title: A targeted ultrasound contrast agent carrying gene and cell-penetrating peptide: preparation and gene transfection in vitro.
abstract: Targeted and high efficient gene delivery is a main issue in gene treatment. Taking advantage of ischemic memory target P-selectin and our previous study-synergistic effects of ultrasound-targeted microbubble destruction (UTMD) and TAT peptide on gene transfection, which were characterized by targeted aggregation and high efficient gene transfection, we set up a 'smart' gene delivery system-targeted ultrasound contrast agent (UCA) carrying gene and cell-permeable peptides (CPP). Such UCA had a strong binding force with DNA which was protected from being hydrolysed by nuclease. Moreover, synergistic effects of UTMD and TAT peptide increased gene transfection. Specifically, the UCA were reacted with an ischemic memory target P-selectin overexpressed by ischemic issues (including ischemic heart disease) and loaded with gene and CPP, which enabled targeted localization and gene delivery to ischemic cells overexpressing P-selectin. We demonstrated their targeting affinity for hypoxia human umbilical vein endothelial cell (HUVEC) and gene transfection in vitro. The results of confocal laser scanning microscopy (CLSM) showed that gene and CPP were distributed on the shell of UCA. Red fluorescence was observed on the surface of targeted UCA using immunofluorescent microscopy, which demonstrated that the antibody was successfully connected to the UCA. The targeted UCA was specifically and tightly binded to hypoxia HUVEC, while there were no or little non-targeted UCA binding around hypoxia HUVEC. 24h after transfection, gene transfection efficiency detected by FCM was higher in targeted group than non-targeted group. Overall, the targeted UCA carrying gene and CPP was prepared successfully. It had a strong target binding capacity to hypoxia HUVEC and high efficient gene transfection, which maybe provide a novel strategy for gene therapy.
authors.affiliation: Institute of Ultrasound Imaging of Chongqing Medical University, Department of Ultrasound, The Second Affiliated Hospital of Chongqing Medical University, No. 76, LinJiang Road, YuZhong District, Chongqing City 400010, China.
authors.forename: Jianli
authors.initials: J
authors.lastname: Ren
authors.affiliation: Institute of Ultrasound Imaging of Chongqing Medical University, Department of Ultrasound, The Second Affiliated Hospital of Chongqing Medical University, No. 76, LinJiang Road, YuZhong District, Chongqing City 400010, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Institute of Ultrasound Imaging of Chongqing Medical University, Department of Ultrasound, The Second Affiliated Hospital of Chongqing Medical University, No. 76, LinJiang Road, YuZhong District, Chongqing City 400010, China.
authors.forename: Ju
authors.initials: J
authors.lastname: Tian
authors.affiliation: Institute of Ultrasound Imaging of Chongqing Medical University, Department of Ultrasound, The Second Affiliated Hospital of Chongqing Medical University, No. 76, LinJiang Road, YuZhong District, Chongqing City 400010, China. Electronic address: zzy16966@163.com.
authors.forename: Zhiyi
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Institute of Ultrasound Imaging of Chongqing Medical University, Department of Ultrasound, The Second Affiliated Hospital of Chongqing Medical University, No. 76, LinJiang Road, YuZhong District, Chongqing City 400010, China.
authors.forename: Xingzhao
authors.initials: X
authors.lastname: Liu
authors.affiliation: Institute of Ultrasound Imaging of Chongqing Medical University, Department of Ultrasound, The Second Affiliated Hospital of Chongqing Medical University, No. 76, LinJiang Road, YuZhong District, Chongqing City 400010, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.affiliation: Institute of Ultrasound Imaging of Chongqing Medical University, Department of Ultrasound, The Second Affiliated Hospital of Chongqing Medical University, No. 76, LinJiang Road, YuZhong District, Chongqing City 400010, China.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D015687:Cell Hypoxia; D057846:Cell-Penetrating Peptides / Q000737:chemistry*; D003287:Contrast Media / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005796:Genes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010316:Particle Size; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics / Q000379:methods*
chemical_list: D057846:Cell-Penetrating Peptides; D003287:Contrast Media; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA
keywords: Cell-penetrating peptide; Gene transfection; Target; Ultrasound contrast agent