Novel Fluorescent Core–Shell Nanocontainers for Cell Membrane Transport Meizhen Yin, † Christoph R. W. Kuhlmann, ‡ Ksenia Sorokina, † Chen Li, † George Mihov, † Eweline Pietrowski, ‡ Kaloian Koynov, † Markus Klapper, † Heiko J. Luhmann, ‡ Klaus Müllen,* ,† and Tanja Weil* ,† Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany, and Johannes Gutenberg-University of Mainz, Institute of Physiology and Pathophysiology, Saarstrasse 21, 55099 Mainz, Germany Received October 14, 2007; Revised Manuscript Received January 27, 2008 The synthesis and characterization of novel core–shell macromolecules consisting of a fluorescent perylene- 3,4,9,10-tetracarboxdiimide chromophore in the center surrounded by a hydrophobic polyphenylene shell as a first and a flexible hydrophilic polymer shell as a second layer was presented. Following this strategy, several macromolecules bearing varying polymer chain lengths, different polymer shell densities, and increasing numbers of positive and negative charges were achieved. Because all of these macromolecules reveal a good water solubility, their ability to cross cellular membranes was investigated. In this way, a qualitative relationship between the molecular architecture of these macromolecules and the biological response was established. Introduction In recent years, there has been a growing interest in the visualization and manipulation of cellular processes down to the single molecule level. 1,2 For this purpose, different kinds of chromophores, 3 quantum dots, 4 tailored macromolecular architectures, 5 or fluorescent proteins 6 have been prepared or expressed in order to gain a better understanding of biological processes such as membrane transport, 7 protein trafficking, 1,2 or the delivery of guest molecules, e.g., nucleic acids. 5 In particular, such macromolecules with sizes below 20 nm are of major interest because they might profit from cellular transport and distribution pathways of classical biomacromolecules of similar sizes, e.g,. proteins. In general, an efficient membrane transport is considered crucial for drug delivery applications 8,9 where drug molecules displaying, e.g., a limited metabolic stability or a low aqueous solubility are successfully absorbed by host macromolecules in order to ensure their beneficial pharmacological effect. 10–12 In addition, there is a growing interest in an improved understanding of the fate of synthetic macromolecules or nanoparticles inside cells. 8 Therefore, the design of complex macromolecular architectures facilitating a fine-tuning of the surface charges, charge densities, and sizes combined with a fluorescent readout are attractive for the visualization of cell transport processes, the uptake and release of guest molecules, as well as for the visualization of their location and interactions inside the cell. Herein, the synthesis of fluorescent core–shell macromol- ecules with different architectures, charges, and charge densities is presented. In addition, their ability to cross cell membranes has been studied by applying optical methods. For this purpose, a set of complex core–shell star polymer architectures consisting of a fluorescent perylene-3,4,9,10-tetracarboxdiimide chro- mophore (PDI) in the center surrounded by two different kinds of polymer shells has been designed. The central PDI chro- mophore allows the detection of cell transport via fluorescence microscopy even at the single molecule level. 13 The next layer consists of a rigid polyphenylene dendrimer shell suited to prevent the PDI chromophore from aggregation in water, which usually leads to reduced fluorescence quantum yields. 14,15 The outer layer is formed by a flexible polymer shell, which enables the introduction of different numbers of positive charges in order to achieve water solubility as well as a variation of the charge densities. Recently, histochemical experiments on fixed cells with structurally similar, however negatively charged core–shell macromolecules were described that revealed a selective staining of the cell nucleus. 16 However, because of the necessity to apply detergents in order to achieve membrane penetration, the application of such macromolecules for cell uptake investigations is rather limited. In contrast, positively charged core–shell macromolecules described herein reveal fast membrane transport without the necessity to apply auxiliary supplies. In addition, such cationic macromolecules reveal a high tendency to interact with negatively charged polyelectrolytes such as DNA, 17 which enables their use as nonviral DNA carriers. In this paper, the synthesis and characterization of fluorescent core–shell macromolecules with different architec- tures and different charge densities is described in detail. Then, these macromolecules are evaluated with respect to their ability to cross cellular membranes, which is essential for their application as drug carriers. We believe that the combination of tailored macromolecular architectures with a high structural complexity and the functional characterization of the resulting biological responses provides a deeper understanding of complex cellular processes such as membrane penetration which paves the way to improved drug delivery systems. Materials and Methods CuBr (Aldrich, 99.999%), 4,4′-di-tert-butyl-2,2′-bipyridine (DTB- bipy) (Aldrich, 98%), N-(2,6-diisopropylphenyl)-9-bromo-perylene-3, * Corresponding authors. E-mail: muellen@mpip-mainz.mpg.de (K.M.); weilt@mpip-mainz.mpg.de (T.W.). † Max Planck Institute for Polymer Research. ‡ Johannes Gutenberg-University of Mainz, Institute of Physiology and Pathophysiology. Biomacromolecules 2008, 9, 1381–1389 1381 10.1021/bm701138g CCC: $40.75  2008 American Chemical Society Published on Web 04/22/2008