title: Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery.
abstract: 1-Ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.
authors.affiliation: Department of Electrical and Computer Engineering, University of Alberta, Edmonton, AB, Canada.
authors.forename: Yuzhi
authors.initials: Y
authors.lastname: Hao
authors.forename: Peng
authors.initials: P
authors.lastname: Xu
authors.forename: Chuan
authors.initials: C
authors.lastname: He
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Yang
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.forename: James
authors.initials: J
authors.lastname: Xing
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D011232:Chemical Precipitation; D004247:DNA / Q000378:metabolism; D000431:Ethanol / Q000737:chemistry; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D008280:Magnetics; D037742:Nanotubes, Carbon / Q000737:chemistry* / Q000648:ultrastructure; D010957:Plasmids / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013388:Succinimides / Q000737:chemistry; D014162:Transfection
chemical_list: D017304:Annexin A5; D037742:Nanotubes, Carbon; D013388:Succinimides; D049452:Green Fluorescent Proteins; D000431:Ethanol; D004247:DNA; D016650:Fluorescein-5-isothiocyanate; C001426:N-hydroxysuccinimide; D005022:Ethyldimethylaminopropyl Carbodiimide