Fluorescence labeled microbubbles for multimodal imaging Åsa Barrefelt a, b, 1 , Ying Zhao a, 1 , Malin K. Larsson c , Gabriella Egri d , Raoul V. Kuiper e , J € org Hamm f , Maryam Saghafian b , Kenneth Caidahl g , Torkel B. Brismar a , Peter Aspelin a , Rainer Heuchel a , Mamoun Muhammed h , Lars D € ahne d , Moustapha Hassan b, i, * a Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet (KI), Stockholm, Sweden b Experimental Cancer Medicine (ECM), Department of Laboratory Medicine, KI, Sweden c Department of Medical Engineering, School of Technology and Health, Royal Institute of Technology (KTH), Stockholm, Sweden d Surflay Nanotec GmbH, 12489 Berlin, Germany e Karolinska Institute Core Facility for Morphologic Phenotype Analysis, Karolinska University Hospital-Huddinge, Stockholm, Sweden f PerkinElmer, 68 Elm St., Hopkinton, MA 01748, USA g Department of Molecular Medicine and Surgery, KI, Dept. Clin Phys, Karolinska University Hospital, Stockholm, Sweden h Department of Materials and Nano Physics, Division of Functional Materials (FNM), Royal Institute of Technology (KTH), Stockholm, Sweden i Clinical Research Center (KFC, Novum), Karolinska University Hospital-Huddinge, Stockholm, Sweden article info Article history: Received 23 June 2015 Accepted 2 July 2015 Available online xxx Keywords: In vivo imaging systems Micro-computed tomography Micro-ultrasound Microbubbles VivoTag 680 Near infrared (NIR) Fluorescence abstract Air-filled polyvinyl alcohol microbubbles (PVA-MBs) were recently introduced as a contrast agent for ultrasound imaging. In the present study, we explore the possibility of extending their application in multimodal imaging by labeling them with a near infrared (NIR) fluorophore, VivoTag-680. PVA-MBs were injected intravenously into FVB/N female mice and their dynamic biodistribution over 24 h was determined by 3D-fluorescence imaging co-registered with 3D-mCT imaging, to verify the anatomic location. To further confirm the biodistribution results from in vivo imaging, organs were removed and examined histologically using bright field and fluorescence microscopy. Fluorescence im- aging detected PVA-MB accumulation in the lungs within the first 30 min post-injection. Redistribution to a low extent was observed in liver and kidneys at 4 h, and to a high extent mainly in the liver and spleen at 24 h. Histology confirmed PVA-MB localization in lung capillaries and macrophages. In the liver, they were associated with Kupffer cells; in the spleen, they were located mostly within the marginal- zone. Occasional MBs were observed in the kidney glomeruli and interstitium. The potential application of PVA-MBs as a contrast agent was also studied using ultrasound (US) imaging in subcutaneous and orthotopic pancreatic cancer mouse models, to visualize blood flow within the tumor mass. In conclusion, this study showed that PVA-MBs are useful as a contrast agent for multimodal imaging. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Nano- and microparticles are on the verge of becoming standard in future medicine. Due to their potential ligation to enhancing agents, such as fluorescent dyes, gold, iron oxide or manganese, and/or antibodies, they have been used as contrast agents and targeted drug delivery vehicles [1e5]. The combination of in vivo imaging modalities such as optical in vivo imaging/magnetic reso- nance imaging (MRI) and optical in vivo imaging/computed to- mography (CT) opens up a new era of non-radioactive agents for functional imaging using a multimodal approach [6]. Adding fluo- rescent markers to injectable contrast agents carrying a combina- tion of superparamagnetic iron oxide nanoparticles (SPION) for detection by MRI or gold nanoparticles for detection by CT enables their multimodal use [7]. Recently, polyvinyl alcohol microbubbles (PVA-MBs) were introduced as a contrast agent for multimodality or hybrid imaging by combining MRI [6,8], single photon emission computed tomography (SPECT) [8] and ultrasound [9e12]. * Corresponding author. ECM, KFC, Novum, Karolinska University Hospital-Hud- dinge, SE-141 86 Stockholm, Sweden. E-mail addresses: asa.barrefelt@ki.se (Å. Barrefelt), ying.zhao.1@ki.se (Y. Zhao), malinl@sth.kth.se (M.K. Larsson), g.egri@surflay.com (G. Egri), Raoul.Kuiper@ki.se (R.V. Kuiper), Jorg.Hamm@perkinelmer.com (J. Hamm), maryam.saghafian@ki.se (M. Saghafian), Kenneth.Caidahl@ki.se (K. Caidahl), torkel.brismar@karolinska.se (T.B. Brismar), Peter.Aspelin@ki.se (P. Aspelin), rainer.heuchel@ki.se (R. Heuchel), mamoun@kth.se (M. Muhammed), l.daehne@surflay.com (L. D€ ahne), Moustapha. hassan@ki.se (M. Hassan). 1 Equal contribution from authors. Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2015.07.017 0006-291X/© 2015 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (2015) 1e6 Please cite this article in press as: Å. Barrefelt, et al., Fluorescence labeled microbubbles for multimodal imaging, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.017