ABSTRACT
We studied internalization of vector nanocarriers loaded with plasmid DNA into C6 glioma cells. For improving selectivity of plasmid delivery, the liposomes were conjugated with monoclonal antibodies to VEGF and its receptor VEGFR2. Flow cytofluorometry and laser scanning confocal microscopy showed more intensive (more than 2-fold) internalization and accumulation of antibody-vectorized liposomes in C6 glioma cells in comparison with the control (liposomes conjugated with non-specific antibodies and non-vectorized liposomes). Using quantitative analysis of fluorescent signal, we showed that cationic immunoliposomes significantly more effective delivered pCop-Green-N plasmid DNA and ensured effective transfection of C6 glioma cells.
Subject(s)
Brain Neoplasms/genetics , Glioma/genetics , Glioma/therapy , Liposomes/chemistry , Plasmids/chemistry , Plasmids/genetics , Animals , Brain Neoplasms/therapy , Cell Line, Tumor , Flow Cytometry , Genetic Therapy , Microscopy, Confocal , Rats , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor Receptor-2/geneticsABSTRACT
cDNA extracellular Ig-like domains I-III of vascular endothelial growth factor receptor 2 (VEGFR2) was cloned in an expressing vector pET_32a. Western blotting showed immunochemical identity of recombinant VEGFR2I-III produced by prokaryotic expression system to the native receptor. BALB/c mice were immunized with VEGFR2I-III for obtaining specific antibodies to VEGFR2. Hybridomas producing monoclonal antibodies were selected by ELISA, Western blotting, and immunocytochemical assay. Thus, we obtained hybridoma producing monoclonal antibodies to VEGFR2 that selectively interact with both recombinant and native extracellular fragment of the receptor.
Subject(s)
Antibodies, Monoclonal/immunology , Vascular Endothelial Growth Factor Receptor-2/immunology , Animals , Female , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Inbred BALB CABSTRACT
Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Vascular Endothelial Growth Factor A/immunology , Animals , Antibodies, Monoclonal/immunology , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Female , Immunohistochemistry , Mice , Mice, Inbred BALB CABSTRACT
cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.
Subject(s)
Biological Assay , Vascular Endothelial Growth Factor A/analysis , Vascular Endothelial Growth Factor Receptor-1/analysis , Biotinylation , Cell Line, Tumor , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Genetic Vectors , Humans , Immobilized Proteins/analysis , Immobilized Proteins/genetics , Immobilized Proteins/metabolism , Kinetics , Ligands , Luminescent Measurements , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sensitivity and Specificity , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-1/metabolismABSTRACT
Brain-specific anion transporter BSAT1 extracellular fragment (451-557) cDNA was cloned in a vector for prokaryotic expression, a producer E. coli strain was obtained, and recombinant extracellular fragment BSAT1(451-557)was purified and used for immunization of BALB/c mice. Splenic cells from mice with verified immune response were used for hybridoma generation. Several hybridoma clones producing monoclonal antibodies to BSAT1 extracellular fragment were selected. Antibody specificity was confirmed by ELISA, immunoblotting with recombinant BSAT1(451-557), and immunofluorescent BSAT1 assay on rat brain sections and cultured HEK293 cells. It was demonstrated that the obtained antibodies specifically bind native rat and human BSAT1 and can be used in both fundamental studies of structures forming the blood-brain barrier and development of targeted transport of diagnostic preparations and drugs across the blood-brain barrier.