title: Molecular magnetic resonance imaging of angiogenesis in vivo using polyvalent cyclic RGD-iron oxide microparticle conjugates.
abstract: Angiogenesis is an essential component of tumour growth and, consequently, an important target both therapeutically and diagnostically. The cell adhesion molecule α(v)β(3) integrin is a specific marker of angiogenic vessels and the most prevalent vascular integrin that binds the amino acid sequence arginine-glycine-aspartic acid (RGD). Previous studies using RGD-targeted nanoparticles (20-50 nm diameter) of iron oxide (NPIO) for magnetic resonance imaging (MRI) of tumour angiogenesis, have identified a number of limitations, including non-specific extravasation, long blood half-life (reducing specific contrast) and low targeting valency. The aim of this study, therefore, was to determine whether conjugation of a cyclic RGD variant [c(RGDyK)], with enhanced affinity for α(v)β(3), to microparticles of iron oxide (MPIO) would provide a more sensitive contrast agent for imaging of angiogenic tumour vessels. Cyclic RGD [c(RGDyK)] and RAD [c(RADyK)] based peptides were coupled to 2.8 μm MPIO, and binding efficacy tested both in vitro and in vivo. Significantly greater specific binding of c(RGDyK)-MPIO to S-nitroso-n-acetylpenicillamine (SNAP)-stimulated human umbilical vein endothelial cells in vitro than PBS-treated cells was demonstrated under both static (14-fold increase; P < 0.001) and flow (44-fold increase; P < 0.001) conditions. Subsequently, mice bearing subcutaneous colorectal (MC38) or melanoma (B16F10) derived tumours underwent in vivo MRI pre- and post-intravenous administration of c(RGDyK)-MPIO or c(RADyK)-MPIO. A significantly greater volume of MPIO-induced hypointensities were found in c(RGDyK)-MPIO injected compared to c(RADyK)-MPIO injected mice, in both tumour models (P < 0.05). Similarly, administration of c(RGDyK)-MPIO induced a greater reduction in mean tumour T(2)* relaxation times than the control agent in both tumour models (melanoma P < 0.001; colorectal P < 0.0001). Correspondingly, MPIO density per tumour volume assessed immunohistochemically was significantly greater for c(RGDyK)-MPIO than c(RADyK)-MPIO injected animals, in both melanoma (P < 0.05) and colorectal (P < 0.0005) tumours. In both cases, binding of c(RGDyK)-MPIO co-localised with α(v)β(3) expression. Comparison of RGD-targeted and dynamic contrast enhanced (DCE) MRI assessment of tumour perfusion indicated sensitivity to different vascular features. This study demonstrates specific binding of c(RGDyK)-MPIO to α(v)β(3) expressing neo-vessels, with marked and quantifiable contrast and rapid clearance of unbound particles from the blood circulation compared to NPIO. Combination of this molecular MRI approach with conventional DCE MRI will enable integrated molecular, anatomical and perfusion tumour imaging.
authors.affiliation: 2. Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Stavros
authors.initials: S
authors.lastname: Melemenidis
authors.affiliation: 1. Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Jefferson
authors.affiliation: 1. Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom.
authors.forename: Neil
authors.initials: N
authors.lastname: Ruparelia
authors.affiliation: 1. Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom.
authors.forename: Asim M
authors.initials: AM
authors.lastname: Akhtar
authors.affiliation: 3. Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, United States.
authors.forename: Jin
authors.initials: J
authors.lastname: Xie
authors.affiliation: 2. Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Danny
authors.initials: D
authors.lastname: Allen
authors.affiliation: 2. Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Alastair
authors.initials: A
authors.lastname: Hamilton
authors.affiliation: 2. Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: James R
authors.initials: JR
authors.lastname: Larkin
authors.affiliation: 2. Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Francisco
authors.initials: F
authors.lastname: Perez-Balderas
authors.affiliation: 2. Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Smart
authors.affiliation: 2. Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Ruth J
authors.initials: RJ
authors.lastname: Muschel
authors.affiliation: 3. Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, United States.
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Chen
authors.affiliation: 2. Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK.
authors.forename: Nicola R
authors.initials: NR
authors.lastname: Sibson
authors.affiliation: 1. Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, United Kingdom.
authors.forename: Robin P
authors.initials: RP
authors.lastname: Choudhury
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D005290:Ferric Compounds / Q000008:administration & dosage* / Q000032:analysis*; D006801:Humans; D008279:Magnetic Resonance Imaging / Q000379:methods*; D008810:Mice, Inbred C57BL; D008863:Microspheres; D009369:Neoplasms / Q000175:diagnosis* / Q000628:therapy; D009389:Neovascularization, Pathologic / Q000000981:diagnostic imaging*; D009842:Oligopeptides / Q000008:administration & dosage* / Q000032:analysis*; D011485:Protein Binding; D011859:Radiography
chemical_list: D005290:Ferric Compounds; D009842:Oligopeptides; C000499:ferric oxide; C047981:arginyl-glycyl-aspartic acid
keywords: DCE.; MRI; Tumour; angiogenesis; cRGDyK; microparticles