Antisense Oligonucleotide Nanocapsules ... - Semantic Scholar

OLIGONUCLEOTIDES 16:158–168 (2006) © Mary Ann Liebert, Inc.

Antisense Oligonucleotide Nanocapsules Efficiently Inhibit EWS-Fli1 Expression in a Ewing’s Sarcoma Model NEDJMA TOUB,1,2 JEAN-RÉMI BERTRAND,2 CLAUDE MALVY,2 ELIAS FATTAL,1 and PATRICK COUVREUR1

ABSTRACT The cytogenetic abnormality of Ewing’s sarcoma is related to the presence of a balanced t(11;22) translocation expressing the EWS-Fli1 chimeric fusion protein. Oligonucleotides (ODNs) are specific compounds that inhibit gene expression at the transcriptional level. They possess a poor bioavailability and are degraded by nucleases very rapidly. Therefore, there is a strong need for the development of ODN drug delivery systems. In the present study, polyisobutylcyanoacrylate nanocapsules entrapping ODNs in their aqueous core were prepared, with high encapsulation yield (99%). Previous studies have demonstrated that such complexes were able to inhibit tumor growth in mice. Nevertheless, no information was available about their mode of action at the cellular level. The aim of this study was to investigate the efficacy of these ODN nanocapsules on cultured tumor cells. We found that nanocapsules were capable of protecting ODN against degradation. Using confocal microscopy, we observed that cell uptake and nuclear accumulation of ODNs were importantly enhanced when ODNs were associated with these nanocapsules. Consequently, a specific cellular growth inhibition and suppression of EWSFli1 fusion gene expression was noticed. In conclusion, it was demonstrated that nanocapsules as nonviral vectors show great potential for the delivery of ODNs to cells.

INTRODUCTION

D

ESPITE ADVANCES IN LIMB-SPARING SURGERY, radiation therapy, and chemotherapy, nearly one half of children with Ewing’s sarcoma succumb to the disease (Arvand et al., 1998; Bailly et al., 1994). Ewing’s family tumors (EFT) (Askin’s tumor of the chest wall, Ewing’s sarcoma, and the primitive peripheral neuroectodermal tumor, neuroepithelioma) share histologic features as well as a recurrent and specific t(11;22) (q24;q12) chromosome translocation present in 85%–90% of these tumors (Desmaze et al., 1992). The predominant t(11;22) translocation causes a chimeric oncogenic transcript fusing the N-terminal domain of the RNA-binding protein EWS with the DNA-binding domain of the ETS family

transcription factor Fli1 (Delattre et al., 1992). Several lines of evidence indicate that related fusion proteins are both necessary and sufficient to induce cell transformation in vivo and in vitro. An essential role of the fusion protein in cellular proliferation was demonstrated by the use of an antisense oligodeoxyribonucleotide (ODN) targeted to EWS Fli-1, which inhibited proliferation and tumorigenicity in Ewing’s sarcoma and PNET cells (Tanaka et al., 1997). In theory, the concept of antisense technology is elegant. Short synthetic ODNs bind selectively to the complementary mRNA transcript of a targeted gene (Stephenson and Zamecnick, 1978). To go from the concept to therapeutic applications, a large field of research has focused on (1) protecting ODN from degradation by

1

Laboratoire de Physicochimie, Pharmacotechnie et Biopharmacie, UMR CNRS 8612, Faculté de Pharmacie, 92286 ChâtenayMalabry Cedex, France. 2 Laboratoire de Vecterologie et Transfert des Gènes, Institut Gustave Roussy, UMR CNRS 8121, 94805 Villejuif Cedex, France. 158

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extracellular nucleases and (2) improving the intracellular delivery of efficient and safe doses of ODNs. ODN drugs are anionic macromolecules that poorly diffuse through biologic cell membranes, resulting in poor bioavailability. This has led, on one hand, to the use of chemically modified ODNs (Levin, 1999) and, on the other hand, to the development of particulate nanocarriers, such as liposomes and polymers. Each approach has its own limitations. Chemical modifications improve mRNA binding (in recent years among the most efficient ones, 2-FANA, 2 MOE, 3 phosphoramidates, locked nucleic acids [LNAs] can be found) (Nakata, 2005). After degradation, however, such chemically modified nucleotides may be toxic if they are incorporated into nucleic acids. Colloidal carriers allow lowering of the percent of modified bases in the ODN, but they require endocytic properties of the target cells. Thus, this study is focused on the intracellular delivery of phosphorothioate ODNs (PS ODNs) targeted toward EWS-Fli1 mRNA by means of polyisobutylcyanoacrylate nanocapsules. These ODN-loaded nanocapsules were developed previously by our group (Lambert et al., 2000b). It has been clearly shown that with this nanotechnology, the ODNs are located in an aqueous nanocore, which is delimited by a thin, biodegradable, polyisobutylcyanoacrylate polymer membrane. Nanocapsules containing EWS-Fli1targeted ODNs were found to trigger significant tumor growth inhibition in an in vivo Ewing’s sarcoma model (Lambert et al., 2000a), although no information was available about their mode of action at the cellular level. Therefore, we used the same previously employed EWSFli1 PS ODN so that data presented here, at the cellular level, would be relevant for interpretation of the animal data published previously (Lambert et al., 2000a). In the present study, EWS-Fli1-targeted nanocapsules were tested for cellular uptake and trafficking and visualized in EWS-Fli1 cells by confocal microscopy, suggesting that this type of noncationic carrier was able to deliver its contents to the cytosol and nucleus, as described previously (Toub et al., 2006), using another cellular model. Cytotoxicity and cellular growth inhibition of ODNloaded nanocapsules were also studied. The efficiency of these aqueous core-containing nanocapsules in delivering antisense ODNs targeted against EWS-Fli1 was measured by quantitative RT-PCR using the SYBR green test.

MATERIALS AND METHODS

quence was specific to the tumor cells, as the chimeric RNA was not expressed in normal cells. We used the antisense ODN corresponding to nucleotides 832–856 of type 1, as used by Tanaka et al. (1997): 5-GAC-TGAGTC-ATA-AGA-AGG-GTT-CTG-C-3. The sense oligonucleotide was used as control: 5-GCA-GAA-CCCTTC-TTA-TGA-CTC-AGT-C-3.

Oligonucleotide radiolabeling The 5 end of the antisense was labeled by T4 polynucleotide kinase (New England Biolabs, Beverly, MA) with 32P-ATP (111 TBq/mmol) (MP Biomedicals, Strasbourg, France) following the manufacturers’ protocols. The purified ODNs were recovered by gel filtration using a Bio-Spin 6 column (Bio-Rad, Richmond, VA) and centrifuged at 2500g for 4 minutes. The purity was controlled by radioactivity analysis using an automatic TLC-Linear Analyzer (EG&G Berthold, Thoiry, France) as described by Aynie et al. (1996).

Nanocapsule preparation An aqueous suspension of nanocapsules containing ODNs was prepared using a modification of the methods of Lambert et al. (2000b) and Watnasirichaikul et al. (2000) as follows. We first prepared a nano-emulsion by mixing 0.1 mL demineralized water containing ODNs at the desired concentration with an oily phase containing 1.125 g Montane 80 (sorbitan monooleate, Seppic, Paris, France) and 1 g Miglyol 812 (medium-chain triglyceride) (Sasol, Hamburg, Germany) using an Ultraturrax for 1 minute at 24,000 rpm. Isobutylcyanoacrylate (IBCA) monomer (10 mg) (Loctite, Hertfordshire, Ireland) was rapidly added to the microemulsion under mechanical stirring at 500 rpm. The system was left for at least 4 hours in order for polymerization to take place. Nanocapsules were collected by ultracentrifugation at 30,000 rpm for 30 minutes at 4°C (Beckman L8-70M Ultracentrifuge, 50 TI rotor) (Beckman Instruments, Sunnyvale, CA). The oily phase, the interface, and the water phase were removed, and the pellet was transferred to a new tube and dissociated in sterile demineralized water under vortex agitation for 1 minute. The product was finally dispersed by fast sonication for 8 seconds, followed by centrifugation at 4000g for 10 minutes at 4°C to remove residual surfactant. This process was repeated twice to ensure complete removal of residues.

Oligonucleotides

Nanocapsule characterization

The antisense and sense full PS ODNs, purified by high performance liquid chromatography (HPLC), were purchased from Eurogentec (Seraing, Belgium). The antisense target sequence was designed at the junction between EWS and Fli1 in the chimeric mRNA. This se-

The particle size and distribution of the nanocapsules containing ODNs were measured after dilution (1:10) in demineralized water using dynamic laser light scattering (Nanosizer N4Plus, Coultronics, Marguency, France). The nanocapsules’ zeta potential was determined as follows:

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200 L of the samples was diluted in 2 mL of a 0.1 mM KCl solution adjusted to pH 7.4 and analyzed with a Zetasizer 4 Malvern (Malvern Instruments, Malvern, U.K.).

with 10% serum was determined by quantification of the band intensity corresponding to the intact ODN.

Cellular trafficking using confocal microscopy Freeze fracture electron microscopy (FFEM) A small drop of the suspension of ODN-loaded nanocapsule containing 30% glycerol as a cryoprotector was deposited on a thin copper plate and rapidly frozen in liquid propane. Fracturing and platinum-carbon replication were performed with a Balzers Baf 301 (Balzers High Vacuum Corp., Balzers, Liechtenstein) freeze-etch. The replicas were washed and examined under a Philips 410 electron microscope.

Determination of oligonucleotide encapsulation yield After 32P-ATP labeling, ODNs were introduced into nanocapsules as described. After the first ultracentrifugation, the radioactivity of the supernatant and pellet was determined by the Cherenkov effect using a liquid scintillation counter (1900TR Packard, Downers Grove, IL). The final encapsulation yield was calculated using the following equation: % Encapsulation Pellet radioactivity 100 Total radioactivity (pellet supernatant)

Colloidal stability of nanocapsule suspension The stability of the nanocapsule-ODN complexes was determined on the particle suspension dispersed in sterile water and then conserved at 4°C. At different times, aliquots were diluted in demineralized water, and particle size, zeta potential, and polydispersity index (PI) were determined as described earlier.

Stability of free and encapsulated PS ODN Nanocapsules were prepared as described using radiolabeled PS ODN. Free ODNs were used as a control for stability studies. Free or encapsulated ODN (150 L) was mixed with 350 L DMEM supplemented with 10% newborn calf serum (NCS) (GIBCO, Grand Island, NY) at 37°C. At 2, 6, 8, and 15 hours of incubation, an aliquot of the mixture was removed and cooled at 4°C to stop the reaction. Hydrolysis of the polymeric nanocapsule was achieved by incubation with NAOH (4 M) for 24 hours at 37°C. PS ODN integrity was assayed after electrophoresis on a 20% polyacrylamide-7 M urea sequencing gel (PAGE), followed by analysis using a phosphorimager (Storm 840 Molecular Dynamics, Little Chalfont, U.K.). The PS ODN half-life (t1/2) in medium supplemented

NIH/3T3 cells stably transfected with the human EWSFli1 gene were a generous gift from Dr. J. Ghysdael (Institut Curie, Orsay, France). We followed the intracellular trafficking of the ODNs by confocal microscopy using FITC-labeled ODNs. Briefly, 105 NIH/3T3 EWSFli1 cells were seeded into 6-well plates containing a coverglass in 1 mL DMEM medium supplemented with 10% heat-inactivated NCS, penicillin, and streptomycin. The cells were incubated overnight at 37°C with 5% CO2 in a moist atmosphere. The medium was discarded and replaced with fresh medium containing 100 nM FITC-labeled ODNs free or encapsulated into nanocapsules for 2, 4, 8, or 15 hours of incubation. The cells were washed with phosphate-buffered saline (PBS) and fixed in PBS containing 2% paraformaldehyde for 10 minutes at ambient temperature. The slides were washed three times with PBS buffer before observation with a confocal microscope (Zeiss LSM 510/Axiovert 200M equipped with a 63/1.4 oil immersion objective) after mowiol (Calbiochem, La Jolla, CA) mounting as an antifading agent.

Cytotoxicity and inhibition of cell growth assay NIH/3T3 cells and NIH/3T3 cells transfected by the EWS-Fli1 oncogene were seeded on 24-well plates at approximately 5 105 viable cells/well with 1 mL fresh DMEM supplemented with 10% NCS and incubated at 37°C in a humidified atmosphere containing 5% CO2. Forty-eight hours later, the medium was discarded, and different nanocapsule preparations were added in fresh medium. Experiments were carried out using different ODN concentrations ranging from 0 to 0.3 M of the antisense ODN nanocapsules (NC ODN-AS) or control ODN nanocapsules (NC ODN-CT), corresponding to doses in weight ranging from 0 to 200 g/mL. After 15 hours of incubation, cell viability was determined by the MTT test: 100 L of 5 mg/mL MTT solution in PBS was added for 2 hours of incubation at 37°C. Then, 1 mL lysis buffer (10 mM HCl, 10% SDS) was added, and the plates were incubated overnight. The formazan that developed was quantified by the OD570/OD630 ratio. All experiments were carried out in triplicate and repeated twice.

EWS-Fli1 detection by RT-PCR NIH/3T3 EWS-Fli1 cells were treated by either free or encapsulated ODN. Total RNA was extracted and purified by chloroform extraction, followed by isopropanol precipitation. RNA concentration and purity were determined by spectrophotometry (Maniatis et al., 1982). The RNA quality was evaluated by 1% agarose gel elec-

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TABLE 1. PHYSICOCHEMICAL CHARACTERISTICS OF POLYISOBUTYL CYANOACRYLATE (PIBCA) NANOCAPSULE SAMPLES WITH DIFFERENT ODN LOADINGa Initial ODN amount Encapsulation (g ODN/mg PIBCA) yield (%) 7.8 170 270 280

Zeta potential (mV)

Polydispersity index

Mean diameter (nm)

28.4 28.1 28.0 28.2

0.1 0.1 0.1 0.1

228 69 221 70 213 66 228 70

70.0 99.5 99.7 95.0

a

Each measurement was done in triplicate and repeated twice.

trophoresis, followed by ethidium bromide staining. The cDNAs were synthesized from 1 g total RNA. The PCR primers and the PCR profile used were: EWS-Fli1 primer sequences: forward, 5-AGC AGT TAC TCT CAG CAG AAC ACC-3; reverse, 5-CCA GGA TCT GAT ACG GAT CTC GCT G-3. The amplification profile was as follows: denaturation at 90°C for 1 minute, annealing at 66°C for 1 minute, and extension at 72°C for 1 minute, for a total of 35 cycles in PCR thermal cycler (MJ Research, Cambridge, MD). Under these conditions, we obtained optimization of the RT-PCR assays in which the amount of each RT-PCR product was directly proportional to that of template RNA. The amplified products were separated by electrophoresis on 2% agarose gel electrophoresis and visualized after staining with ethidium bromide, and their signal intensities were evaluated using an image analyzer (UVP, dual-intensity, transilluminator). PCR quantifications were conducted using the Gene Amp RNA SYBR green PCR kit (Applied Biosystems, Roche, Foster City, CA) according to the manufacturer’s instructions, with 2.5 M primers and a variable amount of DNA standard in a 25-L final reaction volume. Thermocycling was conducted using an ABI System Prism 7000 Sequence Detection System initiated by 15 minutes of incubation at 94°C, followed by 40 cycles of 95°C for 15 seconds, 60°C for 60 seconds, with a single fluorescent reading taken at the end of each cycle. Each run was completed with a melting curve analysis to confirm the specificity of amplification and lack of primer dimers. Internal control 18S RT-PCR standard curves were determined on all samples simultaneously. Each RT-PCR was repeated in duplicate using different preparation of RNA, and representative results are shown as the ratio of the intensity of specific mRNA to that of the 18S mRNA for each sample.

comparisons of the effect of NC ODN-AS and NC ODNCt-treated NIH/3T3T EWS-Fli1 cells to empty nanocapsule-treated cells, using the percentage of inhibition of growth cells as covariate and treatments as factors. Tests were considered significant when p 0.05.

RESULTS Nanocapsule characterization Mean diameter, PI, zeta potential, and encapsulation efficiency of different nanocapsule samples containing 25-mer ODN at different concentrations are reported in Table 1. The mean diameter of the preparation was not affected by the ODN concentration and was around 220

Statistical analysis The p values, obtained by analysis of covariance (ANCOVA) using the ANOVA 2-way test (GraphPad Prism Version 4.01, San Diego, CA), are those of the

FIG. 1. Visualization by electron microscopy after freeze fracture of ODN-loaded nanocapsules. Bar 200 nm.

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nm, showing a unimodal distribution (PI 0.1). The surface charge of these particles was around 28 mV and does not change with ODN loading. The yield of ODN encapsulation was determined under optimal conditions to be 99% using 32P-labeled ODN (see Materials and Methods). These results were confirmed by electron microscopy examination, showing circular, regular particles with a size of around 200 nm (Fig. 1).

Long-term stability of nanocapsules The physicochemical characteristics of the nanocapsule suspensions are strongly dependent on the processing method and storage conditions (Couvreur et al., 1986). To appreciate the time-dependent stability of the nanoparticle samples, measurement of the average size and PI was performed monthly for 1 year. Immediately after preparation and purification, the hydrodynamic diameter of the nanocapsules averaged 235 nm (n 9,

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SD 6.72), with a PI of 0.1. After 12 months of storage at 4°C in sterile water, the suspension appeared stable without any sedimentation or aggregation tendency. The PI and the mean diameter did not change, and the average size values from 12 separate measurements were 234 nm (n 54 measurements, SD 8.08), and the PI was 0.2.

Intracellular localization of ODNs To determine if nanocapsules were able to deliver ODNs into tumor cells, the intracellular distribution of FITC-labeled ODNs was investigated by laser confocal microscopy after incubation of ODNs (free or encapsulated) with NIH/3T3 cells expressing EWS-Fli1 (Fig. 2). At different time intervals, ranging from 2 to 15 hours, incubated cells were fixed by paraformaldehyde and then observed under a confocal microscope. At the early observation (2 hours), we detected ODN fluorescence in both the cytoplasm and the nucleus of the treated cells.

FIG. 2. Confocal microscopy of NIH/3T3 EWS-Fli1 cells transfected by FITC-labeled ODN-loaded nanocapsules after (A) 2, (B) 6, (C) 8, and (D) 15 hours of incubation. The concentration of FITC-labeled ODN nanocapsules was 100 nm, and the corresponding polymer nanocapsule concentration was 100 g/mL. No fluorescence was detected using naked FITC-labeled ODNs under the same conditions. Bar 20 m.


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A

B

FIG. 3. Viability of NIH/3T3 (B) and NIH/3T3 EWS-Fli1 (A) cells treated by increasing concentrations of empty nanocapsules or nanocapsules loaded with antisense or control ODN. Cell growth inhibition was measured after 15 hours of contact using the MTT assay.

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FIG. 4. Antisense effect of different ODN preparations. The viability of the treated cells under nontoxic conditions was determined using the MTT assay. The difference in cell proliferation among NC Empty, NC ODN-AS, NC ODN-Ct-treated cells is shown in the corresponding histograms. The statistical significance of this difference (NC ODN-AS vs. NC ODN-Ct) was checked with the ANOVA 2-way test (p 0.001, mean SD, n 3).

The intensity of the ODN fluorescence increased as a function of the time (2–15 hours), with a preferential accumulation into the nucleus. Under the same conditions, the free ODN fluorescence was not visible in the cells, or there was only a light membrane coloration (data not shown). Thus, the confocal microscopic analysis showed that the intracellular distribution of encapsulated and free ODN was dramatically different. Free ODN added to the culture medium produced a negligible fluorescence localized on the extracellular matrix (data not shown), whereas brightly punctuated FITC fluorescence was observed with the cells treated with ODN nanocapsules (Fig. 2). This green color was located intracellulary in the useful cytoplasmic/nucleus subcellular compartments. This distribution and the time-dependent enhancement of the intracellular signal of fluorescence suggested an endocytotic pathway for nanocapsule uptake of 2–5 hours of incubation.

Cytotoxicity and proliferation studies The cytotoxicity of nanocapsules either empty or loaded with ODNs was measured using the MTT assay. These ODNs were either antisense (NC ODN-AS) or control (NC ODN-Ct) ODNs. Incubation was with either NIH/3T3 EWS-Fli1 or NIH/3T3 cells for 15 hour (Fig. 3). Metabolically active cells have the capacity to transform the tetrazolium salt into formazan (Smith, 1951). Treatment of NIH/3T3 cells by nanocapsules either empty or loaded with antisense or control ODNs (Fig. 3B) showed, after 15 hours incubation, an equivalent dose-dependent effect, with an IC50 of 70 g/mL in terms of polymer (corresponding to a 65 nM ODN concentration). In contrast, treatment of NIH/3T3 EWS-

Fli1 cells with empty nanocapsules and control ODNloaded nanocapsules displayed a bell-shaped curve (Fig. 3A) corresponding to a stimulating effect for the lower concentrations and to a toxic effect afterward (IC50 of, respectively, 165 g/mL and 240 g/mL polymer). However, when NIH/3T3 EWS-Fli1 cells were treated with antisense ODN-loaded nanocapsules, cell stimulation completely disappeared, and a much more pronounced cytotoxic effect (IC50 of 90 g/mL in polymer) was observed, comparable to that obtained on treated NIH/3T3 cells. This specific cell growth inhibition was confirmed in a further study performed at 130 g/mL of nanocapsules, where we obtained an inhibition of 60% of cell growth, with a p value 0.001 in comparison with empty nanocapsules or control nanocapsules (Fig. 4).

EWS-Fli 1 expression To investigate the cellular effect of the encapsulated antisense ODN, we quantified the targeted EWS-Fli1 mRNA expression. After NIH/3T3 EWS-Fli1 cell treatment by antisense ODN or control ODN-loaded nanocapsules, we extracted the total RNA and used RT-PCR for EWS-Fli1 RNA expression detection. We observed an inhibition of EWS-Fli1 expression compared with untreated cells (Fig. 5). The control ODN did not inhibit EWS-Fli1 expression, which confirmed the specific effect of the antisense ODN. A RT-QPCR was done to obtain a quantitative effect. We observed a 40% inhibition of EWS-Fli1 expression in cells treated using NC ODNAS (Fig. 6). This expression was normalized by the 18S rRNA used as an internal standard. No effect was obtained with the NC ODN-Ct treatment.


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FIG. 5. Inhibition of EWS-Fli1 mRNA expression in NIH/3T3 EWS-Fli1 cells by antisense ODN-loaded nanocapsules (RTPCR detection).

DISCUSSION The use of nonviral vectors that facilitate the delivery of short nucleic acids able to be employed as therapeutic agents has been studied extensively. We have developed nanocapsules with an aqueous core containing a potentially therapeutic nucleic acid to be delivered. Earlier, we stated that such nanocapsules were able to efficiently inhibit the tumor growth of mice bearing Ewing’s sarcoma.

Nevertheless, the mechanism of action of these ODNs was not clearly elucidated. For this purpose, we prepared nanocapsules containing ODN in their aqueous core. Using some preparation method modifications, we were able to encapsulate the ODN very efficiently, with a yield close to 98%. These particles had a regular spherical structure (Fig. 1), with a size of 200 nm, quite compatible with cell penetration through the endosomal pathway.

FIG. 6. Quantitative RT-PCR using the SYBR green method was performed in duplicate on total RNA from NIH/3T3 EWS-Fli1 cells.

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In the present study, nanocapsules were found to be stable in the dispersion state, having negative surface charges of 28 mV (Table 1), which is believed to ensure the stability of the suspension by electrostatic repulsion. This value was not modified after ODN encapsulation, which is consistent with the fact that these molecules were efficiently encapsulated and not adsorbed onto the nanocapsule’s surface. The stability of PIBCA nanocapsules containing ODNs was checked by measuring particle size and PI over a 12-month period. As these values did not change, we concluded that no degradation of the polymer occurred during this time. Therefore, the nanosuspension had a homogeneous appearance without any sedimentation or aggregation. Because of the presence of the ODN inside the aqueous core of the nanocapsules, it is expected that (1) the nucleic acids could be protected from nuclease degradation and that (2) ODN liberation from the nanocapsule would be dependent on hydrolysis by the esterases of the polycyanoacrylate-biocompatible capsule. In a previous study (Lambert et al., 2000a), we performed experiments in pure serum showing that nanoencapsulation was able to protect ODNs against degradation by serum nucleases. Of the free ODN, 50% was degraded within 4 hours in serum, whereas 50% of the encapsulated ODN was degraded only after 6 hours. It has been shown that the part that was degraded at time t corresponded to the ODN fraction progressively released as the polymeric envelope was hydrolyzed progressively under the action of serum esterases. Indeed, polyalkylcyanoacrylates are known to be degraded through hydrolysis of the ester side chain of the nanoparticle polymer (Lenaerts et al., 1984; Muller et al., 1990). When the nanocapsules are cell internalized through endocytosis, they are degraded by intracellular lysosomal esterases, thus releasing their ODN content intracellularly into the cytoplasmic and nuclear fraction. Previous publications have addressed this point in detail, using the same polymer as nanospheres (Chavany et al., 1994) or as nanocapsules (Toub et al., 2005). The uptake of FITC-ODN nanocapsules in EWS-Fli1 cells has been investigated using confocal microscopy. Apart from a few discrete points of extracellular fluorescence, the naked ODNs were poorly internalized by cells. When entrapped into nanocapsules, their cellular uptake was dramatically improved (Fig. 2). The transfection level of the cells was high, with cytoplasmic fluorescence and then a time-dependent accumulation into the nucleus. This cytoplasm and nuclear localization is in accordance with the area where antisense inhibition may occur because the target RNA is present in these two subcellular compartments. Although the mechanism of endosomal escape of the ODN nanocapsules is not yet fully understood, deter-

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gents used in their formulation, such as sorbitan monooleate, may play an important role in endosomal membrane destabilization (Torchilin et al., 1993). The cytotoxicity of empty nanocapsules was obviously higher for NIH/3T3 cells compared with NIH/3T3 EWSFli1 cells. This is particularly noticeable for nanocapsule concentrations of 100 g/mL polymer. Considering the curves relating cytotoxicity to nanocapsule concentration as cell phenotypes due to complex phenomena and considering the fact that EWS-Fli-1 expression can deregulate at least 86 genes and probably more (Prieur et al., 2004; Siligan et al., 2005; Hu-Lieskovan et al., 2005), this might explain the difference in the cell lines in the response to nanocapsules. The ODN content of nanocapsules did not modify the phenotype of NIH/3T3 cells. Noteworthy was the reversion of NIH/3T3 EWS/Fli1 cells to the NIH/3T3 phenotype after treatment with the NC ODN-AS. The obvious interpretation is that this was a result of EWS/Fli1 AS inhibition, thus allowing the inference that the phenotype of NIH/3T3 cells expressing EWS-Fli-1 is strictly dependent on EWS-Fli-1 expression. This hypothesis was confirmed by the RT-PCR measurement of EWS-Fli-1 antisense inhibition. According to this measurement, a total inhibition of EWS-Fli-1 at the mRNA level would not be required for the phenotype reversion to occur. Several investigators have used antisense-mediated downregulation of EWS-Fli1 to study the downstream effects of EWS-Fli1 expression (Tanaka et al., 1997; Matsumoto et al., 2001; Prieur et al., 2004). The data presented in this study show that stable, long-term delivery of antisense ODNs by means of nanocapsules promotes the cellular uptake of these molecules and allows maintenance of the intracellular concentrations needed for efficient inhibition of the target mRNA. In conclusion, polyisobutylcyanoacrylate nanocapsules were developed earlier in our laboratory as delivery systems able to render efficiently, in vivo, an antisense ODN targeted toward EWS-Fli1 (Lambert et al., 2000a). The results obtained here show that such nanocapsules were able not only to protect the ODN from nuclease degradation but also to act as a controlled-release system facilitating the cell penetration necessary for RNase Hdependant degradation of the oncogenic targeted mRNA. This study sheds light on the mechanism of action in the cell of the nanocapsule-vectorized ODN and opens the way to further applications of such technology in pharmacology.

ACKNOWLEDGMENTS We thank Mr. Jalil Abdelali (G. Roussy Institute) for excellent technical assistance and Mrs. Ghislaine Frébourg (UMR CNRS 7138) for the electron microscopy experi-


ments. The IBCA monomer was a generous gift from Loctite, Ireland. N.T. is supported by a fellowship from Ia Ligue Nationale Contre le Cancer. This work was supported by the Association de Recherche sur le Cancer (grant 4310), Fondation de l’Avenir, and the European grant PROTHETS.

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TOUB ET AL.

Address reprint requests to: Professor Claude Malvy Laboratoire de Vectorologie et Transfert des Gènes Institut Gustave Roussy UMR CNRS 8121 39, rue Camille Desmoulins 94805 Villejuif Cedex France E-mail: [email protected] Received January 13, 2006; accepted in revised form April 12, 2006.