title: Ultrasound microbubbles combined with the NFκB binding motif increase transfection efficiency by enhancing the cytoplasmic and nuclear import of plasmid DNA.
abstract: Inefficient gene delivery poses a challenge for non‑viral gene therapy. Cytoplasmic and nuclear membrane barriers are responsible for the inefficiency as they restrict the import of exogenous genes. The present study aimed to improve the transfection efficiency using a novel gene delivery system, which consisted of two components: ultrasound microbubbles and the nuclear factor κB (NFκB) binding motif. Ultrasound-targeted microbubble destruction (UTMD) was used to enhance the cytoplasmic import of plasmids and the NFκB binding motif was added to promote the nuclear intake of the plasmid from the cytoplasm. In the present study, human umbilical vein endothelial cells were transfected using UTMD with two different Cy3-labeled plasmids, phSDF-1α and phSDF‑1α‑NFκB. phSDF-1α-NFκB was constructed by inserting a specific DNA targeting sequence (five optimal repeats of the binding motif for the inducible transcription factor NFκB) into phSDF‑1α. The nuclear import and gene expression efficiency of phSDF-1α-NFκB were compared with those of phSDF-1α to investigate the effect of the NFκB binding motif on transfection. The results showed that UTMD significantly increased the cytoplasmic intake of pDNA and maintained high cell viability. The nuclear import and gene expression of phSDF-1α‑NFκB‑transfected cells were significantly higher than those transfected with phSDF-1α. Compared with the NFκB‑free plasmids, the quantity of NFκB plasmids in the nucleus increased 6.5-fold and the expression of SDF-1α was 4.4-fold greater. These results suggest that UTMD combined with NFκB binding motif significantly improve transfection efficiency by enhancing the cytoplasmic and nuclear import of exogenous plasmid DNA.
authors.affiliation: Department of Ultrasound Imaging, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Deng
authors.forename: Jin-Ling
authors.initials: JL
authors.lastname: Chen
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhou
authors.forename: Bo
authors.initials: B
authors.lastname: Hu
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen
authors.forename: Jia
authors.initials: J
authors.lastname: Huang
authors.forename: Rui-Qiang
authors.initials: RQ
authors.lastname: Guo
mesh_terms: D021581:Active Transport, Cell Nucleus; D001665:Binding Sites; D015153:Blotting, Western; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D054377:Chemokine CXCL12 / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage*; D018014:Gene Transfer Techniques; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D045423:Microbubbles; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000008:administration & dosage*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014465:Ultrasonics
chemical_list: C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D016328:NF-kappa B; D012333:RNA, Messenger; D004247:DNA