Nanoparticles Produced by Ring-Opening Metathesis Polymerization

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Nanoparticles Produced by Ring-Opening Metathesis Polymerization Using Norbornenyl-poly(ethylene oxide) as a Ligand-Free Generic Platform for Highly Selective In Vivo Tumor Targeting Fabien Gueugnon,† Iza Denis,† Daniel Pouliquen,† Floraine Collette,‡ Régis Delatouche,§ Valérie Héroguez,‡,∥ Marc Grégoire,† Philippe Bertrand,*,§,∥ and Christophe Blanquart*,†,∥ †

Inserm, U892, CNRS, UMR 6299, and University of Nantes, 8 Quai Moncousu, 44007 Nantes cedex 1, France Laboratoire de Chimie des Polymères Organiques, CNRS, UMR 5629, Bordeaux, 16 Avenue Pey-Berland, F-33607 Pessac, France § Institut de Chimie des Milieux et Matériaux de Poitiers, CNRS, UMR 7582, Poitiers, 4 rue Jacques Fort, B27, F-86022 Poitiers cedex, France ‡

ABSTRACT: We described a norbornenyl-poly(ethylene oxide) nanoparticles ligand-free generic platform, made fluorescent with straightforward preparation by ring-opening metathesis polymerization (ROMP). Our method allowed to easily obtain a drug delivery system (DDS) with facilitated functionalization by means of azide−alkyne click chemistry and with a high selectivity for the tumor in vivo, while cellular internalization is obtained without cell targeting strategy. We demonstrated that our nanoparticles are internalized by endocytosis and colocalized with acidic intracellular compartments in two models of aggressive tumoral cell lines with low prognostic and limited therapeutic treatments. Our nanoparticles could be of real interest to limit the toxicity and to increase the clinical benefit of drugs suffering rapid clearance and side effects and an alternative for cancers with poorly efficient therapeutic solutions by associating the drug delivery in the tumor tissue with an acid-sensitive release system.

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INTRODUCTION Malignant pleural mesothelioma (MPM) and lung adenocarcinoma (ADK) are two aggressive cancer types still requiring innovative therapeutic solutions. MPM in particular is a rare pathology due mostly to asbestos exposure, expected to increase in the coming years,1,2 and difficult to identify, as reliable selective biomarkers3 are missing. Thus, MPM is detected at later stage disease, leading to poor prognostic with median survival around one year. We recently showed by cell populations analysis that MPM and ADK cells can be differentiated and alternative biomarkers used for their identifications.3c From the clinical trials performed for MPM and ADK, approximately 210 and 500, respectively, only modest results were achieved with classical anticancer compounds, alone or in combinations. Liposomes- or albumin-based drug delivery systems (DDS) were investigated in MPM and ADK, and despite the also limited results reported, this strategy is highly investigated for MPM4 or ADK5 © 2013 American Chemical Society

with organic or inorganic carriers. The improved efficacy of anticancer compounds when loaded on DDS is due to the passive tumor targeting based on the enhanced permeability and retention (EPR) effect.6 DDS need long circulation time in plasma requiring stealth property usually obtained by polyethylene oxide (PEO) chains coating. This reduces uptake by the reticulo endothelial system7 and reduces immunogenic responses.8 Since the Ringsdorf’ proposal,9 several organic polymers were investigated as DDS in the past decades, like HPMA (N-(2-hydroxypropyl)methacrylamide),10 PGA (polyglutamic acid),11 or simple PEO.12 The polymerization of norbornene (NB) derivatives by “living” ring-opening metathesis polymerization (ROMP) mediated by Grubbs catalysts or others13 is currently investigated to prepare polymers for Received: April 12, 2013 Revised: May 31, 2013 Published: June 3, 2013 2396

dx.doi.org/10.1021/bm400516b | Biomacromolecules 2013, 14, 2396−2402

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Scheme 1. Synthesis of Fluorescent ROMP Polymer DDS 8a

Reagents and conditions: (i) DPMK, THF, ethyleneoxide, 20 °C, 48 h, then MeOH/HCl, 87%; (ii) (a) MsCl, THF, NEt3, 20 °C, 24 h, (b) NaN3 excess, DMF, 20 °C, 40 h, 74%; (iii) EDC, DMAP, CH2Cl2, HCC−CH2OH, 24 h, overnight, 20 °C; (iv) CuBr, PMDETA, CH2Cl2/EtOH 35/65 v/v; (v) 1 (1% mol), 7 (98% mol), and 4 (1% mol), Grubbs I, CH2Cl2/EtOH 35/65 v/v, 20 °C, 24 h. a

various applications.14 Several cycloalkenes can be used,14b,c but the NB system is more popular for its irreversible ring-opening leading to poly(vinylcyclopentane). ROMP polymers are emerging alternatives as DDS, with the classical multifunctional requirements for tumor targeting, cell recognition, cellular internalization, drug loading, and in vitro/in vivo imaging.15,16 The reported ROMP DDS preparations are more or less complex, with introduction of PEO chains or dyes made after polymerization with different chemistries, but convergent approaches are possible with the Huisgen [3 + 2] cycloaddition (Sharpless’ click concept17).16,18 We previously proposed alternative polynorbornene-based nanoparticles 8 (Scheme 1) prepared by ROMP with the PEO macromonomers 1 and 4 and NB 7.19 This work demonstrated that copolymers can be obtained from the generic macromonomer 1 as a practical strategy for multifunctional ROMP DDS. Monofunctional particles can also be better obtained by click chemistry with azidonanoparticles19a due to the core− shell morphology of the system and the azido groups being positioned at the outer surface and available for post functionalization.16 Our approach offers the advantage of flexibility in design and the ratio of macromonomers used for copolymerization can be adjusted to have particles with the required properties. We also observed the native internalization of NPs 8 in cancer cells,19b the possible mechanism being not established. As the core−shell morphology led to the polynorbornene part in the hydrophobic core and the pendant PEO chains in the hydrophilic shell (Scheme 1),20 we expected such DDS to have intrinsic stealth properties suitable for EPR

effect. In the present contribution we demonstrate that these particles enter cells by endocytosis-based mechanisms and are able to selectively target tumors in vivo with a high selectivity. Preparation of nanoparticles, internalization in MPM and ADK cells, and biodistribution in mice bearing MPM tumors xenografts are presented as a potential innovative strategy for MPM treatments.

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MATERIALS AND METHODS

2.1. Materials. Reagents and solvents were purchased and dried when necessary: NB (99% GC, Aldrich), sodium azide (ReagentPlus, 99.5%, Aldrich), dodecane (99%, Aldrich), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%, Aldrich), Grubbs first generation catalyst Cl2(PCy3)2Ru=CHPh (Aldrich, stored in a glovebox under Argon atmosphere), ethyl vinyl ether (99% stab. with ca. 0.1% N,N-diethylalanine, Alfa Aesar), Na2SO4 (99%, Alfa Aesar), methanesulfonyl chloride (99.5%, Acros Organics), triethylamine (TEA, 99%, Acros Organics), diethyl ether anhydrous (J.T. Baker), and dimethyl formamide (DMF, 99.8%, Panreac) were used as received. CuBr (98%, Aldrich) was purified in acetic acid before use. Ethanol (96%, purissimum grade pur, Xilab) and dichloromethane (purissimum grade pur, Xilab) were degassed before use. Tetrahydrofuran (THF, J.T. Baker) was cryodistilled from sodium benzophenone before use. 2.2. Methods. ROMPs were performed under argon atmosphere using an inert-atmosphere glovebox, at room temperature. 1H NMR studies were completed via a Bruker spectrometer 400 MHz, in CDCl3 at 25 °C. Size exclusion chromatography (SEC) equipment consists of a JASCO HPLC pump type 880-PU, TOSOHAAS TSK gel columns, a Varian refractive index detector, and a JASCO 875 UV−vis absorption detector, with THF as the mobile phase and the calibration curve was 2397


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Figure 1. DLS and TEM determination of the particle sizes. performed using polystyrene standards. The conversion of NB was determined by gas chromatography with dodecane as internal standard. The particle sizes were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). DLS measurements were performed using a MALVERN zetasizer Nano ZS equipped with He−Ne laser (4 mW and 633 nm). Before measurements, latexes were diluted about 800 times to minimize multiple scatterings caused by high concentration. The scattering angle used was 173°. TEM pictures were performed with a JEOL JEM-1230 microscope operating at an accelerating voltage of 80 kV. For the particles size, distribution, and morphology observation, samples diluted about 800 times were deposited on a 200 mesh carbon filmcoated copper grids surface (3 × 5 μL). Fluorescence measurements were done on a Spectramax M2e (molecular devices) UV−visible spectrophotometer. After the coupling reaction, particle dispersions were ultracentrifugated to remove the particles and the concentration of free Rhodamine B in the supernatant was measured by fluorescence spectroscopy. The measurements gave access to the fluorophore coupling efficiency at the particles surface. The calibration curve was established using Rhodamine B solutions of concentrations ranging from 4.10−8 to 3.10−7 M. 2.3. Particles. Compound 8 was synthesized using a previously described procedure19 by copolymerization of 7 (4 × 10−3 mol) with macromonomer 1 (4.08 × 10−5 mol) and macromonomer 4 (4.08 × 10−5 mol) using first-generation Grubbs catalyst as initiator. The polymer obtained exhibits an experimental composition of 332:3:3 (molar ratio in 7:1:4), taking into account the macromonomers conversion (96%, determined by SEC using dodecane as internal standard) and norbornene conversion (>99%, determined by gas chromatography using dodecane as internal standard). The amount of linked rhodamine per NP (nRh/NP) was calculated with the following equation:22

nRh/NP =

were then ultrafiltered during 36 h in order to remove traces of organic solvents and any free molecules, as rhodamine B. 2.4. Cell Culture. The lung adenocarcinoma cell line A549 was obtained from ATCC. The mesothelioma, Meso13, Meso34 and Meso56, and adenocarcinoma (ADK), ADK 72, and ADK 153, cell lines were established from the pleural fluids of mesothelioma or lung ADK patients, respectively (Gueugnon et al.). All cell lines were maintained in RPMI medium (Invitrogen) supplemented with 2 mM L-glutamine, 100 IU/mL penicillin, 0.1 mg/mL streptomycine, and 10% heat inactivated fetal calf serum (FCS; Eurobio).

Figure 2. Determination of NPs 8 toxicity on cells. Cells were seeded in 96-well plates at a density of 5 × 103 cells/well in culture medium. After 24 h, cells were incubated with increasing doses of NPs 8 for 72 h. Cell growth was evaluated using Uptiblue cell counting reagent. Results are the means ± SEM of experiments performed on three different MPM and lung ADK cell lines. 2.5. Determination of Cell Viability (Figure 2). Cell viability was monitored using Uptiblue (Interchim). MPM and ADK cells were seeded in 96-well plates at a density of 5 × 103 cells/well in culture medium. After 24 h, compounds were added for 72 h. Uptiblue reagent (5%, v/v) was then added to the culture medium for 2h at 37 °C. Fluorescence was measured at 595 nM after excitation at 532 nM using a Typhoon apparatus (GE Healthcare). Results are expressed as the mean percentage ± SEM of the untreated cells obtained from experiments performed on three MPM and on three lung ADK cell lines. 2.6. Endocytosis Measurement (Figure 4). Standard concentrations curve was obtained by measuring fluorescence of increasing amount of NPs 8 in black 96-well plates (Berthold Technologies). Fluorescence was measured with a fluoroscan (Thermofischer) at 590

FRh × n4 × π4 × VNP × ρNP m4 × π4 + m1 × π1 + m7

where FRh is the functionalization yield of macromonomer 4 (0.70), n4 is the amount of macromonomer 4 introduced for the NPs synthesis, πi is the conversion ratio of the macromonomer i (0.96), VNP is the volume of a NP (VNP = πDNP3/6; Figure 1), ρNP is the density of the NPs approximated to equal to 1 g/mL, and mi is the weight of the compound i introduced for the synthesis of the NPs. Before any biological test, the dispersion medium was changed: the particles were dispersed into water. Dichloromethane was removed by rotary evaporator, water was then added to particles dispersed in ethanol, and ethanol was removed by rotary evaporator. Dispersions 2398


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Figure 3. Concentration-dependent fluorescence intensities of NPs 8.

Figure 4. Kinetics of NPs 8 endocytosis by MPM and ADK cells; 1 × 103 MPM (Meso 13) or ADK (ADK 153) cells were incubated with 0.43 μg of NPs 8 for different periods of time. Left: expressed in quantity of particles per 1 × 103 cells. Right: expressed in relative percent of particles internalized per 1 × 103 cells. Fluorescence was used to measure the internalization using a fluorimeter as described in the Materials and Methods. Results are expressed as the means ± SEM of the results obtained on three different MPM or lung ADK cell lines; *p < 0.05 and **p < 0.01.

Figure 5. Visualization of endocytosis of NPs 8 in ADK and MPM cancer cells. (A) Columns n and n + 1 μm represent layer n and layer n + 1 μm, with lines 1, 2, and 3: treatment at 37 °C, 4 °C and with cytochalasin D, respectively. Arrows indicate NPs localization. (B) Line 1: particles localization, line 2: clathrin staining, line 3: merging of lines 1 and 2. (C) Line 1: particles localization, line 2: LAMP-1 staining of acidic vesicles, line 3: merging of lines 1 and 2 with yellow color indicating overlap of nanoparticles and acidic vesicles confirming the endocytosis pathway of NPs 8.

nm after excitation at 544 nm (Figure 3). Quantification of endocyted nanoparticles was performed using cells seeded at a density of 1 × 105 per well of 12-well plates and incubated for the indicated times with 0.43 μg/1 × 103 cells of NPs 8, then washed twice with PBS, and lysed in 300 μL of water for 15 min. Lysates were transferred in black 96well plates and fluorescence measured as for standard calibration. 2.7. Internalization Experiments (Figure 5A). ADCA 153 and Meso13 cells were seeded on glass coverslips at a density of 5 × 104 cells/well of 12-well plates in culture medium. After 24 h, three groups of cells were incubated: first group at 37 °C, second group on ice, and third group with cytochalasin D (Sigma-Aldrich; 5 μM for 2 h at 37 °C), respectively. To each group, NPs 8 were added (0.86 μg/1 × 103 cells) for 2 h and 30 min at 37 °C. Cell membranes were labeled using PKH67 (Sigma-Aldrich) according to manufacturer’s conditions. Then, cells were fixed and mounted on microscope slides using ProLong Gold (Molecular Probes) and visualized by fluorescence microscopy using a Zeiss Axiovert 200-M microscope (Zeiss, Le Pecq, France) and ApoTome module (X63 and numerial aperture 1.4). Pictures were acquired using AxioCam MR digital camera and analyzed using the AxioVision 4.6 software. 2.8. Colocalization Experiments (Figure 5B and 5C). Cells were seeded on glass coverslips at a density of 5 × 104 cells/well of 12well plates in culture medium. After 24 h, incubation with NPs 8 (0.43 μg/1 × 103 cells) was performed during 2 h and 30 min in culture conditions. Cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS (15 min at room temperature). Cells were permeabilized with 0.05% Triton X-100 (Merck) /0.05% Tween-20 (Sigma-Aldrich) in PBS (5 min) and incubated with anti-LAMP-1 or anticlathrin antibody at 1 μg/mL (Abcam) in PBS/1% BSA for 1 h. Cells were washed twice in PBS and incubated with the appropriate cyanin-5 or FluoresceinIsoThioCyanate conjugated secondary antibody (Jackson ImmunoResearch Laboratories) for 1 h. After an

additional PBS wash, cell nuclei were stained with 1 μg/mL Hoechst (Sigma-Aldrich; 5 min). Coverslips were mounted in ProLong Gold (Molecular Probes) and fluorescence was visualized by using the Axiovert 200 M microscopy system (Zeiss, Le Pecq, France) with ApoTome module (X63 and numerial aperture 1.4). 2.9. Biodistribution Experiments (Figure 6). The experiments were carried out in compliance with the guidelines of the European Union for the care and use of animals in research protocols. The 3 × 106 AK7 peritoneal mesothelioma cells were administered subcutaneously to seven groups of female nude mice (Elevage Janvier, Le Genest-St-Isle, France). One week after AK7 cells injection, mice from groups 2−6 were injected with 20 μg NPs 8 per gram of mice in the tail vein. Mice from group 1 did not receive any NPs. Mice from groups 1−6 were necropsied 1, 6, 24, 72 h, and 1 week after NPs injection. Before necropsied, mice from Group 4 (24 h) were anesthetized using isoflurane during whole mice imaging analysis. Tumor, liver, ovary, brain, spleen, kidneys, and blood were collected and analyzed for fluorescence emission. Fluorescence was observed at 630 nm using Photon Imager (Biospace Lab) after excitation at 580 nm and pictures were analyzed using PhotoVision+ software (Biospace Lab). Fluorescence was observed on a whole mouse at the indicated times and after necropsies on dissected tumor (Tu), liver (Li), ovary (Ov), brain (Br), spleen (Sp), kidneys (Ki), and blood (Bl) at the 2399


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Figure 6. Biodistribution of NPs 8 in a mouse model of subcutaneous mesothelioma. Nude mice bearing AK7 tumors were injected with 20 μg of NPs 8 per g of mice in the tail vein. (A) Detection of fluorescent NPs 8 over time after injection, observed on whole mice. (B) One week after injection, fluorescence was observed on dissected tumor (Tu), liver (Li), ovary (Ov), brain (Br), spleen (Sp), kidneys (Ki), and blood (Bl). indicated times postinjection. Statistical results were used to establish kinetics of biodistribution (Figure 7).

Regarding the usual 100−200 nm sized particles used for copolymer-based DDS, these particles appeared bigger than the norm. The fluorescent NPs 8 were evaluated for their toxicities, cell penetration, and cellular trafficking with MPM and ADK cancer cells. These two cell lines being from different origins were used in order to evaluate any cell-based-dependent variations in internalization capacities. We first determined the dosedependent cell viability, as shown by Figure 2, for MPM and ADK cell lines and averaged the results for a given concentration, as the viability appeared quite similar for these two cell lines. The inhibition concentration IC50 was 6.47 ± 0.10 μg NPs 8/1 × 103 cells. The fluorescence of NPs 819b was used to quantify the internalization rate, the fluorescence being found dose-dependent in the concentration range tested (Figure 3). Internalizations of NPs 8 (Figure 4) by MPM and ADK were detectable respectively after 120 and 15 min of incubation. A significant difference of internalization capacities between the two types of cell lines was observed. Based on fluorescence response, the quantity of NPs 8 internalized by quantity of cells after 120 min of incubation appeared to be for MPM 0.042 ± 0.028 ng NPs/1 × 103 cells and for ADK 0.629 ± 0.231 ng NPs/1 × 103 cells, showing 15-fold higher capacity of ADK cells to internalize our NPs. After 300 min, this ratio was reduced to 7-fold (MPM: 0.125 ± 0.016 ng NPs/1 × 103 cells and ADK: 0.881 ± 0.226 ng NPs/1 × 103 cells; Figure 4, left scale). Figure 4, right scale, reports efficacy of internalization relative to the amount of NPs 8 used for the experiments (0.43 μg/1 × 103 cells). At 120 min, this gave 0.01% and 0.14% for MPM and ADK, respectively, and, at 300 min, it was 0.03% and 0.20%, respectively. According to our calculations on rhodamine functionalization, it can be said that, at 300 min, in ADK cell lines, with 350000 molecules of rhodamine per NPs, 0.2% of NP internalization (0.881 ng (6.2 × 10−6 nmol)/1 × 103 cells) corresponds to 1.27 × 1015 molecules (2.11 nmol) of rhodamine per 1 × 103 cells (2.11 pmol/cell). For a standard test with 200000 cells in 2 mL using a bioactive compound instead of rhodamine B, this corresponds to an effective concentration around 200 μM. Using NPs with bioactive molecules should thus be possible for compounds with very high activity and low NPs functionalization or with less active compounds with higher functionalization until it does not influence particle stability. Most of the bioactive compounds being active in the micromolar range, such particles are well suited for future development as DDS. Having confirmed and quantified the previously observed internalization of NPs 8 into ADK (ADK 153 cell line) and

Figure 7. Kinetics of NPs 8. Graphic representation of the fluorescence intensities measured in the different organs at the indicated times postinjection. Results are expressed as the means ± SEM of the results obtained on 24 mice (N = 4 per group). Statistical Analysis. Data are expressed as the means ± SEM of at least three experiments. Statistical comparisons were made using oneway Anova followed by a Newman-Keuls Multiple Comparison Test (GraphPad prism, Prism 5 for Windows).

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RESULTS AND DISCUSSION NB derivative 5 (Scheme 1) was used as starting material to give macromonomer 1 by chain extension in basic conditions, the hydroxyl group being then converted to an azide group affording macromonomer 2, useful for generic preparations of functional macromonomers by click chemistry. Macromonomer 2 was converted to fluorescent macromonomer 4 with the rhodamine propargylester 3.21 The functionalization yield of rhodamine FRh = 70% was determined by 1H NMR,19b and no cycloaddition between the azide and the norbornenyl group was observed. Finally, the end-capped NB groups in 1, 4, and 7 allow for copolymerization performed in dispersed media, producing fluorescent nanoparticles 8. SEC gave a numberaverage molecular weight per chain of 142000 g mol−1, giving a 332:3:3 composition for units issued from monomers 7, 1, and 4, respectively. The amount of linked rhodamine per NP (nRh/ NP) was calculated according to a previously reported method.22 By this way, we could determine that 5.72 × 10−19 mol of rhodamine was linked per NP, that is to say, about 350000 molecules of rhodamine per NP. The particles were characterized by DLS and TEM.19b DLS (Figure 1, left) gave an average 390 nm size and TEM 290 nm (Figure 1, right). 2400


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considering the size of our NPs 8, the lack of colocalization with clathrin labeling and the colocalization with acidic vesicles, these NPs are probably internalized through a clathrinindependent macropinocytosis receptor-based mechanism.29,30 NPs 8 being able to enter cells by endocytosis we then evaluated their behavior in vivo, expecting the PEO chains at the outer surface to induce the stealth properties necessary for passive tumor targeting. Experiments were set up to determine the biodistribution of NPs 8 injected in the tail vein of mice bearing a subcutaneous AK7 tumor (murin mesothelioma cancer cells; Figure 6). After 24 h NPs injection, the fluorescence, observed on whole mice, was detected only in the tumor. At 1 week post-injection, analysis of the fluorescence on isolated organs after necropsy of animal groups showed that NPs were mainly concentrated in the tumor. A weak fluorescence was also observed in the liver corresponding to the presence of a small amount of NPs. Kinetic studies (Figure 7) measured after animal groups necropsies showed a high initial plasma concentration, rapidly decreasing after 1 h. The expected stealth behavior of these NPs was thus confirmed. Based on fluorescence analysis, in kidneys the same but lower 1 h peak concentration was observed, followed by rapid decrease, and even lower in ovary. The level detected in spleen and brain was not significant compared to the control. A higher amount was detected in liver but the major result was the high accumulation in tumors after one day, remaining for one week. The rapid decrease in blood compared to the progressive accumulation in tumors can be explained by the overall body distribution at low levels in the mice, quite all the NPs being finally accumulated in tumors. These results demonstrated that our fluorescent NPs 8 were not eliminated by spleen, kidneys, and liver, which represent the main biological barriers for nanoparticles diffusion. The short half-life of these nanovectors in blood (less than 6 h) paralleled with their rapid accumulation in the tumor. This suggests that the structural properties of these NPs allow a highly efficient dissemination in the tumor by EPR effect.

MPM cells (Meso 13 cell line), the effective internalization in cells was first investigated using fluorescence microscopy experiments coupled to cell membrane labeling. The experiments were also conducted by imaging two cell layers noted n and n + 1 μm (Figure 5A) to confirm the spatial localization inside cells. Incubation at 37 °C (Figure 5A, line 1) showed the presence of numerous red dots within cells delimited by a thin green labeled membrane in both row n and n + 1 μm. When cells were incubated on ice (Figure 5A, line 2), in order to stop all the active internalization processes, or with cytochalasin D (Figure 5A, line 3), which inhibits actin polymerization and disrupts actin microfilaments, prior to addition of NPs 8, NPs were mainly localized on membrane and not into cells. This suggests that internalization of the NPs by cells needs an active mechanism and that is not related to a passive diffusion. These results also demonstrated the implication of an endocytosis pathway using the actin network. To determine whether clathrin-dependent endocytosis was implicated in NPs 8 internalization, we performed fluorescent microscopy colocalization experiments using specific anticlathrin antibody labeling and Rhodamine coupled NPs (NPs 8). Clathrin labeling (Figure 5B, line 2) demonstrated the presence on numerous clathrin coated vesicles inside the cells. However, whereas an internalization of several NPs 8 was observed (Figure 5B, line 1), illustrated by the presence of red dots within cells, the absence of yellow color in merge pictures (Figure 5B, line 3) demonstrated that there is no colocalization between clathrin-coated vesicles and NPs. The observed differences in the internalization capacities of these two types of cell lines could be explained by ultrastructural differences of the cellular membranes. Indeed, electron microscopy analyses revealed the presence of long and thin microvilli on mesothelioma cells surface but not on adenocarcinoma cells surface.23 Changes in microvilli structure at the cell surface are responsible for alteration in surface charges24 which could modify interactions with NPs 8 and then, internalization properties. Moreover, primary function of mesothelial cells is to constitute a protective nonadhesive surface,25 whereas adenocarcinoma cells present secretory properties that need a high recycling rate of the cellular membranes. One possible use of such DDS could be the release of bioactive molecules during internalization due to pH variations, in particular, for endocytosis-mediated26 internalization and release based on acidic modifications in endosomes/lysosomes, a well-known strategy for DNA delivery by polyplexes but also for smaller anticancer compounds.27 We thus investigated if internalized NPs 8 were colocalized with intracellular acidic compartments in cancer cells by characterization of the expression of the lysosomal-associated membrane protein 1 (LAMP-1) at their membrane (Figure 5C).28 In these experiments we focused on acidic vesicle labeling, membrane labeling having been reported in Figure 5A. LAMP-1 was labeled by immuno-fluorescence, and once again, the fluorescence of rhodamine was used to determine the colocalization using fluorescence microscopy. In parallel to the confirmation of internalization by cells, LAMP-1 labeling (Figure 5C, line 2) revealed a high number of acidic compartments in the two cell types, suggesting that the acidic-mediated release of bioactive compounds in such cancer cell lines could be efficient. Merged pictures (Figure 5C, line 3) showed colocalization of NPs with acidic compartments, as illustrated by the presence of the additive yellow staining. When

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CONCLUSIONS Azido-PEO macromonomers were prepared as a key building block to be used to synthesize polymeric core−shell nanoparticles as potential DDS. This potential was demonstrated by the synthesis of a fluorescent macromonomer by click chemistry, used in turn to prepare a multifunctional polymeric nanoparticle by ROMP. The behavior of the resulting fluorescent nanoparticles was determined in two different cancer cells selected for the low median survival in clinical cases. The initially observed internalization was confirmed and the probable mechanism is based on endocytosis, as demonstrated by LAMP-1 staining of acidic vesicles. This suggests that such particles can be used for drug loading and internalization, with release based on pH variations. The release could be obtained from pH responsive covalent links between the particles and the drugs or by surface charge modifications as in polyplexes. In the two cell lines used, the high level of acidic compartments could give efficient results with such releasing strategies. A cell type difference in internalization kinetics was observed, that could be correlated to the resistance of cell lines like MPM. A major result appeared to be the high native tumor targeting by EPR effect due to the hydrophilic PEO shell. These nanoparticles have a 300 nm diameter size, pushing the usual limits of the particles sizes used for tumor targeting by EPR and cellular internalization. The effect of the cationic dye and the 2401


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possibility to have a modification in vivo of the core shell structure are under investigation. Our next challenge will be to use acidic responsive linkers to link anticancer compounds and to measure the effect of such functional DDS on the tumor tissues.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (P.R.) and [email protected] (C.B.). Author Contributions ∥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors thank Agence Nationale de la Recherche (ANR) for RD, FG, and FC Grants (ANR-08-PCVI-030), Centre National de la Recherche Scientifique (CNRS), the Ligue Contre le Cancer, committees of Morbihan, Sarthe, Vendée et LoireAtlantique, Poitou-Chanrentes, ARSMESO44, Nantes University Hospital, COST Action TD0905, and Ligue Nationale Contre le Cancer for ID grant.

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REFERENCES

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