Thermostable Luciferase from Luciola cruciate for Imaging of Carbon Nanotubes and Carbon Nanotubes Carrying Doxorubicin Using in Vivo Imaging System Ramy El-Sayed, † Mohamed Eita, ‡ Åsa Barrefelt, † Fei Ye, † Himanshu Jain, ‡ Mona Fares, † Arne Lundin, § Mikael Crona, § Khalid Abu-Salah, ∥ Mamoun Muhammed, ‡ and Moustapha Hassan* ,†,⊥ † Experimental Cancer Medicine, KFC, Novum, Department of Laboratory Medicine, Karolinska Institutet, 141 86 Stockholm, Sweden ‡ Functional Nanomaterials Division, The Royal Institute of Technology (KTH) SE-164 40 Stockholm, Sweden § BioThema AB, Handens stationsvä g 17; 136 40 Handen, Sweden ∥ King Abdulla Institute for Nanotechnology, King Saud University, Riyadh 11451, P.O. Box 2455, Saudi Arabia ⊥ Karolinska University Hospital-Huddinge, 141 86 Stockholm, Sweden * S Supporting Information ABSTRACT: In the present study, we introduce a novel method for in vivo imaging of the biodistribution of single wall carbon nanotubes (SWNTs) labeled with recombinant thermo-stable Luciola cruciata luciferase (LcL). In addition, we highlight a new application for green fluorescent proteins in which they are utilized as imaging moieties for SWNTs. Carbon nanotubes show great positive potential compared to other drug nanocarriers with respect to loading capacity, cell internalization, and biodegradability. We have also studied the effect of binding mode (chemical conjugation and physical adsorption) on the chemiluminescence activity, decay rate, and half-life. We have shown that through proper chemical conjugation of LcL to CNTs, LcL remained biologically active for the catalysis of D-luciferin in the presence of ATP to release detectable amounts of photons for in vivo imaging. Chemiluminescence of LcL allows imaging of CNTs and their cargo in nonsuperficial locations at an organ resolution with no need of an excitation source. Loading LcL-CNTs with the antitumor antibiotic doxorubicin did not alter their biological activity for imaging. In vivo imaging of LcL-CNTs has been carried out using “IVIS spectrum” showing the uptake of LcL-CNTs by different organs in mice. We believe that the LcL-CNT system is an advanced powerful tool for in vivo imaging and therefore a step toward the advancement of the nanomedicine field. KEYWORDS: CNT, luciferase, in vivo imaging, bioluminescence, drug delivery F unctionalized nanomaterials are at the forefront of research interest in nanotechnology, as they have shown great potential specifically in the field of nanomedicine including biosensing, 1 drug delivery, 2 bioimaging, 3 and biocomposites. 4 One major milestone in the field of nanomedicine is the development of advanced carriers capable of delivering therapeutic payloads in significant quantities to specific sites. 5,6 Imaging and/or tracking the nanoparticles and the cargo from administration to the site of delivery is very important. This is the first study to present a novel system designed for in vivo imaging of nanotubes (CNTs) as a nanocarrier using thermostable recombinant luciferase from Luciola cruciata (LcL). We have showed that it is possible to maintain the protein activity by proper chemical conjugation of LcL to CNTs. We have also demonstrated the applicability of this system for imaging the uptake of CNTs by different organs in mice using a commercially available IVIS spectrum instrument. LcL chemically conjugated to CNTs administered intravenously (IV) showed a different organ uptake compared to LcL protein alone. In addition, we studied the effect of immobilization mode (chemical conjugation or physical adsorption) on the bioluminescence activity decay rate and half-life. We have also shown that loading LcL-CNTs with the anticancer agent doxorubicin did not alter the biological activity of LcL. Taken together, these advantages make the use of LcL- CNTs a powerful generic method for in vivo imaging of different nanocarrier drug delivery systems. Received: November 7, 2012 Revised: March 10, 2013 Published: March 22, 2013 Letter pubs.acs.org/NanoLett © 2013 American Chemical Society 1393 dx.doi.org/10.1021/nl304123u | Nano Lett. 2013, 13, 1393−1398