Preperation Of Nanobody-targeted Multifunctional Nanobubbles And Targeting Verification In Renal Cell Carcinoma Cells

BackgroundNanobubbles can enhance the contrast imaging of tissues and release the drugs and theraputic genes loaded in them directionally under ultrasoud irradiation,so they have a high potential value in diagnosis and therapy of renal cell carcinoma.Renal cell carcinoma related antigen,G250,is a transmembrane protein.It is expressed in most of the renal cell carcinoma except the normal kidney tissue.So it can be used as target protein of targeted nanobubbles.Nanobody,which has small volume and high specificity,can be used as ligand to make nanobubbles smaller.ObjectiveThe aim of this work was to develop nanobodies which could specifically combine with human G250 ectodomain by immuning camel and connect them to nanobubbles to prepare targeted nanobubbles.We will provide a potential new way for the diagnosis and treatment of renal cell carcinoma.MethodsThe plasmid harboring the DNA fragment of extracellular domain of human G250 was transiently transfected into HEK 293 F cells for protein expression.Following immunization of two Bactrian camels with the recombinant G250 ectodomain protein,VHH fragments were isolated by nested PCR from lymphocyte cDNA.A VHH nanobody library was developed using phage display technology.Nanobodies specifically recognizing G250 were enriched by biopanning in vitro and identified by monoclonal phage ELISA.Cell binding ability of selected nanobodies was further characterized by cell ELISA and FACS.We prepared blank liposome nanoscale microbubbles(nanobubbles)by mechanical oscillation and connected nanobodies to blank nanobubbles to make targeted nanobubbles.We use immunofluorescence to verify targeted nanobubbles and test specific binding capacity of the two kinds of nanobubbles to the three kinds of cells by targeted cell binding assay.We use FACS to identify expression of G250 antigen in 786-O cells,He La cells and ACHN cells.ResultsThe recombinant DNA fragment encoding human G250 ectodomain was confirmed by DNA sequencing,and the recombinant G250 protein was proved to be successfully expressed by Western blot.After immunization of two Xinjiang Bactrian camels with the recombinant G250 protein,a phage display VHH library containing 2.87×109 in size was constructed.After three rounds of biopanning against the G250 protein in vitro,the G250-specific VHH clones were efficiently enriched with a positive rate of 54.3%.A clone Nb-G5 with the highest cell binding affinity was identified upon three VHH clones selected from the immune phage display VHH library.Nb-G5 was expressed in a soluble form with yield of 1.5mg per liter cell culture and presented high selectivity and specificity to the cells which expressing G250 protein in both cell ELISA and FACS.FACS confirmed Nb-G5 could bind to G250 antigen-positive 786-O cells ? G250 antigen-positive HeLa cells.Immunofluorescence technique confirmed that nanobodies were closely connected to the nanobubbles.The nanobubbles with G250 nanobodies could bind to 786-O cells,He La cells specifically,and could not bind to ACHN cells.No combination was happened between the blank nanobubbles and the three kinds of cells.ConclusionsNanobodies against G250 ectodomain which could combine with G250 protein specically both in molecular and cellular levelwere successfully developed.The G250 nanobody-targeted nanobubbles were prepared and their targeting ability to renal cell carcinoma cells was verified.