ABSTRACT
Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG2 cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG2 cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases.
Subject(s)
DNA/administration & dosage , Gene Targeting/methods , Hepatoblastoma/genetics , Hepatoblastoma/metabolism , Receptors, LDL/metabolism , Sonication , Transfection/methods , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Receptors, LDL/geneticsSubject(s)
Cardiac Pacing, Artificial/methods , Natriuretic Peptide, Brain/blood , Adult , Aged , Female , Humans , Male , Middle Aged , Time FactorsABSTRACT
Although viral vectors are efficient systems to transfer foreign genes into cells or target tissues, safety issues remain in relation to human gene therapy. Microbubbles currently used as ultrasound contrast agents have been applied in transfection of genes. This study was designed to test the transfection efficiency and the expression of exogenous gene mediated by ultrasound irradiation enhanced air filled albumin microbubbles in ECV304 cell line in vitro and the heart of the mouse in vivo. Air filled microbubbles (2.0-4.0 microm in diameter) were created by sonicating the mixture of human albumin, glucose, mannitol and special additive that was designed for stabilization. Plasmid DNA loading the reporter genes was gently mixed with microbubbles. The mixture of plasmid DNA and microbubbles was administrated to cultured ECV304 cells and BALB/c mice (tail vein injection) under different ultrasound/microbubble conditions, and then the transfection and expression efficiency were examined. The results both in vivo and in vitro demonstrated that microbubble with ultrasound irradiation could significantly elevate the exogenous gene expression as compared with microbubble or ultrasound only. Overall, the present study showed that the ultrasound-target microbubble destruction method enhanced the exogenous gene expression in vivo and in vitro, and provided a gene therapy way not only efficient but also easy to be manipulated and carried out in clinical.
