Physicochemical Characteristics and Preliminary in Vivo Biological

Sep 13, 2008 - No difference in size or zeta potential was observed between siRNA NCs that were based on PEG−PCL/MA and empty NCs. Fluorescence ...
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Biomacromolecules 2008, 9, 2881–2890

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Physicochemical Characteristics and Preliminary in Vivo Biological Evaluation of Nanocapsules Loaded with siRNA Targeting Estrogen Receptor Alpha Ce´line Bouclier,† Laurence Moine,† Herve´ Hillaireau,†,§ Ve´ronique Marsaud,† Elisabeth Connault,‡ Paule Opolon,‡ Patrick Couvreur,† Elias Fattal,† and Jack-Michel Renoir*,† Physico-Chimie, Pharmacotechnie, Biopharmacie, Universite´ Paris-Sud, CNRS UMR 8612 and IFR 141, 5 rue Jean-Baptiste Cle´ment, 92296 Chaˆtenay-Malabry, France, and Institut Gustave Roussy, CNRS UNR 8121, Villejuif, France Received June 18, 2008; Revised Manuscript Received July 31, 2008

Specific siRNAs that target estrogen receptor alpha (ERR) were encapsulated in nanocapsules (NCs). We produced small (∼100-200 nm) ERR-siRNA NCs with a water core by incorporating two mixed duplexes of specific ERR-siRNAs (ERR-mix-siRNA) into NCs. The encapsulation yield that was obtained with poly(isobutylcyanoacrylate) (PIBCA) NCs was low, whereas no release of trapped siRNA was observed for poly(ethylene)glycol-poly(D,L-lactide-co-glycolide) (PEG-PLGA) NCs. High levels of ERR-siRNA incorporation into PEGε-caprolactone-malic acid (PEG-PCL/MA) NCs (3.3 µM in a polymer solution at 16 mg/mL) were observed (72% yield). No difference in size or zeta potential was observed between siRNA NCs that were based on PEG-PCL/MA and empty NCs. Fluorescence quenching assays confirmed the incorporation of siRNA into the NC core. A persistent loss of ERR (90% over 5 days) was observed in MCF-7 human breast cancer cells that were exposed to PEG-PCL/MA NCs that were loaded with ERR-siRNA. The intravenous injection of these NCs into estradiol-stimulated MCF-7 cell xenografts led to a significant decrease in tumor growth and a decrease in ERR expression in tumor cells. These data indicate that a novel strategy, based on ERR-siRNA delivery, could be developed for the treatment of hormone-dependent breast cancers.

Introduction RNA interference (RNAi) is a powerful approach to abolishing the production of a given protein. A very large number of small double-stranded RNA molecules, or siRNAs, that bind to mRNAs and prevent their translation into proteins by orchestrating their destruction are now commercially available. Therefore, siRNAs with relevant sequences are thought to have the potential for use in the treatment of cancer. However, poor tissue penetration, nonspecific immune stimulation by siRNAs administered in vivo, and low transfection efficiency have hampered the therapeutic use of these molecules. There is therefore a need to target duplexes of oligonucleotide sequences systemically to tumor sites. A few approaches have been evaluated in animal models and have been described as being potentially feasible in patients, but the delivery of siRNAs by viruses or tumor-targeting nanosytems has been identified as the method of choice.1-6 Estrogen receptor alpha (ERR) is an estradiol E2-activated transcription factor that regulates the proliferation and differentiation of mammary epithelial cells by up- and downregulating the expression of genes that encode proteins that are involved in cell cycle progression. ERR and ERβ are isotypes that display 56% amino acid sequence identity. However, ERR has been identified as the critical therapeutic target for breast cancer and is considered to be responsible for the mitogenic * To whom correspondence should be addressed. Tel: +33 1 46 83 58 31. Fax: +33 1 46 83 58 32. E-mail: [email protected]. † Universite´ Paris-Sud. ‡ Institut Gustave Roussy. § Current address: School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138.

effects of estrogens. The blockade of its activity by antiestrogens (AEs), aromatase inhibitors, or both continues to be the most efficient way to stop tumor cell propagation.7,8 There are two groups of AEs: (1) selective estrogen receptor modulators (SERMs), such as tamoxifen (Tam), which is the most successful of these molecules to date and has been used for more than three decades, and (2) selective estrogen receptor downregulators (SERDs) or pure AEs. SERMs inactivate ER functions but stabilize the expression of both ER isoforms, whereas SERDs, despite acting in a manner similar to that of SERMs by inhibiting E2-induced transcription, rapidly destabilize ERR but stabilize ERβ.9,10 SERDs, such as faslodex (or ICI182,780 (ICI) and RU58668 (RU)), induce the ubiquitin/proteasome-mediated degradation of ERR11-14 through a mechanism that involves the nuclear/cytoplasmic shuttling of the receptor,15 its delocalization in the nuclear matrix,16 and various phosphorylation/ dephosphorylation mechanisms with direct or indirect effects on ERR itself.14 Tam, ICI, and RU induce cell-cycle arrest and apoptosis, but these two phenomena are independent and occur at different concentrations; cell-cycle arrest occurs at nanomolar concentrations, whereas apoptosis occurs at micromolar concentrations. (For a review, see ref 17). The beneficial effects of Tam persist for no longer than 5 years in patients, even in those with an extended response. The long-term use of Tam is limited to 15 months, after which the tumor may become resistant to this drug.18,19 In cases of Tam resistance, ICI is now administered at a concentration of 250 mg by deep intramuscular oil injection.20 This leads to the degradation of ERR from the tumor, which is more likely than that from ERR-positive cells. We therefore hypothesized that the abolition of ERR expression could be used to overcome

10.1021/bm800664c CCC: $40.75  2008 American Chemical Society Published on Web 09/13/2008

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resistance to an AE or an aromatase inhibitor. We therefore selected new specific siRNAs that target ERR without affecting ERβ and tested them on breast cancer (BC) cells. To protect siRNA from degradation and to facilitate cell uptake, we encapsulated siRNA duplexes in NCs. We describe here the synthesis and physicochemical characterization of three different aqueous core NCs that are composed of biodegradable polymers. We assessed the efficiency with which these core NCs incorporated and released siRNAs in vitro. Experiments in MCF-7 ERR-positive BC cells that were exposed to siRNA-loaded stealth NCs composed of poly(ethylene)glycol-co-poly(ε-caprolactone-co-dodecyl β-malate) showed that this treatment resulted in the persistent loss of the receptor, which justifies the assessment of this siRNA-loaded nanodevice in further preclinical studies. In MCF-7 cell xenografts, this formulation decreased E2-induced tumor growth and caused a decrease in the ERR content of tumor cells.

Materials and Methods Chemicals. Iso-butylcyanoacrylate (IBCA) was provided by Loctite (Dublin, Ireland). Caprylic/capric triglycerides (Crodamol GTCC), sorbitan monooleate (Crill 4), and polysorbate 80 (Crillet 4) were provided by Croda (Trappes, France). Caprylic/capric mono/diglycerides (Capmul MCM) were provided by Abitec (Janesville, WI). PEG-PLGA (type RGP d 50105, Resomer) (PLGA (MW ) 45 kDa) consisted of 50% lactic acid units and 50% glycolic acid units; PEG (MW ) 5 kDa)) was obtained from Boehringer (Ingelheim, Germany). Polyethylene imine (PEI) (25 kDa, branched) and polyvinyl alcohol (PVA, wt ) 30 000-70 000) were purchased from Sigma (Saint Quentin Fallavier, France). D,L-malic acid (Aldrich, Saint Quentin Fallavier, France), trifluoroacetic acid anhydride (Aldrich), poly(ethylene glycol)methyl ether 2000 (Aldrich), ε-caprolactone (ε-CL) (Aldrich), stannous octoate (Aldrich), 1-dodecanol (Acros, Halluin, France), triphenylphosphine (Ph3P) (Acros), and diisopropylazo-dicarboxylate (DIAD) (Acros) were used as received. Other chemicals were reagent grade and were of the highest purity. ERR1-siRNAs (sense strand: 5′-GACUUGAAUUAAUAAGUGA-3′, antisense strand: 5′-UCACUUAUUAAUUCAAGUC-3′), ERR2-siRNAs (sense strand: 5′-CCUUUGACCUAUAGGCUAA-3′, antisense strand: 5′-UUAGCCUAUAGGUCAAAGG-3′), and scramble siRNAs were purchased from Qiagen (Courtaboeuf, France), carboxyfluorescein-labeled siRNAs (5′-6-FAM-siRNA) were obtained from Eurogentec (Seraing, Belgium). High-performance liquid chromatography was used for siRNA purification, and purity was then checked by spectrometry (Qiagen). We obtained [γ-33P]ATP (3000 Ci/ mmol) from GE Healthcare (Ve´lizy, France). Synthesis of R,S-β-Dodecyl Malolactonate. We placed 4 g (3 × 10-2 mol) of dry D,L-malic acid in a flask under nitrogen and cooled it in an ice bath. Trifluoroacetic anhydride (TFAA) (2 equiv, 6 × 10-2 mol) was added dropwise, and the mixture was stirred at 0 °C for 1 h and at room temperature for 3 h. The trifluoroacetic acid that formed and the excess TFAA were removed with a rotary evaporator to give the trifluoroacetate of malic acid anhydride as a white solid in a quantitative yield. This compound was degassed with nitrogen, and 1-dodecanol (5.6 g, 1.5 × 10-2 mol) was added. The mixture was stirred for 24 h at room temperature. The resulting oil was dissolved in ethyl acetate and was extracted three times with 1 M NaHCO3. The combined aqueous solutions were acidified to pH 2 with 10 N HCl. The aqueous layer was extracted three times with ethyl acetate, and the organic extract was dried over MgSO4 and evaporated to yield a colorless oil (yield ) 80%). The oil was dissolved in anhydrous THF, and Ph3P (1 equiv) in anhydrous THF was added under nitrogen at 0 °C. A DIAD solution (1 equiv) was then introduced under nitrogen at 0 °C. The mixture was stirred at 0 °C for 1 h and then at room temperature for 12 h. The solvent was evaporated off and the Ph3PdO that formed was precipitated in cold diethyl ether. The precipitate was filtered, and the

Bouclier et al. Et2O was evaporated off. The residue was purified by chromatography on silica gel (pure CH2Cl2). IR (cm-1): 1850 (CO lactone). 1H NMR (CDCl3, 300 MHz, δ): 0.85 (t, 3H, CH3), 1.05-1.2 (m, 20H, 10CH2), 1.7 (t, 2H, CH2), 3.5-3.8 (m, 2H, CH2 lactone), 4.2 (t, 2H, COO-CH2), 4.8 (m, 1H, CH). Synthesis of PEG-co-poly(ε-caprolactone-co-dodecyl β-malate). We added 0.2 g dry PEG 2 k (1 × 10-4 mol), 1.6 g ε-CL (9 × 10-3 mol), 0.29 g dodecyl malolactonate (1 × 10-3 mol), and 15 mg of stannous octoate to a Schlenk flask. After repeated vacuum evacuation and purging with nitrogen gas (three times), the reaction mixture was placed in an oil bath that was preheated to 115 °C, and polymerization was allowed to occur at 115 °C with stirring for 24 h under nitrogen. The reaction mixture was cooled to room temperature and was mixed with CHCl3, and precipitation was then carried out with large amounts of diethyl ether and petroleum ether (50:50). The resulting white powder was dried under vacuum at room temperature for 24 h. 1H NMR (CDCl3, 300 MHz, δ): 0.85 (t, 0.21H, CH3 of MLA), 1.05-1.2 (m, 1.4H, 10CH2 of MA), 1.4 (m, 1.24H, CH2 of ε-CL), 1.55-1.75 (m, 1.24H + 0.14H, CH2 of ε-CL + CH2 of MA), 2.3 (t, 1.24H, CH2 of ε-CL), 2.9 (m, 0.14H, CH2 of MA), 3.4 (s, 0.93, CH3 of PEG), 3.65 (s, 1.24H, CH2-CH2-O of PEG), 4.1 (t, 1.24H, CH2 of ε-CL), 4.4 (m, 0.14H, O-CH2 of MA), 5.5 (m, 0.07H, CH of MA). SEC (THF): Mn ) 12 400, Mw ) 14 900, Ip ) 1.2. Nanocapsule Synthesis. Preparation of PEG-co-poly(ε-caprolactone-co-dodecyl β-malate) or PEG-PLGA Nanocapsules by the Double Emulsion Method. Aqueous-core NCs were prepared by the water-in-oil-in-water solvent evaporation technique described by Perez et al.21 Briefly, 83 µL of an aqueous phase composed of 5 nmol siRNA (ERR-mix-siRNA, FAM-siRNA, or scramble siRNA) and 1 mg/ mL PEI was emulsified in 250 µL of ethyl acetate containing 20 mg of the copolymer PEG-PLGA or PEG-ε-caprolactone-malic acid (PEG-PCL/MA), by vortex mixing for 30 s and sonicating for 1 min (22%, Branson Digital Sonifier, Danbury, CT). We then added 1 mL of a 2% PVA (w/v) solution in water. The mixture was vortex mixed for 30 s, sonicated for 1 min, and poured into 25 mL of 0.3% PVA (w/v). The solution was stirred for 20 min with a magnetic stirrer, and the organic solvent was then evaporated off under a flow of nitrogen. NCs were rinsed twice in H2O MQ by centrifugation at 190 000g for 90 min at 4 °C and were finally dispersed in 1 mL of H2O MQ. Preparation of Polyisobutylcyanoacrylate (PIBCA) Nanocapsules by Interfacial Polymerization. We used the method that was previously developed in our laboratory for the entrapment of nucleoside reverse transcriptase inhibitors.22 Briefly, the aqueous phase (50 µL) that contained 5 nmol siRNA (ERR-mix-siRNA, FAM-siRNA, or scramble siRNA) and 1 mg/mL PEI was added to 380 mg Crodamol GTCC/CapmuL MCM (3:1 w/w) oil mixture and 70 mg of Crillet 4/Crill 4 (3:2 w/w) surfactant mixture. We obtained a microemulsion by vortex mixing the mixture for 1 min and by allowing the mixture to equilibrate during rotation for 10 min on a wheel. We then added 7.5 mg IBCA monomer and vortex mixed the preparation for 1 min. The NCs were rotated on a wheel overnight and were then centrifuged (37 000g, 40 min, 4 °C) on 1 mL of 1:10 PBS. The NCs were rinsed twice in 1:10 PBS and were dispersed in 1 mL of H2O. Physicochemical Characterization. Physical Parameters. We analyzed products by 1H nuclear magnetic resonance (NMR) spectroscopy by using a Bruker DPX300 FT-NMR spectrometer with the solvent peak used as a reference. Analyses were conducted on solutions of polymer in CDCl3. The molecular weight and molecular weight distribution of polymers were determined by size exclusion chromatography (SEC) at 30 °C on a system equipped with a guard column and two GMH HRM columns (Viscotek, Irigny, France) with THF at a flow rate of 1 mL · min-1 as the eluent. A differential refractometer and a double SEC detector (model 270 Dual, Viscotek) with RALS and viscosimeter in series were used to analyze samples. The data obtained were processed with OmniSEC software (Viscotek).

Nanocapsules of Estrogen Receptor Alpha siRNA We checked for the presence of the lactone function in the synthesized monomer by infrared (IR) spectroscopy. Spectra were recorded with an Impact 420 apparatus (Nicolet Instruments, Madison, WI). The size and zeta potential of the NCs were determined by photon correlation spectroscopy (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). Size was determined with samples that were diluted in water (1:50). For zeta-potential measurements, samples were diluted in 1 mM NaCl (1:50). Encapsulation Efficiency. The siRNAs were radiolabeled with 33P by the addition of 13.4 nmol [γ-33P]ATP (1.5 MBq) to 2 nmol ERR-mix-siRNA; T4 polynucleotide kinase (New England Biolabs, Richmond, CA) was used to transfer 33P from ATP to the 5′-OH of each strand of siRNA, according to the manufacturer’s instructions. The siRNAs were then precipitated to eliminate free [γ-33P]ATP. A mixture of [γ-33P]-labeled and unlabeled siRNAs (1:4) was incorporated into the NCs. After centrifugation (190 000g, 90 min, 4 °C) and the rinsing of the NC pellet, the radioactivity of the NCs (50 µL) and of the initial mixture of siRNAs (2 µL) was determined in Pico Fluor 15 (Perkin-Elmer, Courtaboeuf, France) with an LS6000TA liquid scintillation counter (Beckman-France, Villepinte, France). The encapsulation yield (%) corresponds to (radioactivity of NCs from the pellet)/(pellet + supernatant radioactivity) × 100. Determination of the Residual Amount of PVA and Drug Loading. The amount of PVA associated with PEG-PLGA and PEG-PCL/MA NCs was determined by a method that is based on the formation of complexes between two adjacent hydroxyl groups of PVA and an iodine molecule.23 We incubated 25 µL of the NC suspension with 2 mL of 0.5 M NaOH for 15 min at 60 °C. We neutralized the samples by adding 900 µL of 1 N HCl and adjusting the volume to 5 mL. We then added 3 mL of 0.65 M boric acid, 0.5 mL of I2/KI (0.05: 0.15 M), and 1.5 mL of water. Samples were incubated for 15 min, and absorbance was measured at 690 nm. A calibration curve was plotted under the same conditions with various concentrations of PVA. We determined the NC yield, by freeze drying the suspension after centrifugation in a Lyovac GT2 (Leybold-heraeus, Ko¨ln, Germany). We determined the amount of polymer by subtracting the mass of PVA from the total lyophilized mass. Drug loading (% w/w) is expressed as the percentage of the w/w ratio of drug to polymer. For PIBCA-NCs, drug loading was determined as previously described.22 Fluorescence Quenching Experiments. We investigated the location of siRNAs in NCs by carrying out collisional quenching of fluorescence studies with 5′-6-FAM-labeled siRNA and the hydrophilic quenching reagent, potassium iodide (KI), as previously described.24 We measured fluorescence after adding 1 M KI to 10 mM sodium bisulfite so as to obtain concentrations of 0-50 mM in the samples. We kept ionic strength constant and equivalent to a 1 M KI solution by adding potassium chloride (1 M KCl). We measured fluorescence intensity at 524 nm after excitation at 490 nm by using an F-2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Data were plotted according to the Stern-Volmer quenching equation

(F0/F) - 1)KSVQ where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, Q is the quencher concentration, and KSV is the Stern-Volmer quenching constant. Cell Culture. Human MCF-7 ER-positive breast cancer (BC) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Vervier, Belgium) that was supplemented with L-glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50 IU/mL), and 10% fetal calf serum (FCS) and was maintained at 37 °C under a humidified atmosphere containing 5% CO2. Before steroid treatment, cells were grown for 3 days in phenol-red-free DMEM supplemented with 10% charcoal-stripped FCS. Human HERB BC cells were established by a transfection with an expression vector that encoded the full-length human ERβ cDNA in ER-null MDA-MB-231 cells.25 These cells were grown in DMEM/F12 (Gibco, Carlsbad, CA) that was supplemented

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with 10% charcoal-stripped FCS, penicillin (50 IU/mL), streptomycin (50 IU/mL), sodium pyruvate (1 mM), and G418 (200 µg/mL) and was maintained at 37 °C under a humidified atmosphere containing 5% CO2. Transfection Experiments. MCF-7 cells were grown in DMEM that was supplemented with 10% charcoal-stripped FCS. They were transfected with ERR1-siRNA, ERR2-siRNA, ERR-mix-siRNA, or scramble siRNA at concentrations of 10-75 nM by the use of the HiPerfect transfection reagent (Qiagen) according to the manufacturer’s instructions. Total cell extracts were prepared 48 h later, as described below. Cytotoxicity Assay. MCF-7 cells were used to seed 96-well plates (104 cells/well). The plates were incubated for 24 h, and empty NCs were then added at concentrations of 0-500 µg/mL. The plates were incubated for 72 h, and cell viability was determined by MTT assay. Briefly, 20 µL of 5 mg/mL MTT (thiazolyl blue tetrazolium bromide, Sigma-Aldrich, Saint Quentin Fallavier, France) in PBS was added. The mixture was then incubated for 2 h at 37 °C. The medium was removed, and formazan crystals that were dissolved in 200 µL of DMSO were added. We measured the absorbance of converted dye, which is correlated with the number of viable cells, at 570 nm with a background subtraction at 650 nm by using a microplate reader (Metertech Σ 960, Fisher Bioblock, Illkirch, France). Release Kinetics. These experiments were conducted under two different sets of conditions: PEG-PLGA or PEG-PCL/MA NCs loaded with [γ-33P]-labeled siRNA were diluted in Hepes buffer (10 mM Hepes, 145 mM NaCl, pH 7.4) or DMEM, with or without 10% FCS, at a concentration equivalent to 50 nM siRNA. After incubation for various periods of time (1-96 h), samples were centrifuged at 190 000g for 1.5 h at 4 °C to separate NCs from free siRNA. The radioactivity of the buffer, medium, and NCs was measured by scintillation counting (see above). Uptake of Nanocapsules by MCF-7 Cells. MCF-7 cells were used to seed 12-well plates at a density of 5 × 104 cells/well. They were allowed to grow for 24 h, and 50 nM radiolabeled free siRNA or siRNA-loaded NCs were added and incubated with the cells for various periods of time (3-96 h) at 37 °C. The culture medium was then removed, and cells were rinsed twice with 2 mL of PBS each; the culture medium and PBS that were used for washing were then pooled. Cells were lysed by incubation in 500 µL of lysis buffer. The radioactivity in the cells and the medium was then counted. SDS-PAGE and Western Blotting. We obtained total cell extracts by resuspending cell pellets in 100 µL of HGDST lysis buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100) plus protease inhibitors (complete reagent, Roche Diagnostics, Indianapolis, IN) and incubating the mixture for 30 min at 2 °C. Samples were then boiled for 5 min with Laemmli sample buffer. Protein concentration was determined by the Biorad assay, which was modified as previously described for SDS-containing samples with BSA standards to which SDS was added in the same concentration as that in the samples.14 SDS-PAGE was conducted on 8% polyacrylamide gels. The protein bands were then electrotransferred onto a PVDF membrane (ImmobilonP, Millipore, Saint-Quentin en Yvelines, France). The nonspecific binding sites on the membranes were saturated by incubation for 1 h at 37 °C in 10% nonfat milk powder or 5% FCS in PBS that was supplemented with 0.1% Tween 20. ERR was detected with the rabbit HC20 antibody and Hsp70 was detected with the mouse W27 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), both used at a concentration of 0.1 µg/mL, and ERβ was detected with a rabbit polyclonal antibody that was used at a dilution of 1:5000. Appropriate horseradish peroxidase-conjugated secondary antibodies and Luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA) were used for detection. Protein signals were quantified by scanning densitometry with BioProfil V99 BIO 1D software from Vilbert Lourmat (VWR, Fontenay sous Bois, France).

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In Vivo Experiments. Subconfluent MCF-7 cells were treated with trypsin and were resuspended in DMEM at a density of (5-10) × 107 cells/mL. This suspension was mixed with an equal volume of Matrigel (BD Bioscience, Le Pont de Claix, France) and was injected (200 µL total) subcutaneously into the flanks of 7- to 8-week-old female nude mice (Janvier, Le Genest Saint Isle, France). These mice were housed in a room that was maintained at a temperature of 22 °C with a 12 h light/12 h dark light cycle. The mice had ad libitum access to food and water. After the injection, mice were treated weekly with E2 solution in ethanol, which was deposited on the skin (50 µg/mouse). Tumors developed over a period of 5-7 weeks and reached 300-500 mm3 (calculated as 1/2(width × length2)) in size. They were then transplanted (biopsies of 1 to 2 mm3) into recipient mice under the skin in the vicinity of the nipple. Tumors were allowed to grow until they reached 5-8 mm in diameter (6-8 weeks), and the mice were treated with E2 twice per week. Nanocapsule formulations were administered twice weekly by an intravenous injection into the retro-orbital sinus (200 µL, 330 µg/kg/week, 10 mice/group). The tumor volume was measured weekly and at the end of the experiment. The animals were killed, and their tumors were removed, were half-fixed in Finefix (Milestone s.r.l., Sorisole, Italy), and were frozen in liquid nitrogen. The tumor progression was evaluated as the ratio of the volume at each time point and the initial tumor volume (week 0). All experiments were conducted in accordance with the principles of the Helsinki Declaration and French animal welfare legislation. Immunodetection of ERr in Tumors from MCF-7 Cells. Paraffin sections (4 µm thick) were prepared from tumors fixed in Finefix. The paraffin was removed by incubation in xylene, the sections were rehydrated, and the immunohistochemical staining for ERR was performed. Sections were incubated with blocking serum (Biogenex buffer 1:10, San Ramon, CA) for 10 min and then with HC20 antibody (Santa Cruz) at a dilution of 1:50. Antibody binding was detected by incubation with a secondary antirabbit antibody (PowerVision Immunovision Technologies, Microm Microtech, Francheville, France) for 20 min, and the signal was detected with DAB (PowerVision). Slides were then counterstained with Mayer’s hematoxylin, were mounted (Pertex), and were visualized as described in the legend to Figure 8. Statistical Analysis. Student t tests were used to compare the effects of formulated and unformulated drugs. The difference was considered to be statistically significant if the p value was less than 0.05.

Results Efficiency of the Selected siRNAs. We initially transfected MCF-7 human BC cells with two siRNAs against ERR (ERR1-siRNA and ERR2-siRNA) that were designed to recognize two different sites in ERR mRNA, alone or in combination. Receptor levels were analyzed by Western blotting 48 h after transfection. Both siRNAs decreased ERR levels (Figure 1) by 60% at a low concentration (10 nM) (panel A), whereas scramble siRNA (which displayed no sequence matches to any known mammalian gene) did not have an effect at any concentration (panel B). The partial complementarity of siRNAs with mRNA molecules that were unrelated to the target sequence could have nonspecific effects (off-effects), so an equimolar mixture of the two ERR-targeting siRNAs was used to halve the risk of an off-effect. Equimolar amounts of the two siRNAs were mixed to enhance the inhibitory activity and were assayed in MCF-7 cells. At 10 nM, this equimolar mixture of siRNAs (ERR-mix-siRNA) decreased receptor levels by at least 85% (panel B) over a 5 day period, as shown by the scanning densitometry results for 10 different experiments (not shown). We assessed the specificity of ERR species recognition at the concentration that was required for a full effect by transfecting MDA-MB-231 cells that expressed ERβ (HERB) with 10 nM ERR-mix-siRNA (panel C). No change in ERβ expression

Bouclier et al.

Figure 1. Extinction of ERR expression in MCF-7 cells following transfection with siRNAs. MCF-7 cells were transfected (or not, control) with various concentrations (10-75 nM) of ERR1-siRNA or ERR2-siRNA (panel A), or an equimolar mixture of ERR1-siRNA and ERR2-siRNA called ERR-mix-siRNA or an equivalent concentration of scramble siRNA (panel B). HERB cells were transfected (or not, control) with 75 nM ERR-mix-siRNA (panel C). Cell extracts were prepared 48 h later, and ERR and ERβ were immunodetected as described in the Materials and Methods. Hsp70 was detected in parallel as a control for equal protein loading.

was observed, which confirmed the expected ERR specificity of the designed ERR-siRNA. Physicochemical Characteristics of siRNA-Loaded NCs. We investigated the optimal conditions for siRNA encapsulation by polymeric NCs by using two methods: the interfacial polymerization of isobutylcyanoacrylate (IBCA) and the double emulsion of a preformed polymer. In the first method, PIBCA NCs were directly prepared by interfacial emulsion polymerization of the corresponding cyanoacrylate monomer in an aqueous acidic medium in the presence of siRNA. The resulting NCs were heterogeneous in size and formed two distinct populations (Table 1), which was consistent with previous results that were obtained with NCs that were composed of the same copolymer.22 The encapsulation yield was low (11.8%), and the drug loading rate was 0.18% for these PIBCA NCs. Before the NCs were rinsed, 98% of the siRNA was associated with the NCs, as previously reported.4 Unfortunately, the encapsulation rate steeply fell to ∼12% after rinsing. A method for increasing encapsulation yields involves the preparation of NCs by the double emulsion technique that uses a presynthesized polymer. We first used a PEG-PLGA copolymer. A single population of NCs was obtained (∼200 nm in diameter) with this copolymer (Table 1), whatever the initial concentration of siRNA that was used. Larger amounts of siRNA were incorporated into PEG-PLGA NCs (29%) than with the PIBCA method (12%) (Table 1); nonetheless, yields remained too low for efficacy in cells to be expected. We then tried to increase the affinity of siRNA for NCs by synthesizing a new copolymer by random copolymerization of ε-caprolactone (ε-CL) and RS-β-dodecyl malolactonate (Figure 2). A malate unit with a long hanging dodecyl group was introduced to favor encapsulation through hydrophobic interactions. In 2005, ChanPark’s group26 reported the successful copolymerization of ε-CL with benzyl malolactonate at various monomer ratios. We copolymerized dodecyl malolactonate with ε-CL under similar experimental conditions, with poly(ethylene glycol)methyl ether

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Table 1. Characteristics of the Nanocapsule Formulationsa NCs PIBCA PEG-PLGA PEG-PCL/MA

siRNA

mean size (nm)

zeta potential (mV)

+ + +

104 ( 19, 675 ( 143 91 ( 16, 608 ( 120 199 ( 54 196 ( 58 118 ( 55 105 ( 73

-30.3 ( 3.9 30.6 ( 3.9 -3.4 ( 5.2 -3.5 ( 4.2 -9.4 ( 5.7 -9.1 ( 6.1

charge yield (%)

encapsulation (µM)

drug loading (%)

11.8

0.6

0.177

28.6

1.43

0.164

72

3.3

0.8

a

Size, zeta potential, polymer, and encapsulation yield of NC formulations loaded with or without siRNA are shown. Each measurement was carried out in triplicate on at least four preparations. Results are means ( SD.

Figure 2. Synthesis of the PEG-PCL/MA polymer.

2 k as the initiator and stannous octoate as the catalyst at 115 °C. The presence of a PEG sequence at the beginning of the copolymer chain should prevent protein adsorption and result in the formation of a biocompatible polymeric nanocarrier shell, as previously reported.27,28 We determined the relative content of dodecyl malolactonate by 1H NMR spectroscopy by comparing the area of the methine group of the malate unit with the area of the methylene proton of the CL unit. In all cases, the composition of the copolymer was found to be representative of the initial proportions of each monomer. SEC characterization of the resulting copolymer showed a monomodal narrow mass distribution (Mw/Mn ) 1.2). Assuming that each chain was initiated by a PEG group, we calculated an experimental molar mass of 11 900 g · mol-1 on 1H NMR, a value lying within one standard error of the mean of 12 400 g · mol-1 that was determined by SEC. These values are smaller than the predicted molecular weight (Mn,theor ) 15 100 g · mol-1), which is probably because of monomer transfer reactions, as reported for β-lactones.29 The NCs that were obtained with this new copolymer were smaller than those that were obtained with PEG-PLGA (Table 1). However, siRNAs were encapsulated within these new NCs, which were obtained in a high yield (72%). Whether unloaded or loaded, the zeta potential of the various NCs remained stable, which indicates the incorporation of the siRNA within the water core of the nanoparticles rather than adsorption to the surface (Table 1). Cytotoxicity. We investigated the toxicity of NCs. We allowed the MCF-7 cell number to double twice (72 h) and showed that the exposure of these cells to empty PIBCA NCs resulted in high levels of toxicity, as previously observed.30 Indeed, MCF-7 cell viability was strongly affected in a dosedependent manner by incubation with various amounts of PIBCA NCs for only 24 h (Figure 3A), except at the lowest concentration (5 µg polymer/mL).On the basis of the rate of encapsulation of siRNAs into such NCs, this concentration corresponded to 0.6 nM siRNA, a concentration that is too low for any cellular effect to be expected. The other two NC formulations were not toxic in MCF-7 cells because 80% of the cells survived, even after 72 h of incubation (Figure 3B). We used PEG-PCL/MA NCs for most subsequent experiments because these NCs displayed the highest levels of siRNA incorporation. Localization of siRNA in the Aqueous Core of the Nanocapsules. We localized siRNA molecules in NCs by collisional quenching of fluorescence using KI as the quencher, as previously described.24 Stern-Volmer plots revealed that free 5′-6-FAM-siRNA (FAM-siRNA) was accessible to KI because strong fluorescence extinction was observed under these

Figure 3. MCF-7 cell viability following exposure to various NC formulations. MCF-7 cells were used to seed 96-well plates, which were incubated with 0-500 µg/mL polymer for (A) 24 h for PIBCA NCs or (B) 72 h for PEG-PLGA and PEG-PCL/MA NCs. Cell viability was determined by the MTT assay. Results are expressed as the mean ( SEM obtained for at least three independent experiments performed in triplicate.

conditions (Figure 4). This was reflected by a high Stern-Volmer constant, KSV ) 61.5 M-1. If free FAM-siRNAs were mixed with empty PEG-PCL/MA NCs, then no significant difference in KSV constant was observed between free FAM-siRNA and the empty NCs (data not shown). By contrast, significantly lower levels of fluorescence extinction were observed when fluorescent siRNA was incorporated into PEG-PCL/MA NCs, with a KSV of 35 M-1 (Figure 4). In this case, the siRNA was protected within the aqueous core of the NCs and was therefore inaccessible to KI. However, this protection was not complete because KSV was not equal to zero, which suggests that some of the siRNA remained accessible. Nevertheless, siRNA incorporation had no effect on the zeta potential (Table 1), which indicates an absence of detectable siRNA adsorption. Complementary experiments showed that the dilution of NCs in water or in 1 M KCl (conditions similar to those of the quenching experiment) led to the release of 20-25% of the siRNA (data not shown),

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Figure 4. Fluorescence quenching of 6-FAM-siRNA. KI Stern-Volmer quenching of 5′-6-FAM-labeled siRNA was conducted with free siRNA, siRNA-loaded PEG-PCL/MA NCs, and siRNA mixed with empty PEG-PCL/MA NCs. Results are shown as the mean ( SEM of three independent experiments.

which accounts for the fluorescence quenching values that were obtained for siRNA-loaded NCs. Release Kinetics. We studied the release of trapped siRNA from NCs by incubating NCs that were loaded with radiolabeled ERR-mix-siRNAs at 37 °C for various periods of time in Hepes buffer or in cell culture medium with or without 10% FCS. Low levels of siRNA release from the PEG-PLGA NCs were observed in Hepes buffer (Figure 5A; 10% at 96 h). Release levels were higher in the presence of FCS but remained low (30% at 96 h). We concluded that Hepes buffer does not represent optimum conditions, even with protein supplementation, for the release measurement of encapsulated siRNA in this formulation. In DMEM, the addition of proteins again increased the siRNA release, which reached 50% at 96 h (Figure 5B), but in this case, a burst release was obtained during the first hour (up to almost 40%). Encapsulated siRNAs were released more rapidly from PEG-PCL/MA NCs than from PEG-PLGA in both the presence and absence of added proteins (Figure 5C,D). With this formulation, a 1 to 2 h burst release was still observed (from 30 to 70%) which may correspond to the leak of a subpopulation of siRNA that was entrapped at the vicinity of the external polymeric envelope. After this burst, only 10% of the radioactivity was released from NCs up to 96 h regardless of the type of NC that was considered. In results that are not shown here, we observed no increase in siRNA release even after 19 days. Cellular Uptake of Free and Entrapped siRNA. The incorporation of siRNAs into nanovectors should increase their uptake by cells. We checked this by incubating MCF-7 cells with radiolabeled siRNAs, which were free or incorporated into PEG-PCL/MA or PEG-PLGA NCs, for different periods of time. Entrapment in NCs increased the uptake of siRNA (Figure 6). The uptake of PEG-PCL/MA NCs was almost twice as rapid as that of free siRNA, as shown by the values that were obtained at 96 h (12.7 vs 7.6%, respectively). This formulation was more efficient than PEG-PLGA NCs. These observations suggest that PEG-PCL/MA NCs would be more effective at delivering siRNA to cells and would therefore also have a stronger inhibitory effect. Moreover, when comparing the release kinetics with cellular uptake, we found no correlation that suggested that free siRNA are entering cells by a mechanism that is different from that mediated by NCs. Extinction of ERr in MCF-7 Cells Exposed to ERr-siRNALoaded NCs. We then assessed the efficiency with which ERR-mix-siRNA-loaded NCs abolished ERR expression in MCF-7 cells. We did not carry out experiments with PIBCA NCs because of the insufficient loading and toxicity that were observed at concentrations that are required to deliver 50 nM siRNA (Figure 3A). Experiments that were carried out with

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PEG-PLGA NCs showed no ERR loss at concentrations of 10 and 50 nM siRNA (Figure 7A), which is consistent with the slow release of trapped siRNA (Figure 4) and the low rate of cellular uptake (Figure 6). In contrast, ERR-mix-siRNAloaded PEG-PCL/MA-NCs induced a strong decrease in receptor levels at a siRNA concentration of 50 nM, and this receptor loss persisted for >5 days in MCF-7 cells (Figure 7B). This inhibition corresponds to a decrease in ERR levels of at least 85% from initial levels, as deduced from three scanning densitometry experiments (not shown). No decrease in ERR levels was observed with encapsulated scramble siRNA or with a physical mixture of free ERR-siRNA and empty PEG-PCL/ MA NCs (Figure 7C). Again, these data indicate that the cellular captures of free and encapsulated siRNAs are different processes and that NCs facilitate the siRNA uptake. These data are consistent with the long-lasting inhibition by encapsulated ERR-mix-siRNA that were released from PEG-PCL/MANCs and justify the choice of this formulation for further biological evaluation in vivo. Efficiency of ERr-siRNA PEG-PCL/MA NCs in Breast Cancer Cell Xenografts. Estrogen-dependent MCF-7 xenografts were generated in female nude mice. PEG-PCL/MA NCs loaded with either ERR-mix-siRNA or scramble siRNA were intravenously injected into mice twice weekly at a dose of 330 µg/kg/week siRNA. We assessed tumor growth weekly by measurement with an electronic caliper. Tumor growth was not affected by scramble siRNA incorporated into NCs (Figure 8A). In contrast, NCs loaded with ERR-mix siRNA caused a slight slowing of tumor growth, whereas free siRNAs had no effect (data not shown). We note that during the 4 weeks of treatments no deaths occurred in any animal group. ERR immunostaining in tumor cells was strong in the nucleus of most control tumor cells (Figure 8B) and in tumor cells of animals that were treated with scramble siRNA-NCs (Figure 8C). However, significantly lower levels of ER labeling were observed in cells from tumors treated with siRNA-loaded-NCs (Figure 8D), which is consistent with slower tumor growth (Figure 8A). These tumors also contained lower numbers of cells and displayed extensive apoptotic/necrotic areas and large banks of fibrotic tissue.

Discussion Technical challenges remain to be overcome before RNAi can be accepted as a gene-silencing tool. Once effective siRNA sequences have been designed and identified, it is essential to ensure that they deliver the siRNA to the target cells in specific tissues. Several strategies have been developed for introducing siRNAs into cells on the basis of intracellular expression by using small hairpin RNA31 or the direct introduction into the cells by transfection.32-34 The biological efficiency of the transfection method is limited by the instability of siRNA in biological fluids and poor intracellular penetration. Nanocarrier systems can be used to improve the intracellular delivery.2-4 Therefore, we incorporated synthetic siRNAs that target the noncoding region of the ERR gene into various stealth nanosystems. We improved the efficiency of these siRNAs for abolishing ERR expression by mixing two duplexes of oligonucleotides that give persistent and specific inhibition of ERR expression. Because the ERR species is considered to be responsible for the mitogenic activity of estradiol in hormonedependent cancers,35-37 whereas ERβ is thought to act as a tumor suppressor,38,39 it was crucial that the siRNA mixture affected ERR but not ERβ. This specificity was confirmed (Figure 1C).

Nanocapsules of Estrogen Receptor Alpha siRNA

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Figure 5. Kinetics of siRNA release from the different NC formulations. [γ-33P]-labeled ERR-mix-siRNA-loaded PEG-PLGA NCs (panels A and B) or [γ-33P]-labeled ERR-mix-siRNA-loaded PEG-PCL/MA NCs (panels C and D) were incubated for various periods of time (from 1 to 96 h) at 37 °C with 500 µL of Hepes, with (O) or without (b) 10% FCS (panels A and C) or with 500 µL of DMEM with (0) or without (9) 10% FCS (panels B and D). At the indicated times, radioactivity was measured in Hepes buffer, cell culture medium, and intact NCs that were obtained by ultracentrifugation. The percent radioactivity release is plotted. Each point is the mean ( SEM of three measurements from two independent experiments.

Figure 6. Uptake of NCs by cells. MCF-7 cells (5 × 104/well) were incubated with 50 nM radiolabeled siRNAs that were incorporated into NCs that were composed of PEG-PLGA (9) or PEG-PCL/MA (2) or were incubated with free radiolabeled siRNAs (b) for various periods of time (3-96 h). Radioactivity was then measured in cell culture medium and in cells. The amount of siRNA (pmol/5 × 104 cells) that was measured in pelleted cells is plotted. Each point is the mean from two independent experiments. The values indicate the percentages of radiolabeled siRNA that were incorporated at 96 h.

Our attempts to encapsulate these ERR-siRNAs had various degrees of success. Although they were initially designed to incorporate siRNAs that target the EWS-Fli1 oncogene in sufficient amounts to inhibit tumor growth in xenografts,4 PIBCA-NCs incorporated our ERR-siRNA mixture with a low level of efficiency. Moreover, this formulation was highly toxic to MCF-7 BC cells, which precluded further studies. Nanocapsules that were based on PEG-PLGA incorporated higher concentrations of siRNAs. However, pegylated copolymers that contained ERR-siRNA had no effect on the expression of the receptor (Figure 7A) on MCF-7 cells. This lack of effect may have been due to low levels of siRNA release, low levels of uptake by cells, or both and thereby prevented siRNA from accumulating in cells in sufficiently large amounts to induce a detectable effect. We therefore focused on a new PEG-PCL/ MA copolymer that contained a long hanging hydrophobic tail

Figure 7. Expression of ERR in MCF-7 cells that were exposed to ERR-siRNA-loaded NCs. (A) MCF-7 cells were or were not exposed to PEG-PLGA NCs loaded with ERR-mix-siRNA to a concentration that was equivalent to 10 or 50 nM siRNA for 72 h. (B) MCF-7 cells were or were not exposed to PEG-PCL/MA NCs loaded with ERR-mix-siRNA or scramble siRNA for 48-144 h. (C) ERRmix-siRNA incorporated into PEG-PCL/MA NCs or mixed with empty PEG-PCL/MA NC was incubated with MCF-7 cells for 72 h. Equivalent amounts of cell extract (30 µg protein equivalent) were run on denaturing gels. ERR and hsp70 were detected as described in the experimental procedures.

with the aim of increasing the incorporation of larger amounts of siRNA through hydrophobic interactions. With this formulation, 2 to 3 times as much ERR-siRNA was incorporated into the capsules as with PEG-PLGA, which resulted in sufficiently high concentrations for a biological effect to be expected. Furthermore, the PEG-PCL/MA copolymer displayed no signs of toxicity when it was incubated with MCF-7 cells for 6 days (not shown). We assessed the ability of NCs to deliver the incorporated siRNA efficiently by evaluating their release capacities in different media. In all cases, the presence of proteins increased

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Figure 8. Efficiency of ERR-mix-siRNA-loaded PEG-PCL/MA NCs in BC cell xenografts. (A) Mice bearing MCF-7 xenografts received two intravenous injections per week (O) of ERR-mix-siRNA (9) or scramble siRNA (2) incorporated into PEG-PCL/MA NCs (330 µg/kg/week). Tumor growth (tumor volume vs initial tumor volume) ( SEM is shown (*p ) 0.0008 as compared with the untreated group). ERR immunostaining of tumor sections after 4 weeks of injections, or in the absence of injection (panel B), of scramble siRNA (panel C) or ERR-mix-siRNA (panel D) incorporated into PEG-PCL/MA NCs was carried out as described in the experimental procedures. Sections were observed under a microscope at 100× original magnification.

siRNA release. As observed in Figure 5, approximately 35% of the total siRNA loaded within the PEG-PCL/MA NCs is released over the 96 h incubation period in the absence of serum. Noteworthily, the addition of serum accelerates the release to 75% with no significant higher release at 19 days (data not shown). It has already been observed in various studies40,41 that the presence of serum proteins may influence the release of drug from the nanocarriers as a result of protein/drug interactions. Apparently, the serum proteins act as scavengers that bind the released siRNA and thus accelerate its diffusion from the NCs. Whatever the medium used, ERR-siRNA release rates were always higher from PEG-PCL/MA than from PEG-PLGA NCs. This difference may be due to the smaller size of PEG-PCL/MA NCs (∼100 nm) than of PEG-PLGA NCs (∼200 nm). Indeed, for the same amount of polymer, PEG-PCL/ MA NCs should have a larger surface area to volume ratio than PEG-PLGA NCs, which facilitates an exchange with the external medium. Moreover, the higher encapsulation rate that is obtained with PEG-PCL/MA NCs than with PEG-PLGA NCs would also be expected to be associated with a higher release rate. A burst of siRNA release followed by a period of slower release was observed with both PEG-PLGA and PEG-PCL/ MA NCs containing radiolabeled ERR-siRNA (Figure 5, panels B-D). This behavior may result from the formed NCs not having a core/shell structure with a single water core and instead being formed by a multitude of droplets containing the aqueous phase with the siRNA inside. This morphology has already been observed for PLGA microspheres that were synthesized by multiple emulsion techniques.42 It has not yet been possible to

demonstrate such a morphology for our NCs because of their small size (