Polymeric Nanocarriers for Magnetic Targeted Drug Delivery

Oct 29, 2013 - University of Palermo, Via Archirafi, 32 90123 Palermo, Italy. ‡. Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”,...
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Polymeric Nanocarriers for Magnetic Targeted Drug Delivery: Preparation, Characterization, and in Vitro and in Vivo Evaluation Mariano Licciardi,† Cinzia Scialabba,† Calogero Fiorica,† Gennara Cavallaro,† Giovanni Cassata,‡ and Gaetano Giammona*,†,§ †

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Department of Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Laboratory of Biocompatible Polymers, University of Palermo, Via Archirafi, 32 90123 Palermo, Italy ‡ Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, 90129 Palermo, Italy § IBF-CNR, via Ugo La Malfa, 153, 90143 Palermo, Italy S Supporting Information *

ABSTRACT: In this paper the preparation of magnetic nanocarriers (MNCs), containing superparamagnetic domains, is reported, useful as potential magnetically targeted drug delivery systems. The preparation of MNCs was performed by using the PHEA-IB-p(BMA) graft copolymer as coating material through the homogenization−solvent evaporation method. Magnetic and nonmagnetic nanocarriers containing flutamide (FLU-MNCs) were prepared. The prepared nanocarriers have been exhaustively characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and magnetic measurements. Biological evaluation was performed by in vitro cytotoxicity and cell uptake tests and in vivo biodistribution studies. Magnetic nanocarriers showed dimensions of about 300 nm with a narrow size distribution, an amount of loaded FLU of 20% (w/w), and a superparamagnetic behavior. Cell culture experiments performed on prostate cancer cell line LNCaP demonstrated the cytotoxic effect of FLU-MNCs. In vivo biodistribution studies carried out by the application of an external magnetic field in rats demonstrated the effect of the external magnet on modifying the biodistribution of FLU-MNCs. FLU-MNCs resulted efficiently internalized by tumor cells and susceptible to magnetic targeting by application of an external magnetic field. The proposed nanocarriers can represent a very promising approach to obtain an efficient magnetically targeted anticancer drug delivery system. KEYWORDS: magnetic nanocarrier, magnetic targeting, flutamide, superparamagnetic nanoparticles

1. INTRODUCTION One of the most important challenges in cancer chemotherapy is the design of a more efficient drug delivery system with the objective of targeting drugs to specific organs or tissues of the body to improve the therapeutic index and minimize or eliminate the undesirable side effects. In this regard, a particularly attractive technology, that in the past few years has showed a growing interest, is the magnetic drug delivery system.1 Several types of magnetic materials, such as iron oxides (Fe2O3 and Fe3O4), metal alloys (Fe, Co, and Ni), and iron cobalt alloy, have been widely studied for magnetic drug delivery.2 Among these materials, magnetite (Fe3O4, single domains of about 5−20 nm), a common magnetic iron oxide, is a very promising candidate for its biocompatibility and biodegrability. It was demonstrated that magnetic nanoparticles (MNPs), after in vivo administration, can be metabolized, and the released free iron ions included to the physiologic iron turnover (for example incorporated in erythrocytes hemoglobin) and thus eliminated by the normal iron recycling pathways.3,4 The main advantages of MNPs, compared to © 2013 American Chemical Society

their bulk counterparts, are not only their high specific surface areas, low sedimentation rate, and reduced magnetic dipole− dipole interaction, but especially their magnetic behavior. In fact, MNPs having dimensions below 20 nm exhibit superparamagnetic behavior (superparamagnetic iron oxide nanoparticles, SPIONs). They magnetize strongly when an external magnetic field is applied, but no residual magnetic forces exist between the particles upon the removal of the magnetic filed.5 This behavior makes them suitable for use experimentally for numerous in vitro applications, such as cell separation experiments,6,7 and in vivo applications such as magnetic resonance imaging (MRI) contrast enhancement,8,9 hyperthermia, and drug delivery2,10 and targeting.11 Moreover, although MNPs are highly biocompatible, their high superficial area and hydrophobic surfaces may result in Received: Revised: Accepted: Published: 4397

December 19, 2012 August 22, 2013 October 29, 2013 October 29, 2013 dx.doi.org/10.1021/mp300718b | Mol. Pharmaceutics 2013, 10, 4397−4407

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(SPIONs) (10 ± 1 nm) in toluene (magnetization emu/g, at room temperature under 4500 Oe), polyvinyl pyrrolidone (PVP), glyceryl monostearate, sodium dodecyl sulfate (SDS), flutamide (FLU) hydroxyflutamide (FLU−OH), dihydrotestosterone (DHT), L-ascorbic acid sodium salt, bovine serum albumin, ferrozine (3-(2-pyridyl)-5,6-bis(phenyl sulfonic acid)1,2,4-triazine), and neocuproine (2,9-dimethyl(1,10-phenanthroline)) were purchased from Aldrich (Italy) and were used as received. Potassium thiocyanate was purchased from Carlo Erba Reagents (Italy). SpectraPor dialysis tubing was purchased from Spectrum Laboratories, Inc. (Italy). 2.1. Synthesis of α,β-Poly(N-2-hydroxyethyl)-D,L-aspartamide-co-(N-2-ethylen-isobutirrate)-graf t-poly(butyl methacrylate) (PHEA-IB-p(BMA) Copolymer). Derivatization of PHEA with 2-bromoisobutyryl bromide (BIB) to obtain a PHEA-BIB multifunctional macroinitiator was carried out using the previously described protocol.29 The product was obtained with a yield of 95 wt %, based on the starting PHEA. The degree of derivatization (DD), determined by 1H NMR spectroscopy in D2O and calculated according to the method reported elsewhere,29 was 30 mol %. The homopolymerization of butyl methacrylate, using PHEA-BIB as the macroinitiator, was carried out according to a previously reported procedure,30 by modifying some reaction parameters. Briefly, the reaction of PHEA-BIB with butyl methacrylate (being molar ratio between butyl methacrylate and BIB residue equal to 10) was carried out in a previously degassed 1:1 DMF/water (v/v) solvent mixture at 50 °C for 20 h; CuIBr catalyst (25.5 mg, being the molar ratio between CuIBr and BIB linked group equal to 1) and bpy ligand (101 mg, being the molar ratio between bpy ligand and BIB linked group equal to 4) were then added to the flask under argon. Reaction was stopped by keeping reaction mixture in contact with air oxygen until the complete oxidation of copper. The reaction mixture was added dropwise into double-distilled water, and the resulting solid residue was washed twice in a 1:1 H2O/MeOH solvent mixture. The white residue, obtained after centrifugation, was suspended in double-distilled water and purified through exhaustive dialysis using a SpectraPor dialysis tubing with 12 000−14 000 molecular weight cutoff. After dialysis the dispersion was freeze-dried from water. Obtained PHEA-IB-p(BMA) copolymer was characterized by 1H NMR, and spectroscopic data were in agreement with the previous results.30 1H NMR (300 MHz, DMSO-d6, 25 °C, TMS, δ): 0.77 (m, 3H, CH3)BMA, 0.90 (m, 3H, CH3)BMA, 1.29 (m, 6H, CH3)IB, 1.36 (m, 2H, CH2)BMA, 1.56 (m, 2H, CH2)BMA, 1.88 (s, 6H, CH3)BIB, 2.70 (m, 2H, CH2)PHEA, 3.16 (m, 2H, CH2)PHEA, 3.41 (m, 2H, CH2)PHEA, 3.90 (m, 2H, CH2)PHEA, 4.13 (m, 2H, CH2)PHEA, 4.59 (m, 1H, CH)PHEA. 2.2. Preparation of PHEA-IB-p(BMA) Superparamagnetic Nanocarriers (MNCs). MNPs were prepared starting from PHEA-IB-p(BMA) graft copolymer by the homogenization−solvent evaporation method. An organic phase was prepared by dispersing PHEA-IB-p(BMA) graft copolymer (typical concentration: 40 mg/mL) in 1 mL of chloroform containing glyceryl monostearate (40 mg) and 10 nm Fe3O4 SPIONs stabilized with SDS (5 mg). Non magnetic nanocarriers were prepared in the same conditions but without Fe3O4 SPIONs. This organic solution was then added under stirring to 50 mL of an aqueous phase, containing PVP (1.5% w/v) and Pluronic F68 (0.25% w/v), and a stable o/w emulsion was obtained by using an Ultra-Turrax (T 25, Janke and Kunkel Ika−Labortechnik) for 20 min at 24 000 rpm. The

aggregation and cause also their uptake by the body reticuloendothelial system. Therefore, it is necessary to coat MNPs with a suitable polymer to improve their stability and their circulation time. MNP coatings to date explored comprise several polymer materials, including both synthetic and natural polymers.5 Recent examples of synthetic polymers used to coat MNPs and increase their stability, circulation half-life and biocompatibility are poly(ethylene imine)-g-poly(ethylene glycol),12 poly(lactic acid), poly(ethylene glycol), polyvinyl alcohol,13 and poly(ethyl-2-cyanoacrylate).14,15 Meanwhile natural polymers include gelatin,16 dextran, chitosan,13 and pullulan.13,17 The polymer coating not only stabilizes the nanoparticles but also provides them active functional groups for controllable bioconjugation of targeting ligands.18 The targeting by drug delivery systems containing iron oxides such as polymeric vesicles, 19 magnetic nanoparticles, 20,21 magnetic liposomes,22−24 and magnetic lipid nanoparticles25,26 have been studied by several research groups. All of these magnetic systems are usually composed of a magnetic core and a biodegradable shell. The magnetic core consisting of iron oxide particles, which are responsible for the magnetic property, could target the system to a specific site by the action of the external magnetic field. However, the polymeric shell could entrap drug molecules to transport and release them during its biodegradation process. Thus, the side effects of the drug could be reduced due to its lower level in the general circulation. In this paper, a novel approach to prepare polymeric magnetic nanocarriers, MNCs, containing superparamagnetic domains into a polymeric matrix is reported. PHEA-IBp(BMA) was used as biocompatible copolymer to constitute the matrix containing Fe3O4 superparamagnetic domains by emulsifying a chloroform polymer solution in the simultaneous presence of SPIONs in water. FLU-MNCs were prepared by adding the chloroform drug solution to the polymer solution in the same solvent. The obtained nanocarriers were characterized by TEM, DLS analysis, and magnetic measurement studies. The cytotoxicity profile and in vitro ability to deliver drug into human metastatic prostate adenocarcinoma (LNCaP) cells were also evaluated. Finally, in vivo biodistribution of drug and Fe2O3 loaded into nanoparticles, in rats subjected to an external magnetic field, was also assessed.

2. MATERIALS AND METHODS α,β-Poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was prepared and purified according to the previously reported procedure.27,28 Spectroscopic data (FT-IR and 1H NMR) were in agreement with attributed structure: 1H NMR (300 MHz, D2O, 25 °C, δ): 2.82 (m, 2H, −CH−CH2−CO−NH−), 3.36 (t, 2H, −NH−CH2−CH2−OH), 3.66 (t, 2H, −CH2− CH2−OH), 4.72 (m, 1H, −NH−CH−CO−CH2−).27,28 The PHEA average molecular weight was 48.0 kDa (Mw/Mn = 1.66) based on PEO/PEG standards, measured by size exclusion chromatography (SEC). The 1H NMR spectra were recorded in D2O or DMSO-d6 (Aldrich) using a Bruker Avance II 300 spectrometer operating at 300 MHz. Centrifugations were performed using a Centra MP4R IEC centrifuge. Triethylamine (TEA), SEC polyethylene glycol standards, and methanol (MeOH) were purchased from Fluka (Switzerland). 2-Bromoisobutyryl bromide (BIB), butyl methacrylate (BMA), 2,2′-bipyridine (bpy, 99%), copper(I) bromide (CuIBr 99.999%), dimethylacetamide (DMA), dimethylformamide (DMF), iron oxide (Fe3O4) superparamagnetic nanoparticles 4398

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membrane filter. The intensity-average hydrodynamic diameter and polydispersity index (PDI) were obtained by cumulant analysis of the correlation function. The zeta potential (mV) was calculated from the electrophoretic mobility using the Smoluchowsky relationship and assuming that Ka ≫1 (where K and a are the Debye−Hückel parameter and particle radius, respectively). FT-IR Analysis. FT-IR spectra of MNCs copolymer, MNCs, and solid iron oxide nanoparticles were recorded in KBr pellets in the frequency range of 4000−400 cm−1 by using a PerkinElmer Spectrum RX I FT-IR System spectrophotometer. Spectra were recorded in transmittance scale (%T) with a resolution of 1 cm−1 and a number of scans = 100. 2.4. Determination of Loaded Drug Amount into Magnetic Nanocarriers and Drug Release Studies. The amount of FLU loaded into FLU-MNC and FLU-NC samples was determined by HPLC analysis, using a Waters Breexe System Liquid Chromatograph equipped with a Waters 717 Plus Autosampler (40 μL injection volume) and using a Shimadzu UV−vis HPLC detector on line with a computerized workstation, monitored at 300 nm. As column a reversed-phase Gemini C18 Phenomenex (5 μm, 4.6 × 250 mm column with a precolumn H5ODS-10CS) was used. The used mobile phase was: H2O/acetic acid/triethylamine/methanol (38/1/0.02/61 v/v), flow 1 mL/min. Before analysis, the drug was extracted by disperding known amount of nanocarrier samples in methanol. A calibration curve of standards of FLU was used for drug determination. For drug release studies, an appropriate amount (5 mg) of dried FLU-MNC, FLU-NC samples and FLU alone (1 mg, as positive control) were suspended in bidistilled water (20 mL) and transferred inside of a Spectra/Por dialysis membrane (MWCO 12 000−14 000 Da). This dialysis membrane was immersed into PBS at pH 7.4 (80 mL) and incubated at 37 °C under continuous stirring (100 rpm) in a Benchtop 808C Incubator Orbital Shaker model 420. At scheduled times, aliquots of external medium (1 mL) were withdrawn from the outside of the dialysis membrane and replaced with equal amount of fresh medium. The withdrawn samples were analyzed by HPLC in order to determine the released drug amount. Profile releases were determined by comparing the amount of released drug as a function of incubation time with the total amount of drug loaded into the nanoparticles. Data were correct taking in account the dilution procedure. Each experiment was carried out in triplicate, and the results were in agreement within ±5% standard error. 2.5. In Vitro Cytotoxicity and Cellular Uptake of DrugLoaded Magnetic Nanocarriers. Human metastatic prostate adenocarcinoma (LNCaP) cells were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS), 1 mM sodium pyruvate, 1 mM glutamine, and 1% antibiotics (50 mg/mL penicillin and 50 mg/mL streptomycin), at 37 °C in a humidified atmosphere and 5% CO2 in air. For hormone responsiveness experiments, fetal bovine serum charcoal stripped (ZSD), a commercially processed serum of bovine origin, was used instead of FBS. In ZSD the concentration of steroids is reduced by charcoal filtration by approximately 20-fold respect to regular FBS. For the experiments in the presence of dihydrotestosterone (DHT), LNCaP cells were previously growth in 96-well plates at a density of 1 × 105 cells/mL in RPMI-1640 with 5% (v/v) ZDS for 96 h. After 96 h cell growth the medium was replaced with RPMI-1640 with 5% (v/v) ZDS and cell incubated with

temperature was maintained near 3 °C by means of a ice bath. This emulsion was broken by evaporating the organic phase under reduced pressure at room temperature, and as a consequence of this process, nanoparticle hardening occurred. Obtained nanoparticles were purified by dialysis for 48 h, using a dialysis tube with cutoff 100 kDa. Finally, nanoparticles were dried by means of a Modulyo freeze-dryer (Labconco Corporation, Kansas City, MO). An analogous procedure was adopted for the preparation of FLU-loaded magnetic (FLU-MNCs) and FLU-loaded nonmagnetic (FLU-NCs) nanocarriers. In this case FLU (180 mg) was dissolved in 1 mL of chloroform, and this solution was added to the polymeric organic phase before obtaining the primary microemulsion. 2.3. Characterization of MNCs. Transmission Electron Microscopy (TEM) Analysis. The aqueous dispersion of the nanocarriers, in distilled water, was put on a copper grid, and the samples were let to dry spontaneously at room temperature overnight. The samples were analyzed using a JEM-2100 LaB6 transmission electron microscope operating at an accelerating voltage of 200 kV, equipped with a Multi Scan CCD camera. Total Iron Determination. The iron content in the nanocarriers was determined spectrophotometrically using a method elsewhere reported based on the formation of the highly colored complexes iron−thiocyanate ion.16 First, 5 mg of nanoparticles was completely dissolved in 1 mL of 30% (v/v) HCl for 2 h at 60 °C. Then, 0.1 mL of hydrogen peroxide (H2O2) water solution (35% w/w) was added to oxidize the ferrous ions (Fe2+) present in the above solution to ferric ions (Fe3+), and subsequently, 1.5 mL of 0.1 M solution of potassium thiocyanate was added to this solution to form the red colored iron−thiocyanate complex. The iron concentration was determined by recording absorbance at 478 nm using a Shimadzu UV-2401PC spectrophotometer. For quantization, a standard curve for the iron complex was made under identical conditions using known amount of Fe3O4 standard nanocarriers. Magnetic Properties. Magnetization measurements were performed by using two commercial SQUID magnetometers (Cryogenic Ltd. S600 and Quantum Design MPMS) operating in the 1.8−350 K temperature range with applied field up to 6.5 T and recorded as a function of temperature and using a magnetic field. Measurements were performed both on the lyophilized sample and on the water dispersion (1% w/w of magnetite). All of the data were corrected for the magnetism of the sample holder, which was separately measured, and for the diamagnetic contribution of water. The temperature dependence of the magnetization was measured after zero field cooling (ZFC) and field cooling (FC) procedures with an applied magnetic field of μ0H = 5 mT in the temperature range 5−300 K. ZFC curves were obtained after cooling samples until starting temperature (5 K) in the absence of an external magnetic field. On the contrary, FC curves were obtained by cooling samples from 300 to 5 K and applying an external magnetic field of 5 mT. Dynamic Light Scattering (DLS) Measurement and ZPotential Analysis. DLS studies and Z-potential measurements (mV) were performed at 25 °C using a Malvern Zetasizer NanoZS instrument, fitted with a 532 nm laser at a fixed scattering angle of 173°. Aqueous dispersions of each nanoparticle sample prepared in bidistilled water at a final copolymer concentration of 0.2 mg/mL were analyzed as prepared and also after filtration through a 5 μm cellulose 4399

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different samples such as DHT (10−7 and 10−8 M), MNCs (9.4 and 27.6 μg/mL), or FLU-MNCs at two concentrations corresponding to 34 or 100 μM of total loaded drug amounts. Moreover, in other experiments LNCaP cells were cotreated with DHT and free FLU (only at a concentration of 10 μM which corresponds to the maximum solubility of the drug in aqueous medium) or with DHT and FLU-MNCs. Free FLU and FLU-MNCs were dispersed in PBS pH 7.4, whereas DHT was dissolved in DMSO. Untreated or DMSO treated cells were used as controls. The incubation time for all experiments was 72 h. After 72 h of incubation cell viability was measured by MTS assay (PROMEGA). MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)2H-tetrazolium] was utilized according to the manufacturer’s instructions. In particular, 20 μL of the MTS solution were added to each well and additionally incubated at 37 °C for 4 h. The absorbance was read at 490 nm on the Microplate reader Wallac Victor 2 1420 Multilabel Counter (Perkin-Elmer). Results were expressed as the percentage reduction of cell viability in comparison with the control cells. All culture experiments were repeated at least three times, and each experiment was performed in triplicate. In proliferation experiments, the mean value of the data measured in the control group was set at 100%, and all other values were expressed in relation to those mean values. LNCaP cells were maintained in Dulbecco’s modified essential medium (DMEM) containing 10% (v/v) FBS, 1 mM glutamine, and 1% antibiotics (10 000 U/mL penicillin, 10 mg/mL streptomycin) and 2% amphotericine B (0.25 mg/mL), at 37 °C in a humidified atmosphere and 5% CO2 in air, in a 24 well plates at density of 5 × 105 cells/mL for 96 h. After 96 h cell growth the medium was replaced with fresh medium (500 μL), and cells incubated with further 500 μL of DMEM containing different amounts of FLU-MNCs (nanocarrier concentration per well ranging from 0.09 to 0.61 mg/mL) and free FLU (drug concentration per well ranging from 0.018 to 0.122 mg/mL) for 4, 24, and 48 h. Following the incubation period, the medium was removed, and the cell monolayer was washed twice with Dulbecco’s modified Eagle’s medium (DPBS). The cell monolayer was then treated with an appropriate volume (500 μL) of bidistilled water. After incubation for 30 min on a shaker, cells were scraped and the cell lysate collected into Eppendorf tubes. For iron determination, aliquots (100 μL) of cell lysates were placed in Pyrex tubes and mixed with 100 μL of a 1.4 M HCl solution. These mixtures were incubated for 2 h at 60 °C within a water bath. After this time to each mixture, cooled to room temperature, 140 μL of 1 M NaOH solution, 100 μL of acetate buffer at pH 4.5, and 50 μL of the iron-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, and 1 M ascorbic acid dissolved in acetate buffer at pH 4.5) were added. After 10 min, 200 μL of each sample solution was transferred into a well of a 96-well plate, and the absorbance was measured at 560 nm on a microplate reader (Thermo, UK). The iron content of the sample was calculated through a calibration curve obtained by recording absorbance of standard solutions containing FeCl3 in the range concentration between 0.05 and 5 μg/mL in 10 mM HCl and using 50 μL of detection reagent. The intracellular iron concentration determined for each well of each cell lysates was corrected against blank and normalized against the cell number per well. For the quantization of the internalized drug amount cell lysates were analyzed by HPLC after filtration through 0.2 μm

filters. All culture experiments were repeated at least three times, and each experiment was performed in triplicate. 2.6. In Vivo Experiments. Animals. For in vivo studies healthy male Winstar rats (300−350 g body weight) obtained from the Istituto Zooprofilattico della Sicilia, Palermo, Italy were used. All animal experiments were conducted according to the protocols approved by the National Bioethical Committee. Magnetic Field Experiments. Rats were kindly anesthetized by inhalation of 1% halothane. On two groups of six rats, a cubic permanent magnet (0.5 cm3) with a maximum magnetic flux density of 0.3 T was fixed securely to the abdominal region of treated rats, by means of a biocompatible ethyl-2cyanoacrylate mastic (EPIGLU, Meyer-Haake). A volume of 0.25 mL of isotonic dispersions containing 20 mg/mL (i.e., ∼15 mg/kg) of FLU-MNCs or FLU-NCs were slowly injected intraperitoneally to each group of rats. Another group of six rats without a fixed magnet was treated with the same amount of FLU-MNPs as a control. The rats were sacrificed 24 or 48 h after the injection of the nanocarriers, previous anesthesia by intraperitoneal administration of ketamine (100 mg/kg). Blood samples were collected prior animal sacrifice; then organs (lungs, liver, kidneys, spleen, and brain) were collected immediately after animal sacrifice, washed twice with normal saline, and lyophilized. Blood samples were centrifuged and plasma collected in a sterile centrifuge tube. Determination of FLU, FLU−OH, and Iron Oxide in Rat Organs. For drug determination, the lyophilized organs and plasma were homogenized (15 min, 24 000 rpm) in the presence of HPLC grade methanol (10 or 20 mL in function of organ weight) and organ suspensions centrifuged at 9800 rpm for 10 min. The surnatants were collected and concentrated under vacuum until a final volume of 1 mL. FLU and its metabolite FLU−OH were quantized by HPLC analysis using a Waters Breexe System Liquid Chromatograph equipped with a Waters 717 Plus Autosampler (40 μL injection volume) and a Shimadzu UV−vis HPLC detector online with a computerized workstation monitoring at 250 nm. The used column was a reversed-phase Gemini C18 Phenomenex (5 μm, 4.6 × 250 mm column with a precolumn H5ODS-10CS). The used mobile phase was: MeOH/PBS pH = 8.7 (80:20 v/v), flow 1 mL/min. For iron detection, magnetic nanocarriers were first extracted from the organ suspension by using an external permanent magnet applied at the tube bottom for 96 h. Organ homogenate was then eliminated, and magnetic nanocarriers remained adhered to the tube wall were completely dissolved in 2 mL of 30% (v/v) HCl heating for 2 h at 60 °C. Then, obtained solutions were treated overnight with 0.1 mL of H2O2 solution (35% w/w) to oxidize the total ferrous ions and to decolorize the solution. Subsequently, 2 mL of 0.1 M solution of potassium thiocyanate was added to this solution to form the red colored iron−thiocyante complex. The iron concentration was determined by recording absorbance at 478 nm using a Shimadzu UV-2401PC spectrophotometer. For quantization, a standard curve for the iron complex was made under identical conditions using a known amount of Fe3O4 standard nanoparticles. 2.7. Statistical Analysis. A one-way analysis of variance (ANOVA) was used to evaluate group comparison. If the group by each time interaction was significantly different (P < 0.05), differences between groups were compared within an a posteriori Bonferroni t test. All of the values are reported as the average ± standard deviation or mean ± s.e.m. 4400

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3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of PHEA-IBp(BMA)-Coated MNCs. PHEA-IB-p(BMA) is a brush copolymer with structural and hydrophobic characteristics that make it counted among smart copolymers. Some of these features were already reported, regarding the copolymer ability to arrange into microfibers.29 Actually, the smart features of this copolymer are strictly correlated to its chemical structure and hydrophobicity; thus slight variations of these characteristics should modify its self-assembling properties, for example, an increase of hydrophobicity should allow preferentially the formation of colloidal aggregates in aqueous medium. For this aim, experimental conditions adopted in this study to synthesize PHEA-IB-p(BMA) copolymer were adjusted in order to obtain significant hydrophobicity increase of final PHEA-IB-p(BMA) graft copolymer and total loss of its water solubility.30 In fact, a mean number of BMA monomer residues equal to 7 for each poly(BMA) chain are shown to be sufficient to makes PHEA-IB-p(BMA) freely soluble in nonpolar solvents, such as chloroform, and this feature made possible the production of nanoparticles in aqueous media. Reaction conditions and molecular parameters of synthesized PHEA-IB-p(BMA) copolymer are reported in Table 1.

PHEA-IB-p(BMA) graft copolymer dissolved in chloroform, alternatively mixed with colloidal superparamagnetic Fe3O4 and/or FLU, by homogenization into an aqueous phase containing PVP as stabilizer and Pluronic F68 as surfactant. Actually, the dimension of primary (o/w) emulsion determined the particle size upon chloroform evaporation under reduced pressure. TEM images of freeze-dried obtained nanocarriers are shown in Figure 2. No significant differences were observed in particle shape and size between MNCs (a) and FLU-MNCs (b) nanocarrier samples, the majority of observed nanocarriers in these two samples being spherical, with a diameter smaller than 300 nm. The FLU-NCs sample image (c) showed medially a spherical shape as well and size values between 250 and 300 nm. DLS measurements indicated hydrodynamic size values in water in agreement with dimensions observed with TEM, even after 24 h of incubation in aqueous medium (DLS data at 24 h are reported in Table 2), being the measured hydrodynamic diameter of 290 nm for MNCs (PDI 0.21), of 320 nm for FLUMNCs (PDI 0.32), and of 300 nm for FLU-NCs (PDI 0.35). Sample filtration did not change the nanocarriers polydispersity. All nanocarrier aqueous dispersions had a negative zeta potential with values ranging from −15.8 mV for MNCs to −8.5 mV for FLU-MNCs and FLU-NCs (Table 2). The less negative zeta potential values in the latter cases should be the result of the presence of a part of FLU molecules partially exposed in the nanocarriers surface that determines a shielding of the surface charge. The FTIR spectra of solid samples of PHEA-IB-p(BMA) copolymer and MNCs (Figure 3) showed the characteristic broad band of Fe−O stretching at 575 cm−1, that is absent in the PHEA-IB-p(BMA) spectrum. This datum evidences the presence of magnetite in the nanocarrier samples. On the other hand, comparing the FTIR spectra of MNCs and those of coating polymer are also clearly evidence of vibrating bands attributable to CO stretching (1651 and 1727 cm−1) of the polymer. These data confirm the existence of a polymer matrix coating magnetite domains. 3.2. Determination of Magnetite Content and Evaluation of Magnetic Behavior of MNCs and FLUMNCs. The number of magnetic nanometric domains (10 nm) contained into PHEA-IB-p(BMA) polymeric matrix of MNCs and FLU-MNCs was evaluated indirectly by determining the total iron oxide amount per nanocarrier mass unit. For this evaluation, iron oxide contained into the MNCs or into FLUMNCs was completely dissolved by HCl solution. Then, ferrous ions present in the solution were oxidized to ferric ions by hydrogen peroxide before reacting with thiocyanate salt to form the iron−thiocyanate complex. The concentration of the complex and, hence, iron content in the nanocarriers was determined spectrophotometrically. Iron oxide content in the nanocarriers was found to be 1.5% (w/w) in MNCs and 3.4% in FLU-MNCs. It was calculated that a total of 3.37 × 1015 iron oxide magnetic domains is present in 1 g of MNCs and each polymer nanocarrier should contain about 30 magnetic domains. These data were in good agreement with magnetic field measurements. The magnetic behavior of prepared MNCs was already evidenced macroscopically by the effect of an external magnetic field on a MNCs water dispersion. In Figure 4a and b, the photographs of MNCs dispersion before and after the application of an external magnetic field are shown. It can be observed that the MNCs dispersion, in which nanocarriers are initially homogeneously dispersed (a), moved toward the

Table 1. Reaction Conditions and Molecular Parameters of PHEA-IB-p(ButMA) Copolymer reaction temperature (°C)

reaction time (h)

DDBIB%a (mol %)

nb

Mwc (kDa)

50

20

30

7

380

DDBIB% = (linked BIB residues/PHEA repeating units) × 100 (mol/ mol). The derivatization degree (DD) for the obtained PHEA-BIB copolymer was determined by 1H NMR in DMSO-d6, comparing the integral of the peak corresponding to protons at 1.88 ppm assigned to methyl groups belonging to linked BIB with the integral of the peak related to protons at 2.7 ppm assigned to CH2, belonging to PHEA. bn = mean monomer residue for each p(BMA) chain. cMw values were determined by SEC using two organic phase columns (Phenogel 104R and 103R, Phenomenex) connected to a Water 2410 refractive index detector. DMF containing 0.01 M LiBr was used as eluent with a flow of 0.8 mL/min. The column temperature was set at 50 °C. Poly(ethylene oxide) standards were used for calibration. a

The chemical structure of PHEA-IB-p(BMA) is reported in Figure 1. The synthesized copolymer was than used to prepare three different nanocarrier samples: magnetic nanocarriers (MNCs), flutamide-loaded magnetic nanocarriers (FLU-MNCs), and flutamide-loaded nanocarriers (FLU-NCs). The amount of loaded FLU was 20% (w/w). Preparation was performed by emulsifying a previously prepared organic phase, consisting of

Figure 1. Chemical structure of PHEA-IB-p(BMA) graft copolymer. 4401

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Figure 2. TEM images (a, b, c) and DLS (d, e, f) characterization of MNCs (a, d), FLU-MNCs (b, e), and FLU-NCs (c, f) samples.

Table 2. Summary of DLS Analysis Data in Deionized Water after 30 min and 24 h sample

hydrodynamic diameter (nm)

PDI

zeta potential (mV)

incubation time (h)

MNCs FLU-MNCs FLU-NCs MNCs FLU-MNCs FLU-NCs

290 320 300 292 319 304

0.21 0.32 0.38 0.19 0.31 0.39

−15.8 ± 4.7 −8.5 ± 2.8 −8.6 ± 3.3 −15.4 ± 5.1 −8.4 ± 3.7 −8.7 ± 4.2

0.5 0.5 0.5 24 24 24

magnet as the dispersion stood in the presence of that overnight (b). This result demonstrated that the greater part of prepared nanocarriers contains encapsulated Fe3O4 nanometric domains. Magnetic Measurements. The magnetic properties of MNCs sample were investigated as a function of the magnetic field at high and low temperatures, respectively, 300 K and 5 K. Measurements were performed either on MNC and FLU-MNC dispersions in water and on the same samples as dried powder. No significant differences were observed between dispersion and dried samples, suggesting that magnetic properties of nanocarriers are not affected by the presence of physical environment and interactions with drug molecules. The M vs H curves at room temperature (Figure 5a) of MNCs and FLUMNCs, are perfectly superimposable and do not show coercivity, confirming that all the samples are in the superparamagnetic regime. The saturation magnetization value, MS, estimated by fitting the curve to the empirical law M = MS + a/H + b/H2, is 0.9 Am2/kg.31 The MS values are smaller than that observed for bulk magnetite (∼60 Am2/kg; Figure 5c): this reduction is commonly observed in MNCs in which magnetite is coated by a polymeric matrix,32 and it is attributed to the presence of a disordered spin layer on the

Figure 3. FTIR spectra of magnetite (Fe3O4), PHEA-IB-p(BMA) copolymer, and MNCs (PHEA-IB-p(BMA) + Fe3O4).

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surface,33 due to increased surface-to-volume ratio. The M vs H curves recorded at low temperature (5 K), reported in Figure 5b, show a hysteresis loop with a low coercive field μ0HC = 7 mT and MS value of 1.1 Am2/kg (see the magnification of magnetization curve of Figure 5b, reported in the Supporting Information section). The remaining magnetization, MR = M0T/M5T, evaluated as the ratio between the value of magnetization at zero and 5 T, is 0.23. This value is slightly lower than the typical one, 0.5, expected for a set of isolated uniaxial nanoparticles whose easy axes are isotropically orientated, confirming the presence of a portion of ultrasmall nanoparticles that relaxes faster than the average size ones. This observation is consistent with the presence of a polymer wall between magnetite domains that increase the means distance between them with a consequent significant reduction of dipolar interactions. This hypothesis is also in agreement with the reduction of the blocking temperature observed in the zerofield cooled (ZFC) and field-cooled (FC) experiments. Also in the case of ZFC and FC magnetization curves, no differences were observed between MNC and FLU-MNC samples. As an example, the temperature dependences of the ZFC and FC magnetizations of MNCs and magnetite sample dispersion in water are reported in Figure 6a and b,

Figure 4. Photographs of MNCs water dispersion before (a) and after the application of an external magnetic field overnight (b).

Figure 6. Zero-field cooled (ZFC) and field-cooled (FC) curves of MNCs (a) and magnetite (b) sample dispersions.

respectively. The MNC ZFC curve (Figure 6a) was acquired up to 250 K in order to avoid the melting of the solvent. The sample shows the typical thermal irreversibility characteristic of an ensemble of weakly interacting single domain nanoparticles, which can be described within the framework of the Néel model,34 where the relaxation time of the magnetic moment reversal of the nanoparticles, τ, is given by τ = τ0 eKV/kBT, where K is the anisotropy constant, V is the particle volume, τ0 is the attempt time, and kB is the Boltzmann constant. The

Figure 5. Room temperature (a) and 5 K (b) magnetization curves of MNCs and FLU-MNCs and bulk magnetite alone (c).

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MNCs containing an amount of drug corresponding to a concentration of 34 μM (a and a′) showed a drastic inhibition (∼ 80% of inhibition) of the DHT proliferation effect and a quite complete inhibition of cell viability when administered at a concentration corresponding to 100 μM of FLU (b and b′). In these two cases we have to consider that the final FLU concentration is approximately three (34 μM) and ten (100 μM) times higher than that used on cells treated with FLU alone (10 μM) and that the greater antiproliferative effect should be attributed to the greater final drug concentration. Moreover, the antiproliferative effect of FLU-MNCs is also visible in the absence of DHT (d and e). Actually, data suggest that the pharmacological activity of FLU is not reduced by its loading into magnetic nanoparticles. These results suggest the hypothesis that PHEA-IB-p(BMA) copolymer constituting nanocarriers can be able to interact with cell membrane and promote the uptake of encapsulated drug, upon nanocarrier endocytosis and drug diffusion. The enhanced drug uptake could be related also to nanocarrier adhesion on cell membranes attributable also to the hydrophobic structure of PHEA-IB-p(BMA). This fact was already reported for PHEAIB-p(BMA) microparticles.29 An important factor to reach the optimal pharmacological effect of FLU is the internalization ability of magnetic nanocarriers into tumor cells, because the action site of the antitumor drug FLU is inside cellular cytoplasm.37 For this purpose, LNCaP cells were incubated with FLU-MNCs for 4, 24, and 48 h, in order to evaluate the cellular uptake of FLU and Fe3+ (expressed as Fe3O4) loaded into magnetic nanocarriers. Figure 8 shows these results, in terms of nanograms of FLU (Figure 8a) and Fe3O4 (Figure 8b) per cell, as a function of FLU-MNCs concentration. The amounts of FLU and Fe3O4 in cell lysate, detected by HPLC and UV analysis respectively, were dependent on the nanocarriers concentration and on the incubation time; however, no significant differences in uptake values of FLU and Fe3O4 were observed between 24 and 48 h. Therefore, it seems that a plateau in the nanocarriers internalization is achieved after an incubation time comprised between 24 and 48 h. To evaluate the ability of FLU-MNCs to release loaded drug in vitro, drug release studies were performed in physiological pH mimicking conditions. For comparison a drug dissolution curve under the same experimental condition was also reported. In Figure 9 the percentage of released FLU from FLU-MNCs in comparison with the dissolution curve of the free FLU in the same experimental conditions is shown. As it can be seen during the first day of incubation the released drug was about 70% of loaded FLU. This release profile changes (release rate becomes constant and lower) in the time interval between 24 and 48 h, being released after 48 h about 85% of loaded FLU. Finally, the comparison with the dissolution profile of free FLU confirmed that really the drug release rate is influenced by the incorporation into FLU-MNCs. 3.4. In Vivo Biological Evaluation of PHEA-IB-p(BMA)Coated Magnetic Nanocarriers. The effect of an external magnetic field on the biodistribution of nanoparticles was assessed by administrating magnetic (FLU-MNCs) and nonmagnetic (FLU-NCs) nanocarriers, the latter used as negative control, in rats subjected to an external magnetic field. FLUMNC samples were also administered to a control animal group without external magnetic field in order to exclude effects on biodistribution not attributed to the external magnetic field. Then the amount of Fe3O4, FLU, and its metabolite FLU−OH,

temperature at which the measuring time is equal to the relaxation time, τ, is the blocking temperature TB which, assuming τ0 to be constant, is directly proportional to the anisotropy barrier KV and corresponds to the temperature at which most of the nanoparticles relax. In real systems TB is commonly identified with the temperature corresponding to the maximum of the ZFC curve, while the difference between TB and the temperature at which the ZFC and FC collapse gives an estimate of the energy barrier distribution. The average TB of magnetite (Figure 6b) is 50 K, consistent with superparamagnetic maghemite/magnetite nanoparticles with an average size of 10−20 nm.35 Differently, MNCs show a TB of 15 K which is consistent with the nanoparticle average size well above 20 nm and thus in agreement with the presence of polymer coating. However, a TB of 15 K is well below room temperature, indicating that the sample is always in the superparamagnetic regime at physiological temperature, as required for in vivo applications. The splitting temperature between the ZFC and FC curves is well above TB, at ca. 250 K, suggesting that the particle size is not perfectly monodisperse. 3.3. In Vitro Biological Evaluation of PHEA-IB-p(BMA)Coated Magnetic Nanocarriers. The antiproliferative effect of prepared FLU-MNCs was studied by MTS assay on human prostate adenocarcinoma (LNCaP) cells. Moreover, the effect of dihydrotestosterone (DHT), which is a proliferative agent for prostate cancer cells,36 was also evaluated on this cell line. In this case, LNCaP were coincubated for 72 h with DHT (at two concentrations, 10−8 and 10−7 M) and an amount of FLUMNCs corresponding to a drug concentrations of 34 (a, a′) and 100 (b, b′) μM; free FLU was used as positive control at concentrations of 10 μM (c, c′) that correspond to the maximum aqueous solubility of this drug. The results, in terms of cell viability (%) as a function of sample concentration, are shown in Figure 7. As expected, the incubation with DHT produced a significant increase of cell viability that depends on the concentration of the DHT (DHT 10−8 and DHT 10−7 M) on LNCaP cells; on the contrary, this effect decreased (∼ 20% of inhibition) when the cells were cotreated with FLU at 10 μM (c and c′), confirming the antiandrogen action of FLU. In contrast, FLU-

Figure 7. LNCaP viability after 72 h of incubation with: DHT 10−8 and DHT 10−7 M; FLU-MNCs 34 μM (a) and 100 μM (b) in the presence of DHT 10−8; FLU-MNCs 34 μM (a′) and 100 μM (b′) in the presence of DHT 10−7; free FLU in the presence of DHT 10−8 (c) and 10−7 M (c′); FLU-MNCs corresponding to a drug concentration of 34 μM (d) and 100 μM (e) in the absence of DHT; MNCs at concentrations corresponding to (9.4 μg/mL) (f) and (27.6 μg/mL) (g) in the absence of DHT. The cell proliferation was determined by an MTS colorimetric assay, and the standard deviation values (±SD) were calculated on the basis of three experiments conducted in multiples of six. 4404

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Figure 8. LNCap uptake of FLU (a) and Fe3+ (expressed as μg of Fe3O4) (b) after 4, 24, and 48 h of incubation with FLU-MNCs. Free FLU after 48 h of incubation was reported as a control. Standard deviation values (±SD) were calculated on the basis of three experiments conducted in multiples of six.

Figure 10. Biodistribution of FLU (a) and its metabolite FLU−OH (b) 24 h postinjection, in the organs of rats subjected to an external magnetic field (FLU-NCs and FLU-MNCs) and control rats (FLUMNCs no ext. magnet). The internal photo of part a indicates the magnet position and the site of administration. Data are presented as mean ± s.e.m. (n = 6 animal per group). Significance = p < 0.05.

Figure 9. Percentage of released FLU from FLU-MNCs in comparison with the dissolution curve of free FLU under the same experimental conditions (PBS pH 7.4, 37 °C). Standard deviation values (±SD) were calculated on the basis of three experiments.

Figure 11. Biodistribution of iron (expressed as μg of Fe3O4) 24 h postinjection, in the organs of rats subjected to an external magnetic field (FLU-NCs and FLU-MNCs) and control rats (FLU-MNCs no ext. magnet). Data are presented as mean ± s.e.m. (n = 6 animal per group). Significance = p < 0.05.

accumulated in the principal organs (liver, spleen, lungs, kidney, brain, and plasma), was quantified 24 and 48 h postinjection. Biodistribution data collected 24 h postinjection are shown in the Figures 10a, b and 11. Overall, the biodistribution of FLUMNCs in rats subjected to an external magnetic field was conspicuously different than that found in the rats treated with FLU-NCs and in control rats either in the absence of an external magnetic field, 24 h postinjection. In effect a

significantly high concentration of FLU was found in those organs, such as the kidney, physiologically closer to the magnet (see the internal photo of Figure 10a), rather than in the other organs. Therefore we can suppose that the presence of the external magnet forced the magnetic nanocarriers to remain near the side of administration and retarded their elimination 4405

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postinjection. On the contrary, the uptake by the reticuloendothelial system of the liver, spleen, and lungs was significantly reduced. Actually, the obtained system results in efficiently internaliation by tumor cells and is susceptible to magnetic targeting by application of an innocuous external magnetic field; therefore it can offer a very good approach to obtain an efficient magnetically targeted anticancer drug delivery system.

from the animal body (only at 48 h postinjection the FLU amount in the kidney was analogue to that found in the other organs; data not reported). Differently, a not altered drug biodistribution was observed in the animals whose FLU-NCs were administered or in the animals that received FLU-MNCs but in the absence of the external magnet. On the other hand, the absence of an high amount of the metabolite FLU−OH in the kidney (Figure 10b) indicated that the presence of the magnet prevented the nanocarrier passage from the liver, where FLU is rapidly transformed in FLU−OH.38 This finding further supports the hypothesis that the presence of the external magnet reduced the escape of administered magnetic nanocarriers from the area involved by the magnet and consequently the capture of these nanosystems by that organs acting in the reticuloendothelial system, such as liver, spleen, and lungs, notably responsible for removing nanoparticulates from circulation. As counterproof, the amount of magnetite accumulated in the same organs 24 h postinjection was also determined and results are reported in Figure 11. As expected, it was found an high amount of magnetite in the kidney, corresponding to the presence of magnetic nanocarriers here accumulated. However, an appreciable amount of iron (expressed as Fe3O4) was found also in liver and spleen, even if lower than that found in the kidney. These data can be explained considering that the iron oxide nanoparticles that arrive to these organs are not rapidly metabolized differently from FLU, and therefore 24 h post injection an appreciable amount can be still detected. On the contrary in the absence of the external magnetic field a massive accumulation of magnetic nanocarriers measured as μg of Fe3O4 was found in the liver of the rats, indicating that this is the main organ in that these nanocarriers are accumulated. Finally, the amount of Fe3+ detect in animal organs after administration of nonmagnetic nanocarriers (FLU-NCs) is presumably correspondent to the Fe3+ amount physiologically present in these organs.

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ASSOCIATED CONTENT

* Supporting Information S

Magnification of the magnetization curve of Figure 5b, SEM images of MNCs, and the XRD spectrum of MNCs and FLUMNCs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Department STEBICEF, University of Palermo, via Archirafi 32, 90123, Palermo, Italy. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors thank the University of Palermo for financial support of the Doctorate scholarship in “Tecnologie delle Sostanze Biologicamente Attive” for Dr. Cinzia Scialabba. Authors thank Dr. Claudio Sangregorio, Laboratory of Molecular Magnetism (LAMM) at University of Florence for scientific support in the magnetic characterization. Thanks are also due to Angela Cusimano, Giuseppe La Corte, and Giovanni D’angelo, Istituto Zooprofilattico della Sicilia, “A. Mirri”, of Palermo, for technical assistance during animal experiments.

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REFERENCES

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4. CONCLUSIONS In this paper, magnetic nanocarriers, constituted by a PHEAIB-p(BMA) matrix and containing a superparamagnetic iron oxide domains of 10 nm have been prepared using the homogenization−solvent evaporation method. The same magnetic nanocarriers were also prepared in the presence of the anticancer drug flutamide (FLU), obtaining a significantly high drug loading (20% w/w). The prepared magnetic nanocarriers (FLU-MNCs) have been exhaustively characterized in vitro by various physicochemical means, including DLS, TEM, magnetic measurements, Fe3O4 and FLU contents, and drug release studies. Magnetic nanocarriers showed dimensions of about 300 nm with a narrow size distribution, a characteristic superparamagnetic behavior, and an high attraction susceptibility by an external magnetic field. The results of cell culture experiments have indicated that PHEA-IB-p(BMA) magnetic nanocarriers were nontoxic as empty magnetic nanocarriers (MNCs), but they act as drug delivery system of FLU on prostate cancer cell line LNCaP, since highly internalized by these cells. This property can be attributed to the presence of a polymer made surface that may enhance the cellular uptake. The in vivo biodistribution studies revealed that application of an external magnetic field over the abdominal region in rats drastically modified the biodistribution of FLU-MNCs. The nanocarriers were taken up predominantly by the kidney 24 h 4406

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