Amphiphilic Copolymer for Delivery of Xenobiotics: In ViWo Studies in a Freshwater Invertebrate, a Mesostominae Flatworm Laetitia De Jong, ‡ Xavier Moreau, ‡ Alain Thiéry, ‡ Guilhem Godeau, †,3 Mark W. Grinstaff, § and Philippe Barthélémy* ,†,3 UMR-CNRS 6116, Institut Méditerranéen d’Ecologie et de Paléoécologie - IMEP (Biomarqueurs & Bioindicateurs Environnementaux), case 17, Université de Provence, 3 place Victor Hugo 13331 Marseille Cedex 3, France, INSERM U869, Bordeaux, F-33076, France, Université Victor Segalen Bordeaux 2, Bordeaux, F-33076, France, and Department of Biomedical Engineering, Metcalf Center for Science and Engineering, Boston University, Boston, Massachusetts 02215. Received November 21, 2007; Revised Manuscript Received February 1, 2008 The synthesis of an amphiphilic polymethacrylate copolymer containing cholesterol hydrophobic moieties and rhodamine as a fluorescent probe, the formation of microspheres, and the uptake of these microspheres in an invertebrate are reported. The cholesterol-derived methacryloyl monomer, which was prepared Via a one-step synthesis, was copolymerized with methacrylic acid and methacryloxyethyl thiocarbamoyl rhodamine B in the presence of AIBN as initiator. The obtained dye-labeled copolymer was characterized by 1 H NMR and UV–vis spectroscopy. Fluorescence and TEM microscopies studies show that this amphiphilic copolymer aggregates to give microspheres with diameters ranging from 3 to 11 μm. The in ViVo study in a freshwater invertebrate, a Mesostominae flatworm (Rhabdocoela, Thyphloplanidae), indicates that the microspheres enter the cells by endocytosis. The data collected demonstrate that the rhodamine B covalently attached to the amphiphilic copolymers is bioaccumulated without being translocated out of the cell by the multixenobiotic resistance (MXR) transporters. As the MXR system is similar to the multidrug resistance (MDR) first observed in tumor cell lines resistant to anticancer drugs, the present data confirm the significant role that amphiphilic copolymers can play in the ongoing development of drug delivery strategies to overcome multidrug resistance. These investigations illustrate a promising approach for the development of new medical and ecotoxicological tools that can deliver specific molecules within cells. INTRODUCTION Within the past four decades, there has been increased interest in the design of macromolecules for applications ranging from drug delivery (1–3) to biological imaging technologies (4). Polymer chemistry and engineering have had a direct impact on enhancing drug loading of therapeutic agents as well as creating stable biosensors for novel therapeutic (5) and diag- nostic devices (6). With regard to polymeric microspheres, there are two broad categories: reservoir devices and polymer conjugates. The former involves carriers where a drug or a dye is physically incorporated into the polymeric matrix, whereas in the latter system, the drug is covalently linked to the polymer. Polymers under investigations include proteins, nucleic acids, and polysaccharides as well as synthetic polymers. Among the various options, the synthetic polymers, including polyanhy- drides, polyesters, polyacrylic acids, polymethacrylates, and polyurethanes provide important avenues for research, primarily because of their ease of processing and the ability to control their chemical and physical properties. Synthetic polymers are also advantageous due to high physical stability, possibility of sustained drug release, and potential for functionalization. In the polymer conjugates area, the bioactive molecule and/ or the dye is covalently bound and transported to the tissues and cells. These macromolecular carriers should ideally be water-soluble, biocompatible, nontoxic, and nonimmunogenic (7), as well as degraded and/or eliminated from the organism (8). The biological rationale for the use of water-soluble copolymers as drug carriers is based on their unique biological behaviors in terms of pharmacokinetics properties and cellular uptake. It is well-known that high-molecular-weight polymers can accumulate in solid tumors due to the differences in the biochemical and physiological characteristics of healthy and malignant tissues (9). This feature termed “enhanced perme- ability and retention” (10-12) (EPR effect) was described by Maeda and his co-workers (13). The cellular uptake mechanism of a molecule is also known to depend on its molecular weight. Whereas most low-molecular-weight molecules penetrate into the cell Via simple diffusion thought the cell membrane, macromolecules are taken up by the cell through endocytosis (14). During this step, a significant drop in the pH value takes place from the physiological values (7.2–7.4) in the extracellular space to pH 6.5–5.0 in the endosomes and finally to about pH 4.0 in primary and secondary lysosomes. Recently, Leroux and co-workers reported that synthetic polyanions derived from polycarboxylates can take advantage of this drop in pH to overcome the endosomal/lysosomal barrier and deliver macro- molecular drugs (15–17). Moreover, it was reported that increasing the copolymer hydrophobicity by introducing hy- drophobic monomers induced a destabilization of cell mem- branes facilitating the endosomal escape (18). With the aim to further increase the hydrophobicity of polymethacrylate polyanions, an amphiphilic polymethacrylate copolymer has been synthesized. In this study, we have selected (i) the cholesterol as a highly hydrophobic species and (ii) rhodamine as a fluorescent marker as well as a substrate of the multixenobiotic resistance (MXR) pump transporter, which * Corresponding author. Philippe.Barthelemy@bordeaux.inserm.fr. ‡ UMR-CNRS 6116. † INSERM U869. 3 Université Victor Segalen Bordeaux 2. § Boston University. Bioconjugate Chem. 2008, 19, 891–898 891 10.1021/bc700425x CCC: $40.75  2008 American Chemical Society Published on Web 03/15/2008