• Linfeng Zheng, Zhuoli Zhang, Khashayarsha Khazaie, Saurabh Saha, Robert J Lewandowski, Guixiang Zhang, and Andrew C Larson.
• Department of Radiology, First People's Hospital, Shanghai Jiaotong University, Shanghai, China; Department of Radiology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.
• Plos One. 2014 Jan 1; 9 (12): e116204.

ObjectivesTo validate the feasibility of labeling Clostridium novyi-NT (C.novyi-NT) anaerobes with iron-oxide nanoparticles for magnetic resonance imaging (MRI) and demonstrate the potential to use MRI to visualize intra-tumoral delivery of these iron-oxide labeled C.novyi-NT during percutaneous injection procedures.Materials And MethodsAll studies were approved by IACUC. C.novyi-NT were labeled with hybrid iron-oxide Texas red nanoparticles. Growth of labeled and control samples were evaluated with optical density. Labeling was confirmed with confocal fluorescence and transmission electron microscopy (TEM). MRI were performed using a 7 Tesla scanner with T2*-weighted (T2*W) sequence. Contrast-to-noise ratio (CNR) measurements were performed for phantoms and signal-to-noise ratio (SNR) measurements performed in C57BL/6 mice (n = 12) with Panc02 xenografts before and after percutaneous injection of iron-oxide labeled C.novyi-NT. MRI was repeated 3 and 7 days post-injection. Hematoxylin-eosin (HE), Prussian blue and Gram staining of tumor specimens were performed for confirmation of intra-tumoral delivery.ResultsIron-oxide labeling had no influence upon C.novyi-NT growth. The signal intensity (SI) within T2*W images was significantly decreased for iron-oxide labeled C.novyi-NT phantoms compared to unlabeled controls. Under confocal fluorescence microscopy, the iron-oxide labeled C.novyi-NT exhibited a uniform red fluorescence consistent with observed regions of DAPI staining and overall labeling efficiency was 100% (all DAPI stained C.novyi-NT exhibited red fluorescence). Within TEM images, a large number iron granules were observed within the iron-oxide labeled C.novyi-NT; these were not observed within unlabeled controls. Intra-procedural MRI measurements permitted in vivo visualization of the intra-tumoral distribution of iron-oxide labeled C.novyi-NT following percutaneous injection (depicted as punctate regions of SI reductions within T2*-weighted images); tumor SNR decreased significantly following intra-tumoral injection of C.novyi-NT (p<0.05); these SNR reductions were maintained at 3 and 7 day follow-up intervals. Prussian blue and Gram staining confirmed presence of the iron-oxide labeled anaerobes.ConclusionsC.novyi-NT can be labeled with iron-oxide nanoparticles for MRI visualization of intra-tumoral deposition following percutaneous injection during bacteriolytic therapy.

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