Biomaterials 27 (2006) 2820–2828 Uptake of functionalized, fluorescent-labeled polymeric particles in different cell lines and stem cells Myriam Ricarda Lorenz a,b , Verena Holzapfel b , Anna Musyanovych b , Karin Nothelfer a , Paul Walther c , Hendrik Frank b , Katharina Landfester b , Hubert Schrezenmeier a , Volker Maila¨nder a,Ã a Department of Transfusion Medicine, Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, University of Ulm, Helmholtzstr. 10, 89081 Ulm, Germany b Department of Organic Chemistry III, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany c Central Facility for Electron Microscopy, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany Received 28 September 2005; accepted 30 December 2005 Available online 23 January 2006 Abstract Labeling of cells with particles for in-vivo detection is interesting for various biomedical applications. The objective of this study was to evaluate the feasibility and efficiency labeling of cells with polymeric particles without the use of transfection agents. We hypothesized that surface charge would influence cellular uptake. The submicron particles were synthesized by the miniemulsion process. A fluorescent dye which served as reporter was embedded in these particles. The surface charge was varied by adjusting the amount of copolymerized monomer with amino group thus enabling to study the cellular uptake in correlation to the surface charge. Fluorescent-activated cell sorter (FACS) measurements were performed for detecting the uptake of the particles or attachment of particles in mesenchymal stem cells (MSC), and the three cell lines HeLa, Jurkat, and KG1a. These cell lines were chosen as they can serve as models for clinically interesting cellular targets. For these cell lines—with the exception of MSCs—a clear correlation of surface charge and fluorescence intensity could be shown. For an efficient uptake of the submicron particles, no transfection agents were needed. Confocal laser scanning microscopy and transmission electron microscopy (TEM) revealed differences in subcellular localization of the particles. In MSCs and HeLa particles were mostly located inside of cellular compartments resembling endosomes, while in Jurkat and KG1a, nanoparticles were predominantly located in clusters on the cell surface. Scanning electron microscopy showed microvilli to be involved in this process. r 2006 Elsevier Ltd. All rights reserved. Keywords: Particle; Fluorescence; Cell uptake; Surface modification; Mesenchymal stem cell; FACS; SEM; TEM; Confocal microscopy 1. Introduction Materials in the submicron level are increasingly used for biomedical applications exploiting specific effects on this level. As an example the superparamagnetic effect of iron oxide particles makes them interesting contrast agents in magnetic resonance tomography [1]. These particles are also used as non-viral vehicles for gene therapy [2–4], drug delivery [5–7], immunization [8–11] and detoxification [12]. Understanding the interactions of these materials with cells is crucial for improving their behaviour in-vivo and in-vitro. Especially uptake into cells and degradation in intracellular compartments is of high importance. In order to increase the rate of intracellular uptake of polymeric particles transfection agents can be used [13]. These transfection agents are mostly cationic, positively charged molecules [14]. These are toxic and not approved for clinical use. Hence applications in human trials and therapeutic interventions are prohibited up to now. For small molecules and macromolecules, routes of uptake into cells have been described [15,16]. Particles in the range of a few to several hundred nanometers are much larger and conditions and mechanisms of uptake involving several possible mechanisms like pinocytosis, non-specific endocytosis, receptor-mediated endocytosis, and for larger ARTICLE IN PRESS www.elsevier.com/locate/biomaterials 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2005.12.022 Ã Corresponding author. Fax: +49 731 150-500. E-mail address: v.mailaender@blutspende.de (V. Maila¨nder).