In Vivo Studies in a Freshwater Invertebrate, a Mesostominae Flatworm

Bioconjugate Chem. 2008, 19, 891–898

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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 1H 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 diagnostic 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 polyanhydrides, 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 * Corresponding author. [email protected]. ‡ UMR-CNRS 6116. † INSERM U869. 3 Université Victor Segalen Bordeaux 2. § Boston University.

(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 permeability 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 macromolecular drugs (15–17). Moreover, it was reported that increasing the copolymer hydrophobicity by introducing hydrophobic monomers induced a destabilization of cell membranes 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

10.1021/bc700425x CCC: $40.75  2008 American Chemical Society Published on Web 03/15/2008

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Figure 1. Photomicrography of a Mesostominae flatworm (Rhabdocoela, Thyphloplanidae). P: pharynx. Note the transparency of the organism.

removes xenobiotic molecules or drugs from the cell. These two functional monomers were prepared containing either cholesterol or rhodamine and copolymerized with methacrylate in the presence of AIBN to give the corresponding amphiphilic labeled copolymer. Based on the chemical features of this macromolecule (i) drug/dye polymer conjugate, (ii) amphipatic structure, (iii) pH-sensitive groups (carboxylates), we hypothesize that this carrier could play a significant role for drug delivery applications and/or imaging cells. A goal of this work is to investigate this new macromolecular carrier for in ViVo delivery to a simple freshwater invertebrate: a Mesostominae flatworm (Platyhelminthes, Rhabdocoela, Thyphloplanidae) (Figure 1). Flatworms are acoelomata organisms, known as simplest triploblastic animals. They possess a simple body plan organization lacking respiratory and circulatory organs. The pharynx and epidermis are in contact with surrounding aquatic media. These flatworms also carry eggs within their body. The aquatic biota of these animals is convenient for copolymer incubation. Indeed, these organisms tolerate laboratory conditions of temperature, pH, and conductivity. Furthermore, their small size (between 2 mm to 3.5 mm long) and transparent soft body, devoid of cuticle, allows in toto epifluorescence microscopy observations. Aquatic invertebrates are also often used as environmental indicators for environmental risk assessment in the presence of xenobiotics such as pollutants (19). In general, the term xenobiotic is given to all chemicals, including drugs or pollutants that are foreign to the organism under investigation. To study the in ViVo uptake of the amphiphilic copolymer as well as its role in the delivery of a xenobiotic, we used the fluorescent marker, rhodamine B, covalently attached to the copolymer. Rhodamine B is a multixenobiotic resistance (MXR) pump transporter substrate; thus, animals were also incubated in a rhodamine B solution, with or without a MXR transporter inhibitor (verapamil). The MXR defense system, reported in many aquatic invertebrates, is thought to work in a similar manner to the multidrug resistance (MDR) P-glycoprotein (Pgp) observed in cancer cells (20, 21). It has been shown that MXR actively transports a variety of xenobiotics across the cell membrane (22). In this paper, we describe a new amphiphilic copolymer labeled with rhodamine B, and report the results from in ViVo studies in a freshwater invertebrate. The in ViVo uptake of free rhodamine B and rhodamine B covalently attached to the amphiphilic copolymer carrier either in the presence or in the absence of the MXR inhibitor verapamil is discussed.

MATERIALS AND METHODS Materials. General Experiments and Analytical Conditions. Unless noted otherwise, all starting materials were obtained from Aldrich, and were used without further purification; the solvents

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were redistilled on calcium chloride, calcium hydride, potassium hydroxide, or sodium according to the solvent used. All compounds were characterized using standard analytical and spectroscopic techniques such as 1H spectroscopy (apparatus Bruker Avance DPX-300, 1H at 300.13 MHz) and mass spectrometry (instrument JEOL SX 102, NBA matrix). The NMR chemical shifts are reported in ppm relative to tetramethylsilane using the deuterium signal of the solvent (CDCl3, DMSO-d6 or D2O) as a heteronuclear reference for 1H. The 1H NMR coupling constants, Js, are reported in Hz. TEM microscopy experiments were performed on a Hitachi H 7650 (negative staining with ammonium molybdate 1% in water, Ni carboncoated grids). Fluorescence microscopy experiments were realized on a Zeiss axiovert 200. UV spectroscopy measurements were carried out on a Lambda 25 (Perkin-Elmer, France) with Peltier temperature programmer PTP-6. Silica gel 60 (particle size 40–60 µm) was used for flash chromatography. Thin-layer chromatograms were performed with aluminum plates coated with silica gel 60 F254 (Merck). Varian silica gel reverse-phase C18 MEGA BE-C18, 2GM 12 mL, and Sephadex LH-20 (25–100 µm) were used for quantitative chromatography. Synthesis. N-(6-Cholesterylcarbonylaminohexyl)-2-methylacrylamide (Compound 1). Triethylamine (0.6 mL, 4.03 mM, 2.1 equiv) was added under nitrogen dropwise to N-Bochexylammonium chloride (0.500 g, 1.94 mM, 1 equiv) in 50 mL of methylene chloride at 0 °C. Then, cholesterolchloroformiate (0.961 g, 2.14 mM, 1.1 equiv), dissolved in 20 mL of dry methylene chloride, was added to the reaction at 0 °C. The reaction mixture was stirred at 0 °C for 1 and 2 h at room temperature. The resulting mixture was washed 3 times with 20 mL of water and then dried over sodium sulfate. After removing the solvent, the crude material was dissolved in 20 mL of a 50/50 trifluoroacetic acid/dichloromethane mixture and stirred for 2 h. The solvent is removed under high vacuum. The trifloroacetate salt was suspended in DCM, and 0.5 mL of TEA was added to the reaction flask. Then, 0.1 mL of methacryloyl chloride was added to the solution at 0 °C. This solution was stirred for 1 h at 0 °C and 2 h at room temperature. Purification on a silica gel column (MeOH/DCM, 3/97) yielded 0.240 g of 1. (Yield: 21%). 1H NMR (CDCl3) δ in ppm: 0.65–2.26 (m, skeleton of cholesterol, CH2 decyl amine, CH3 methacrylate), 3.2 (m, 4H, 2CH2NH), 4.4 (m, 1H, HCO cholesterol), 4.6 (m, 1H, NH), 5.2 (s, 1H, CH methacryl), 5.3 (m, 1H, CH cholesterol), 5.6 (s, 1H, CH methacryl), 5.9 (m, 1H, NH). High resolution MS: [M + H]+, theoritical m/z ) 597.4917, observed m/z ) 597.5007. Anal. Calcd. (M + 1/2 H2O) C, 75.32%; H, 10.81%; N, 4.62%. Found C, 74.99%; H, 10.19%; N, 4.83%. Copolymerization (Compound 2). Copolymer 2. Compound 1 (0.02 g, 0.033 mM, 1 equiv), methacrylic acid (0.100 g, 1.15 mM, 35 equiv), methacryloxyethyl thiocarbamoyl rhodamine B (N-[9-(2-carboxy-x-methacryloxyethylthiocarbamoylphenyl)6-diethylamino-3H-xanthen-3-ylidene]-N-ethylethanaminium chloride; PolyFluor 570) (0.005 g) and AIBN (0.005 g, 0.03 mM) were dissolved in 40 mL of dry methanol. The reaction mixture was heated under nitrogen for 24 h. Methanol was evaporated, and the resulting material was dissolved in a minimum amount of MeOH/DCM 50/50 and purified on Sephadex LH20. 43 mg of copolymer 2 was isolated. (conversion 81%). 1H NMR (CD3OD) δ in ppm (broad peaks): 1H NMR (CDCl3) δ in ppm: 0.65–2.26 (m, skeleton of cholesterol, CH2 hexyl amine, CH2 and CH3 methacrylate, CH2 and CH3 of the rhodamine), 3.2 (m, CH2NH), 4.1 (m, HCO cholesterol), 5.3 (m, CdCH cholesterol). Content % of each monomer was determined by measuring the integration between 0.65 and 2.26 ppm (cholesterol, CH2 of hexyl amine, CH2 and CH3 of the rhodamine and polymer backbone), integration at 5.43 ppm (cholesterol CdCH)

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Table 1. Experimental Design for in ViWo Uptake of the Rhodamine B (RhB) or of the Rhodamine B-Labeled Amphiphilic Copolymer (RhB-amphiphilic copolymer) solutions of incubation mineral water (control) RhB (5 µg.mL-1) Rh B (5 µg.mL-1) + Verapamil (10 µM) RhB-amphiphilic copolymer (5 µg.mL-1) RhB-amphiphilic copolymer (5 µg.mL-1) + Verapamil (10 µM)

incubation period at 13 °C

incubation period at 4 °C

10 h 1 h 30, 4 h, 10 h 1 h 30, 4 h, 10 h 1 h 30, 4 h, 10 h

10 h

1 h 30, 4 h, 10 h

10 h

and at 7.83 ppm (corresponding to one aromatic rhodamine H), x ) 4%, y ) 95.6%, z ) 0.4%. Transmission Electronic Microscopy (TEM). The copolymer was dispersed in either methanol or distilled water for TEM experiments. Typically, 1 mg of copolymer was dispersed in 1 mL of methanol. This solution was left for 3 h at RT prior to examination. Fluorescence Microscopy. The samples were prepared by dispersing 5 mg of copolymer in 1 L of water. The pH of the solutions was adjusted by addition of either HCl (1 M) or NaOH (1 M). Prior to examination, these solutions were heated at 70 °C for 1 min and sonicated at RT for 5 min and left for 1 h at RT. UV–Vis Spectroscopy. UV–vis spectroscopy experiments were carried out on a Lambda 25 (Perkin-Elmer, France). The absorbance of the control samples containing free rhodamine was measured at 554 nm on aqueous solutions of free rhodamine at different concentrations (5 × 10-4 to 5 × 10-3 mg/mL. The UV–vis spectrum of the polymer was recorded on aqueous suspensions containing 0.1 mg/mL of polymer in water. Material and Methods for in ViWo Studies. Collection and ConserVation of Biological Material. Flatworms were collected in a temporary pound in the south of France with a hand net (125 µm mesh). Limnological parameters were the following: water temperature 15.3 °C; conductivity (C20) 252 µS/cm; pH 7.02; dissolved oxygen 3 mg/L and 31% of saturation. Animals were brought back alive to the laboratory and maintained at 13 °C in a commercial mineral water (C20 ) 200 µS/cm, pH 7.11). Water was refreshed daily and animals were fed every day with 1 mL of Teramin (Tetra Werke, Melle, Germany) dissolved in the commercial water (4 g/L). Protocol for in ViVo Uptake of the Amphiphilic Copolymer. Solutions were prepared with the same mineral water used for the laboratory maintenance of flatworms. Flatworms were incubated in the dark either with rhodamine B alone (5 µg/mL) or with the rhodamine B-labeled amphiphilic copolymer (5 µg/ mL). These experiments have been performed in the absence or in the presence of 10 µM of verapamil, a MXR transporter inhibitor. The temperature was 4 or 13 °C and pulse period 1 h 30, 4 h, or 10 h, followed by a 1 h chase period at room temperature to allow the expulsion of nonabsorbed material from the digestive tract. For each experimental condition, 5 flatworms were used. The experimental design is summarized in Table 1. Animals were then fixed in phosphate buffer containing paraformaldehyde 1% (PB-PFA 1%). After rinsing in phosphate buffer, animals were mounted in antifading medium (Gel/Mount, BiØmeda corp., Foster city, CA, USA) and observed under an epifluorescence microscope (Leica M1560). EVidence of MXR Membrane Proteins in Flatworms. The presence and the density of MXR membrane proteins in flatworms were evaluated using a ELISA assay. A monoclonal primary antibody (mAb1) raised against the human Pgp (C219 clone) was produced in mouse (Interchim, Montluçon, France). The C219 probe recognizes a sequence expressed by all MXR

genes whose sequence is known, from bacteria to man (23). Fixed flatworms in PB-PFA 1% were weighted and then homogenized in Tris-HCl 0.05 M, pH 7.4, and centrifuged 20 min at 4 °C (14 000 g). The supernatant was discarded and membrane pellets were resuspended in PB (0.1 M, pH 7.2). The microplate wells were coated with 200 µL of this solution overnight at 4 °C (8 mg wet weight/well). Overnight coated wells were washed five times in 0.01% Triton X100-PB, then saturation was performed 1 h at 25 °C with BSA 0.1% diluted in 0.01% Triton X100-PB. Wells were incubated (1 h at 25 °C) with mAb1 (1:10) and rinsed three times in 0.01% Triton X100PB. Then, the wells were incubated in alkaline phosphataseconjugated goat antimouse IgG (Ab2, 1:1000) (Promega, Madison, WI, USA) in 0.01% Triton X100, 0.1% BSA in PB (1 h, at 25 °C). After five washes in 0.01% Triton X100-PB, the enzymatic reaction is initiated with the addition of 100 µL of p-nitrophenyl phosphate (p-NPP) dissolved in diethylethanolamine (Sigma, St Quentin-Fallavier, France). The optical density (OD405), that reflects the membrane density in MXRrelated proteins, was measured in triplicate at 10, 20, 30, and 40 min after the initiation with a microplate reader (ELX800 with KC4 software, Biotek Instrument) at 23 °C. The negative controls were done by incubating the mAb1 and all the components except the membrane homogenates. The other controls consisted of (i) incubating all the components without the primary antibody for background studies, (ii) incubating Ab2 alone, and, finally, (iii) incubating Ab2 with 0.01% Triton X100 and 0.1% BSA in PB.

RESULTS AND DISCUSSION The following three constituting monomeric building blocks were selected to prepare the amphiphilic copolymer: methacrylic acid (water-soluble part), methacryloyl cholesterol (hydrophobe), and methacrloyl rhodamine (fluorescent probe). Cholesterol was used as a hydrophobic moiety based on its known aggregation properties and its role in the lipid raft-mediated endocytosis (24). This natural lipid is also used in many cationic delivery systems including lipoplexes to both improve DNA delivery and increase the stability of formulations in the presence of serum (25–27). Recently, rhodamine was used as a probe to follow the release of a bioactive molecule entrapped in polymeric structures (28). This dye is also a known substrate of the multixenobiotic resistance (MXR) pump transporter, which removes xenobiotic molecules or drugs from the cell. Consequently, to easily follow the cellular uptake of our amphipatic copolymer structure, rhodamine was selected as a water-soluble model of a xenobiotic. Synthesis. The amphiphilic copolymer was prepared as shown in Scheme 1. After synthesis of the cholesterol benzyloxycarbonyle (Boc) diamino derivative in basic conditions, this intermediate was treated with TFA in methylene chloride to afford the deprotected amine. A coupling reaction with methacryloyl chloride at 0 °C for 1 h and then at room temperature for 3 h yielded the expected methacryloyl cholesterol monomer 1. The copolymerization reaction was followed by thin-layer chromatography (TLC) on silica gel (methanol/DCM 5/95). The free radical copolymerization (29) of the three methacryloyl monomers is facile. Specifically, the reaction mixture was heated under nitrogen for 24 h in the presence of AIBN and the resulting crude polymer purified on exclusion gel chromatography. Importantly, the synthetic strategy developed allows easy access in four steps to the cholesterol rhodamine labeled copolymer. The percentage of each monomer unit in the polymer (x ) 4.0%, y ) 95.6%, z ) 0.4%) was determined by measuring the integrations of CH2 backbone and CH2NH via 1H NMR. Physicochemical Studies. The UV–vis spectra of the copolymer labeled with rhodamine (Figure 2, blue) and the free rhodamine (Figure 2, red) in the region 500–600 nm are shown

894 Bioconjugate Chem., Vol. 19, No. 4, 2008 Scheme 1a

a (a) (1) TEA in DCM, 1 h 0 °C, then 2 h at RT. (2) TFA/DCM 50/50, 2 h at RT. (3) Methacryloyl chloride, TEA in DCM, 1 h 0 °C then 3 h at RT (yield: 21%). (b) AIBN, MeOH, reflux, 72 h, conversion: 81%.

Figure 2. UV–vis spectra (absorbance vs wavelength in nm) of the copolymer labeled with rhodamine (blue) and the free rhodamine (red).

in Figure 2. The red shift observed for the labeled copolymer (λmax ) 562 nm) compared to free rhodamine in water (λmax ) 554 nm) can be attributed to the rhodamine being located in a more hydrophobic microenvironment of the copolymer structure and a resulting decrease in local polarity (30). The amount of rhodamine grafted to the polymeric structure was also determined by UV–vis spectroscopy. The absorbance measured at λmax ) 562 nm for a solution of the labeled copolymer at a concentration of 0.1 mg/mL corresponds to a concentration of rhodamine equivalent to 2 × 10-3 mg/mL (weight fraction of rhodamine ) 2%). Note that a weight fraction of 2.% in rhodamine gives a mole fraction of 0.35%, similar to the value obtained from 1H NMR analysis (z ) 0.4%). As expected, the amphiphilic character of copolymer 2 tends to promote the formation of aggregates in both organic solutions and aqueous media. Amphiphilic copolymer (typical concentrations: 1 mg/mL) dissolved in methanol exhibits three populations of aggregates. The smallest particles are a few hundred nanometers in size, whereas the two other populations show

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hydrodynamic radii of a few micrometers and a few tens of micrometers, respectively. Note that the formation of aggregates in both organic and aqueous solutions (water, methanol, DMF) prohibits the study of the isolated macromolecules. Consequently, despite numerous trials, including size exclusion chromatography (SEC) and dynamic light scattering (DLS) experiments, it was impossible to determine the average molecular weight of the copolymers, since the polymer exhibited an aggregated state even at low concentrations. Microscopy Studies of the Aggregates. The size and morphology of the copolymer aggregates in solution were examined by fluorescence and electronic microscopies (TEM). As shown in Figure 3, the amphiphilic copolymer self-assembles in water to give large spherical aggregates with diameters of approximately 5 µm. These aggregates can be observed by using both light (Figure 3a) and fluorescence microscopies. The formation of aggregates was also confirmed by TEM (Figure 3b,c). At higher magnifications, TEM images (Figure 3c) show the presence of hollow microspheres similar to the ones reported by Caruso et al. (31). The folds and creases seen in the polymer microspheres are a result of evaporation of the solvent by airdrying. The size distribution of the objects obtained from TEM images reveals a relatively homogeneous population of microspheres with diameters ranging from 3 to 11 µm (Figure 4 blue). Note that the population of the microspheres observed by TEM (Figure 4 blue) is similar to the population of the same microspheres observed by in ViVo by fluorescence microscopy (see the in ViVo Studies section) suggesting that the microspheres remain intact even after cellular uptake. In ViWo Studies. Uptake of Rhodamine B. The accumulation and efflux of model P-glycoprotein (Pgp) substrates such as fluorescent dyes (rhodamine B, rhodamine 123, etc.) is a method routinely used to follow the activity of the multixenobiotic resistance (MXR) defense mechanism in aquatic invertebrates (32), especially in the biomarker research field. Figure 5 presents the in ViVo uptake of free rhodamine B by flatworms after 10 h of incubation at 13 °C either in the absence (Figure 5a) or in the presence (Figure 5b) of a MXR inhibitor, verapamil. The higher fluorescence intensity observed in the presence of verapamil indicates that, without verapamil, the molecules of rhodamine B inside the cells are actively expulsed in ViVo by the MXR membrane efflux pump proteins. To verify the presence of MXR transporters in these flatworms, an ELISA assay has been developed and performed. MXR transporters have been detected by the C219 antibody in membrane homogenates of flatworms. The optical density at 405 nm (OD405) that reflects the membrane density in MXRrelated proteins reaches 3.890 ( 0.005 for 8 mg wet weight of flatworms (Figure 6). These results showing the absence of rhodamine B in flatworms in the absence of verapamil and presence of rhodamine B in flatworms in the presence of verapamil and the semiquantification of MXR-transporter membrane density demonstrate the presence of the MXR system in these Mesostominae flatworms. MXR genes have been highly conserved through the phylogenesis as demonstrated by their presence in microorganisms (33), plants (34), aquatic invertebrates (35), and vertebrates (36). In fact, the MXR system is similar to the multidrug resistance (MDR) first observed in tumor cell lines resistant to anticancer drugs (20, 37–38). One important mechanism of MDR involves the multidrug transporter, P-glycoprotein, which confers upon cells the ability to resist lethal doses of certain cytotoxic drugs by pumping them out of the cells, and thus reduce their cytotoxicity (39–41). Uptake of Rhodamine B-Labeled Amphiphilic Copolymers. In light of the physicochemical properties of the copolymer and

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Figure 3. (a) Fluorescence microscopy image of copolymer aggregates in aqueous media at pH ) 7.3 (concentrations ) 5 mg/L, bar represents 10 µm). The insets left and right are light and fluorescence microscopy images of the same sample, respectively (bars represent 5 µm). (b,c) TEM images visualizing the polymer microspheres at different magnification (bars represent 10 and 2 µm for (b) and (c), respectively).

Figure 4. Size distributions of the microspheres (number of objects in percent vs size) obtained from (i) TEM images (blue) and (ii) fluorescence microscopy images (red) collected from the in ViVo studies (copolymers in flatworm egg cells after 4 h incubation at 13 °C. See in ViVo Studies section for details).

Figure 5. Uptake of rhodamine B by Mesostominae flatworms in the absence (a, -Ver) or in the presence (b, +Ver) of the MXR inhibitor verapamil after 10 h of incubation at 13 °C. Note the higher fluorescence intensity in (b) compared to (a). E: resting egg. P: pharynx. Bars ) 200 µm.

the in ViVo data collected with free rhodamine, we hypothesize that the amphiphilic macromolecule, which bears rhodamine B as a water-soluble model of xenobiotic, can be taken up by flatworms. In principle, the copolymer can be used as a carrier to improve the delivery of xenobiotics. In this context, two key issues must be addressed: (i) How is the copolymer taken up by the flatworms? (ii) Is the macromolecular carrier translocated out of the cells by the MXR system? Endocytosis is a cellular uptake process that is coupled with temperature-dependent metabolic activities (42). To determine whether the rhodamine B-labeled amphiphilic copolymer is incorporated into the cells by endocytosis, the organisms were observed at two different temperatures, 13 and 4 °C, under an epifluorescence microscope. The lowest temperature corresponds to winter cold temperature tolerated by these organisms and the higher to spring water temperature of the temporary pond

Figure 6. Optical density measured at 405 nm reflecting the density of MXR transporters in membrane homogenates of Mesostominae flatworms read at 40 min after the initiation.

where they live. A control sample showing flatworms by fluorescent microscopy is presented in Figure 7a. For each experimental condition, rhodamine B-labeled amphiphilic copolymers are observed in flatworm cells by fluorescence microscopy. For a given incubation condition (same time and same temperature), no difference was noticed between samples treated with or without verapamil. At 13 °C, flatworms were exposed to rhodamine B-labeled amphiphilic copolymers for 1 h 30, 4 h, or 10 h. After 1 h 30 of exposure time, the labeled copolymer is observed in a few organelles, named rhabdites, which are located in some flatworm epidermal cells (Figure 7b). The rhabdite initially appears as a large Golgi saccule enclosed by an extensive system of microtubules. Golgi vesicles and vacuoles fuse with the limiting membrane of the rhabdite and contribute to enlarge this structure. Microtubules mediate movement of vesicles containing secretory material along the rhabdite length. Rhabdites are known to be acidophilic and contain predominantly proteins, which may be combined with a purine component and mucopolysaccharides (43, 44). Rhabdite cells are parenchyma-derived cells that migrate into the epidermis. Once in the epidermis, the rhabdite cells become part of the epithelium. It has been proposed that most if not all of the epidermal cells originate from rhabdite cells or from other parenchyma-derived cells (43). After 4 h of incubation, the labeled copolymer is observed in several different cell types of the worm. For example, Figure 7c,d shows fluorescent objects within egg cells. The size distribution of the objects obtained from fluorescence microscopy images (Figure 7c,d) reveals a population of microspheres with a mean diameter of 4.25 ( 1.51 µm. Note that size distribution of the fluorescent objects observed in ViVo (Figure 4, red graph) corresponds to the population of the microspheres obtained spontaneously with copolymer 2 (Figure 4, blue graph). Homogeneous dots in the

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Figure 7. Fluorescence microscopy images showing the in ViVo uptake of rhodamine B-labeled amphiphilic copolymers (5 µg/mL) by Mesostominae flatworms at 13 °C. (a) Control. (b) Arrowheads indicate a few labeled rhabdite. (c) Arrowheads indicate the uptake of the labeled copolymers into some cells in a resting egg; * indicates cells without labeling. (d) Detail of (c) showing labeled structures (mean diameter ( SEM ) 4.25 ( 1.51 µm). (e) General view of the pharynx. Arrows indicate labeling into glandular cells. (f) Detail of a labeled pharyngeal glandular unit. (g-j) Uptake of the copolymers into rhabdite cells; note the increase of the uptake between 4 h (g) and 10 h (h); (i) longitudinal view of labeled rhabdites; (j) transverse view of labeled rhabdites. Bars ) 50 µm.

Figure 8. In ViVo uptake of rhodamine B-labeled amphiphilic copolymers (5 µg.mL-1) by Mesostominae flatworms at 4 °C. (a-c) Localization of rhodamine B-labeled amphiphilic copolymers in rhabdite cells: (a) Fluorescence view showing the localization of the labeled copolymer. (b) Autofluorescence view of (a) showing the localization of unlabeled rhabdites. (c) Merged image of (a) and (b). Note that the copolymers are located nearby the rhabdites. (d) View of a small cluster of labeled rhabdite. Note the scarcity of labeled rhabdites at 4 °C compared with their abundance at 13 °C (cf. Figure 7h). Bars ) 50 µm.

pharyngeal glandular cells (Figure 7e,f) and numerous sticklike structures, which are typical mature rhabdites (Figure 7g), are also observed in the fluorescence microscopy images. After 10 h of exposure, numerous rhabdites are labeled (Figure 7h-j). In the experiments performed at 4 °C for 10 h, labeled copolymers are observed near unlabeled rhabdites (Figure 8a-c). Some labeled rhabdites are also observed (Figure 8d).

As the mean length of the rhabdites is 19.98 ( 3.52 and their mean diameter is 3.00 ( 0.80 µm, rhabdites are large enough to accumulate the amphiphilic copolymers. Furthermore, incorporation of the rhodamine B-labeled amphiphilic copolymers is time- and temperature-dependent. At 13 °C, the number of labeled rhabdites per surface unit is 1.83-fold higher at 10 h than at 4 h of incubation. For 10 h of

Amphiphilic Copolymer for Delivery of Xenobiotics

incubation, the number of labeled rhabdites per surface unit is 7.25-fold higher at 13 °C than at 4 °C. When flatworms were exposed to rhodamine B-labeled amphiphilic copolymers 2 for 10 h at 4 °C, the number of labeled rhabdites per surface unit represents only 13% of the maximum level measured after 10 h at 13 °C. As has been recently reported by Suenaga et al. for a copolymer carrier featuring hydrophobic lauryloyl groups (45), cold temperature induces a remarkable decrease in the copolymer-rhodamine uptake, indicating that the polymer is internalized Via an endocytosis mechanism. After a 10 h incubation at 4 °C, labeled copolymers observed near unlabeled rhabdites are probably endocytotic vesicles that are about to fuse with rhabdites (Figure 8a-c). The following observations can be made from the above data: (i) Rhodamine B-labeled amphiphilic copolymers are in ViVo uptaken Via an endocytosis mechanism in the early phases of incubation in the invertebrate, and its localization is then observed in different cell types (pharyngeal glandular cells, resting egg cells, epidermis cells). (ii) After incorporation into epidermis cells, rhodamine B-labeled amphiphilic copolymers are concentrated in storage reserve structures, the rhabdites. This storage is observable after 4 h of incubation at 13 °C. The endocytotic pathways (receptors and membrane microdomains) responsible for the uptake of the amphiphilic copolymers need to be investigated further. In ViWo Study Perspectives. In an ecotoxicological study, Horvat et al. observed histological changes including a decrease in the number of rhabdites in the Platyhelminthe Polycelis felina (Daly.) after exposure to norflurazon herbicide (46). The in ViVo uptake experiments described above show that the rhodamine B-labeled amphiphilic copolymer is taken up by the flatworm, enters the cells, and is accumulated in rhabdites. This pattern of amphiphilic copolymer uptake could be used as an ecotoxicological tool to measure cellular activity when animals are exposed to a xenobiotic(s). Moreover, the amphiphilic copolymer could be used as an in ViVo carrier of specific molecules that could react with pollutants accumulated in cells and subsequently produce a detectable signal. In the present study, we demonstrate that the bioaccumulation of the MXR substrate rhodamine B was dramatically increased when the rhodamine B was covalently attached to the amphiphilic copolymer and a resulting microsphere was formed. The translocation out of the cell by the MXR system is avoided in comparison to the free rhodamine. As the MXR system is similar to the MDR one, the in ViVo uptake results strongly suggest that our amphiphilic copolymers can play a significant role in the field of carriers for drug delivery. This finding represents a potential therapeutic option that should be considered during the ongoing development of strategies to overcome multidrug resistance in cancer studies, as it was suggested for the treatment of human ovarian and endometrial cancers that express receptors for luteinizing hormone-releasing hormone with agents such as AN-152 bypassing their Pgp extrusion (47).

CONCLUSION In this study, we have synthesized a new rhodamine-labeled amphiphilic copolymer featuring cholesterol moieties for in ViVo delivery of xenobiotics and/or drugs. The amphiphilic copolymer structure was prepared from cholesterol-derived methacryloyl monomer, methacrylic acid, and methacryloxyethyl thiocarbamoyl rhodamine B. Fluorescence and TEM microscopies studies indicate that this amphiphilic copolymer aggregates to give microspheres. The in ViVo experiments carried out on a simple triploblastic aquatic organism, a Mesostominae flatworm, clearly demonstrate that the copolymer/microsphere is bioaccumulated without being translocated out of the cell by the

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multixenobiotic resistance (MXR) transporters. Synthetic polyanions such as poly(methacrylic acid) are finding utility in enhancing the delivery of nucleic acids to cells, for example, as recently reported by Leroux and co-workers (15). Consequently, these and similar polyanionic polymers and copolymers are likely to be of further interest for the in Vitro and in ViVo delivery of oligonucleotides, small molecule drugs, and imaging agents. These investigations further support the synthesis and evaluation of new polyanion-based drug delivery vehicles and ecotoxicological tools.

ACKNOWLEDGMENT The authors would like to thank Dr. Christophe Schatz and Pr. Sébastien Lecommandoux from the LCPO - ENSCPB of Bordeaux for kindly helping us with DLS and SEC experiments.

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