molecule is monoclonal antibody or single-chain antibody, and molecular weight is 20～180k dalton;Described coupling agent is polyethyleneglycol derivative, described polyethyleneglycol derivative is selected from PEG-DSPE or PEG-DSPE-maleimide PEG derivant, and the molecular weight of the PEG of coupling agent is 1000～4000;Described preparation method comprises the following steps:1) prepare the phospholipid thin layer that PEG modifies; The concrete grammar of the phospholipid thin layer that described preparation PEG modifies comprises: with chloroform, POPC, didodecyldimethylammbromide bromide, PEG-DSPE 2000 and PEG-DSPE 2000-maleimide are mixed with respectively to the respective concentration of 20mg/mL, 5mg/mL, 20mg/mL and 10mg/mL, and are mixed in sample bottle by the amount of 18.6 μ mol, 0.6 μ mol, 0.6 μ mol and 0.2 μ mol; After fully mixing, logical nitrogen current is 10min at least, removes organic solvent, and continuous rotary sample bottle, until form phospholipid thin layer; And be placed on vacuum evaporation 2h in Rotary Evaporators;2) pack plasmid/medicine/image forming material into, prepare nanometer liposome granule; Described plasmid/medicine/the image forming material that packs into, the concrete grammar of preparing nanometer liposome granule comprises: toward the TRIS-HCL buffer 0.2mL that adds 50mM, pH=7.0 in sample bottle described in step 1), after slight rotation and concussion, put water-bath ultrasonoscope and process 3min, abundant resuspended mixture of phospholipids should be avoided the generation of bubble simultaneously as far as possible; Dropwise add successively ethanol and 200mM CaCl that material to be loaded 350 μ g, final concentration are 40%～80% 2solution, and make the final concentration of ethanol reach 35%(V/V), CaCl 2final concentration reach 4mM; By bottle cap screwing sealing, the multigelation that carries out 5 circulations, obtains plasmid liposome, and wherein, each freeze-thaw cycle comprises that 5min liquid nitrogen-2min37 ℃ of water-bath-4min room temperature is standing; Material described to be loaded is selected from least one in the plasmid, anti-tumor medicine, tumor tissues image forming material that carries antitumor genes of interest;3) homogenization of nanometer liposome granule and purification;4) sulfhydrylation Endoglin antibody; The concrete grammar of described sulfhydrylation Endoglin antibody comprises; By Traut ' the s Reagent of Endoglin antibody 1.6mg and 200 μ L10mM, at lucifuge room temperature mercaptolation 1h, ultrafiltration subsequently 3 times, when removing Traut ' s Reagent, is replaced as HEPES buffer by buffer; Described Endoglin antibody can be monoclonal antibody or single-chain antibody, and molecular weight is 20～180k dalton;5) Endoglin antibody molecule is coupled on the nanometer liposome of PEG modification.
- 2. the liposome of Endoglin antibody coupling as claimed in claim 1, it is characterized in that in step 3), the homogenization of described nanometer liposome granule and the concrete grammar of purification comprise: by step 2) the plasmid liposome that obtains is successively by 400nm, 100nm in film extruder and the double-deck microporous filter membrane of 50nm each 20 times, 20 times and 21 times; The 2h that dialyses in HEPES buffer reclaims the plasmid liposome obtain, during change dialysis solution once.
- 3. the liposome of Endoglin antibody coupling as claimed in claim 1; it is characterized in that in step 5); the described concrete grammar that 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; under room temperature, in nitrogen protection environment, rock coupling reaction and spend the night.
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