Specific labelling of cell populations in blood with targeted immuno-fluorescent/ magnetic glyconanoparticles Juan Gallo a , Isabel García a, b , Nuria Genicio a , Daniel Padro c , Soledad Penadés a, b, * ,1 a Laboratory of GlycoNanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE, Paseo Miramón 182, E-20009 San Sebastián, Spain b Biomedical Research Networking Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain c Molecular Imaging Unit, CIC biomaGUNE, P o de Miramón 182, 20009 San Sebastian, Spain article info Article history: Received 22 July 2011 Accepted 4 September 2011 Available online 21 September 2011 Keywords: Fluorescent/magnetic nanoparticles Specific labelling Human blood cells Magnetic resonance imaging Fluorescence microscopy abstract Current performance of iron oxide nanoparticle-based contrast agents in clinical use is based on the unspecific accumulation of the probes in certain organs or tissues. Specific targeted biofunctional nanoparticles would significantly increase their potential as diagnostic and therapeutic tools in vivo. In this study, multimodal fluorescent/magnetic glyco-nanoparticles were synthesized from gold-coated magnetite (glyco-ferrites) and converted into specific probes by the covalent coupling of protein G and subsequent incubation with an IgG antibody. The immuno-magnetic-fluorescent nanoparticles were applied to the specific labelling of peripheral blood mononuclear cells (PBMCs) in a complex biological medium, as human blood. We have been able to label specifically PBMCs present in blood in a percentage as low as 0.10e0.17%. Red blood cells (RBCs) were also clearly labelled, even though the inherent T 2 contrast arising from the high iron content of these cells (coming mainly from haemoglobin). The labelling was further assessed at cellular level by fluorescence microscopy. In conclusion, we have developed new contrast agents able to label specifically a cell population under adverse biological conditions (low abundance, low intrinsic T 2 , high protein content). These findings open the door to the application of these probes for the labelling and tracking of endogenous cell populations like metastatic cancer cells, or progenitor stem cells that exist in very low amount in vivo. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Nanoparticles have long been proposed to play an important role in future medicine [1], [2]. One of the most advanced application of nanocrystals so far is the use of superparamagnetic iron oxide nanoparticles (SPIOs) as probes for magnetic resonance imaging that provide enormous possibilities for improving the efficiency of medical diagnosis [2], [3]. In this multidisciplinary field, discovering, developing and understanding the underlying chemistry which controls particle size, surface functionality and behaviour into complex biological media are of the outmost importance. Magnetic resonance imaging (MRI) is gaining importance in medical diagnosis as technology improves (higher fields and stronger gradients). New smart contrast agents are being devel- oped to increase the resolution and sensitivity of MRI [3]. Low sensitivity is one of the main drawbacks of MRI when working in vivo, while resolution is the problem to be solved on going down to the cellular level. These inherent disadvantages do not enable MRI technique to compete with fluorescence microscopy in in vitro cellular experiments. The resolution obtained by both methodolo- gies is still not comparable (mm to mm for MRI vs w200 nm for fluorescence microscopy), although great efforts are being done in the development of micro-imaging systems for MRI. This reason explains why multimodal contrast agents with both magnetic properties and fluorescence emission are attracting so much interest from the scientific community [2,4], [5], [6]. For in vivo applications, the physical/chemical characteristics of non-targeted nanoparticles, especially hydrodynamic size, govern their bio- distribution and toxicology. Passive targeting iron oxide nanop- robes named SPIO (hydrodynamic size 40 nm) are typical liver contrast agents [7], [8], and nanoparticles smaller than 40 nm, called USPIO, have been widely developed to detect lymph node metastases [9], [10], and inflammatory and degenerative diseases due to their unspecific uptake by macrophages [11]. However, disease detection through iron oxide-enhanced MRI, especially for early tumour detection, is being gradually shifted from passive * Corresponding author. Laboratory of GlycoNanotechnology, Biofunctional Nanomaterials Unit, CIC biomaGUNE, Spain. Tel.: þ34 943005307; fax: þ34 943005301. E-mail address: spenades@cicbiomagune.es (S. Penadés). 1 Dedicated to Prof. Manuel Martin-Lomas on the occasion of his 70th birthday. Contents lists available at SciVerse ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.09.010 Biomaterials 32 (2011) 9818e9825