title: In vitro toxicity in long-term cell culture of MR contrast agents targeted to cartilage evaluation.
abstract: OBJECTIVE
  Contrast-enhanced magnetic resonance (MR) imaging methods have been proposed for non-invasive evaluation of osteoarthritis (OA). We measured cell toxicities of cartilage-targeted low-generation dendrimer-linked nitroxide MR contrast agents and gadopentetate dimeglumine (Gd-DTPA) on cultured chondrocytes.
  
  
  METHODS
  A long-term Swarm rat chondrosarcoma chondrocyte-like cell line was exposed for 48-h to different salts (citrate, maleate, tartrate) and concentrations of generation one or two diaminobutyl-linked nitroxides (DAB4-DLN or DAB8-DLN), Gd-DTPA, or staurosporine (positive control). Impact on microscopic cell appearance, MTT spectrophotometric assays of metabolic activity, and quantitative PicoGreen assays of DNA content (cell proliferation) were measured and compared to untreated cultures.
  
  
  RESULTS
  Chondrocyte cultures treated with up to 7.5 mM Gd-DTPA for 48-h had no statistical differences in DNA content or MTT reaction compared to untreated cultures. At all doses, DAB4-DLN citrate treated cultures had results similar to untreated and Gd-DTPA-treated cultures. At doses >1 mM, DAB4-DLN citrate treated cultures showed statistically greater DNA and MTT reaction than maleate and tartrate DAB4-DLN salts. Cultures exposed to 5 mM or 7.5 mM DAB8-DLN citrate exhibited rounded cells, poor cell proliferation, and barely detectable MTT reaction. Treatment with 0.1 μM staurosporine caused chondrocyte death.
  
  
  CONCLUSIONS
  Long-term exposure, greater than clinically expected, to either DAB4-DLN citrate or Gd-DTPA had no detectable toxicity with results equivalent to untreated cultures. DAB4-DLN citrate was more biocompatible than either the maleate or tartrate salts. Cells exposed for 48-h to 5 mM or 7.5 mM DAB8-DLN salts demonstrated significant cell toxicity. Further evaluation of DAB8-DLN with clinically appropriate exposure times is required to determine the maximum useful concentration.
authors.affiliation: Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: S
authors.initials: S
authors.lastname: Midura
authors.affiliation: Imaging Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA; NitroSci Pharmaceuticals, LLC, New Berlin, WI 53151, USA.
authors.forename: E
authors.initials: E
authors.lastname: Schneider
authors.affiliation: Imaging Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: F A
authors.initials: FA
authors.lastname: Sakamoto
authors.affiliation: NitroSci Pharmaceuticals, LLC, New Berlin, WI 53151, USA; Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.
authors.forename: G M
authors.initials: GM
authors.lastname: Rosen
authors.affiliation: Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA; Imaging Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA. Electronic address: winalsc@ccf.org.
authors.forename: C S
authors.initials: CS
authors.lastname: Winalski
authors.affiliation: Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Midura
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D003287:Contrast Media / Q000008:administration & dosage / Q000633:toxicity*; D004247:DNA / Q000032:analysis; D050091:Dendrimers / Q000008:administration & dosage / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D019786:Gadolinium DTPA / Q000008:administration & dosage / Q000633:toxicity; D008279:Magnetic Resonance Imaging; D051381:Rats; D019311:Staurosporine / Q000008:administration & dosage / Q000633:toxicity; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D003287:Contrast Media; D050091:Dendrimers; D004247:DNA; D019311:Staurosporine; D019786:Gadolinium DTPA
keywords: Cartilage; Chondrocyte; Contrast agent; Cytotoxicity; Dendrimer; Magnetic resonance imaging