title: In vitro characterization and in vivo ultrasound molecular imaging of nucleolin-targeted microbubbles.
abstract: Nucleolin (NCL) plays an important role in tumor vascular development. An increased endothelial expression level of NCL has been related to cancer aggressiveness and prognosis and has been detected clinically in advanced tumors. Here, with a peptide targeted to NCL (F3 peptide), we created an NCL-targeted microbubble (MB) and compared the performance of F3-conjugated MBs with non-targeted (NT) MBs both in vitro and in vivo. In an in vitro study, F3-conjugated MBs bound 433 times more than NT MBs to an NCL-expressing cell line, while pretreating cells with 0.5 mM free F3 peptide reduced the binding of F3-conjugated MBs by 84%, n = 4, p < 0.001. We then set out to create a method to extract both the tumor wash-in and wash-out kinetics and tumor accumulation following a single injection of targeted MBs. In order to accomplish this, a series of ultrasound frames (a clip) was recorded at the time of injection and subsequent time points. Each pixel within this clip was analyzed for the minimum intensity projection (MinIP) and average intensity projection (AvgIP). We found that the MinIP robustly demonstrates enhanced accumulation of F3-conjugated MBs over the range of tumor diameters evaluated here (2-8 mm), and the difference between the AvgIP and the MinIP quantifies inflow and kinetics. The inflow and clearance were similar for unbound F3-conjugated MBs, control (non-targeted) and scrambled control agents. Targeted agent accumulation was confirmed by a high amplitude pulse and by a two-dimensional Fourier Transform technique. In summary, F3-conjugated MBs provide a new imaging agent for ultrasound molecular imaging of cancer vasculature, and we have validated metrics to assess performance using low mechanical index strategies that have potential for use in human molecular imaging studies.
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Ingham
authors.affiliation: Department of Environmental Health and Safety, University of California, Davis, CA, 95616, USA.
authors.forename: M Karen J
authors.initials: MK
authors.lastname: Gagnon
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Mahakian
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.
authors.forename: Jingfei
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.
authors.forename: Josquin L
authors.initials: JL
authors.lastname: Foiret
authors.affiliation: Department of Radiology, Stanford University, Stanford, CA, 94304, USA.
authors.forename: Juergen K
authors.initials: JK
authors.lastname: Willmann
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA. Electronic address: kwferrara@ucdavis.edu.
authors.forename: Katherine W
authors.initials: KW
authors.lastname: Ferrara
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D051379:Mice; D045423:Microbubbles; D057054:Molecular Imaging / Q000379:methods*; D009374:Neoplasms, Experimental / Q000000981:diagnostic imaging* / Q000378:metabolism*; D010455:Peptides / Q000493:pharmacokinetics*; D010750:Phosphoproteins / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014463:Ultrasonography; D000097796:Nucleolin
chemical_list: D010455:Peptides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin
keywords: Angiogenesis; In vivo; Molecular imaging; Nucleolin; Peptide; Ultrasound contrast agents