title: Focused ultrasound-induced blood-brain barrier opening for non-viral, non-invasive, and targeted gene delivery.
abstract: Focused ultrasound (FUS) exposure in the presence of microbubbles can temporally open the blood-brain barrier (BBB) and is an emerging technique for non-invasive brain therapeutic agent delivery. Given the potential to deliver large molecules into the CNS via this technique, we propose a reliable strategy to synergistically apply FUS-BBB opening for the non-invasive and targeted delivery of non-viral genes into the CNS for therapeutic purpose. In this study, we developed a gene-liposome system, in which the liposomes are designed to carry plasmid DNA (pDNA, containing luciferase reporter gene) to form a liposomal-plasmid DNA (LpDNA) complex. Pulsed FUS exposure was delivered to induce BBB opening (500-kHz, burst length=10ms, 1% duty cycle, PRF=1Hz). The longitudinal expression of luciferase was quantitated via an in vivo imaging system (IVIS). The reporter gene expression level was confirmed via immunoblotting, and histological staining was used to identify transfected cells via fluorescent microscopy. In a comparison of gene transduction efficiency, the LpDNA system showed better cell transduction than the pDNA system. With longitudinal observation of IVIS monitoring, animals with FUS treatment showed significant promotion of LpDNA release into the CNS and demonstrated enhanced expression of genes upon sonication with FUS-BBB opening, while both the luciferase and GDNF protein expression were successfully measured via Western blotting. The gene expression peak was observed at day 2, and the gene expression level was up to 5-fold higher than that in the untreated hemisphere (compared to a 1-fold increase in the direct-inject positive-control group). The transfection efficiency was also found to be LpDNA dose-dependent, where higher payloads of pDNA resulted in a higher transfection rate. Immunoblotting and histological staining confirmed the expression of reporter genes in glial cells as well as astrocytes. This study suggests that IV administration of LpDNA in combination with FUS-BBB opening can provide effective gene delivery and expression in the CNS, demonstrating the potential to achieve non-invasive and targeted gene delivery for treatment of CNS diseases.
authors.affiliation: Medical Imaging Research Center, Institute for Radiological Research, Chang Gung University/Chang Gung Memorial Hospital, Taoyuan 333, Taiwan.
authors.forename: Chung-Yin
authors.initials: CY
authors.lastname: Lin
authors.affiliation: Department of Electrical Engineering, Chang Gung University, Taoyuan 333, Taiwan.
authors.forename: Han-Yi
authors.initials: HY
authors.lastname: Hsieh
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, UT 84602, United States.
authors.forename: William G
authors.initials: WG
authors.lastname: Pitt
authors.affiliation: Department of Neurosurgery, Chang Gung Memorial Hospital, Linkou Medical Center and College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
authors.forename: Chiung-Yin
authors.initials: CY
authors.lastname: Huang
authors.affiliation: Department of Neurosurgery, Chang Gung Memorial Hospital, Linkou Medical Center and College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
authors.forename: I-Chou
authors.initials: IC
authors.lastname: Tseng
authors.affiliation: Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 300, Taiwan.
authors.forename: Chih-Kuang
authors.initials: CK
authors.lastname: Yeh
authors.affiliation: Department of Neurosurgery, Chang Gung Memorial Hospital, Linkou Medical Center and College of Medicine, Chang Gung University, Taoyuan 333, Taiwan. Electronic address: kuochenwei@cgmh.org.tw.
authors.forename: Kuo-Chen
authors.initials: KC
authors.lastname: Wei
authors.affiliation: Department of Electrical Engineering, Chang Gung University, Taoyuan 333, Taiwan; Health Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan. Electronic address: haoliliu@mail.cgu.edu.tw.
authors.forename: Hao-Li
authors.initials: HL
authors.lastname: Liu
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000235:genetics; D008081:Liposomes; D008156:Luciferases / Q000235:genetics; D008297:Male; D008807:Mice, Inbred BALB C; D045423:Microbubbles; D009457:Neuroglia / Q000378:metabolism; D010957:Plasmids; D000069453:Ultrasonic Waves
chemical_list: D051100:Glial Cell Line-Derived Neurotrophic Factor; D008081:Liposomes; D004247:DNA; D008156:Luciferases
keywords: Blood-brain barrier opening; Focused ultrasound; GDNF; Gene delivery; Liposomes; Luciferase