Polymeric Nanocarriers for Magnetic Targeted ... - ACS Publications

Subscriber access provided by LAURENTIAN UNIV

Article

POLYMERIC NANO-CARRIERS 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 Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 29 Oct 2013 Downloaded from http://pubs.acs.org on November 1, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

POLYMERIC NANO-CARRIERS FOR MAGNETIC TARGETED DRUG DELIVERY: PREPARATION, CHARACTERIZATION AND IN VITRO AND IN VIVO EVALUATION

Mariano Licciardia, Cinzia Scialabbaa, Calogero Fioricaa, Gennara Cavallaroa, Giovanni Cassatab, Gaetano Giammonaa,c

a

Department of Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Laboratory of

Biocompatible Polymers, University of Palermo, Via Archirafi, 32 90129 Palermo, Italy.

b

c

Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri” Palermo, Italy.

IBF-CNR, via Ugo La Malfa, 153, 90143 Palermo, Italy.

Corresponding author's coordinates: Gaetano Giammona, Department STEMBIO, University of Palermo, via Archirafi 32, 90123, Palermo, Italy. e-mail: [email protected]

1 Environment ACS Paragon Plus


Page 2 of 39

ABSTRACT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In this paper is reported the preparation of magnetic nanocarriers (MNCs), containing superparamagnetic domains, 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 non-magnetic nanocarriers containing flutamide (FLU-MNCs) were prepared. The prepared nanocarriers have been exhaustively characterized by DLS, TEM and magnetic measurements. Biological evaluation was performed by in vitro cytotoxicity and cell uptake tests and in vivo biodistribution studies. Magnetic nanocarriers showed dimension of about 300 nm with a narrow size distribution, an amount of loaded FLU of 20% w/w and a superparamagnetic behaviour. 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 represents 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 challenge in cancer chemotherapy is the design of more efficient drug delivery system with the objective of targeting drugs to specific organs or tissues of the body to improve therapeutic index and minimize or eliminate the undesirable side effects. At this regard, a particularly attractive technology, that in the past few years have been showed a growing interest, is the magnetic drug delivery system1. Several types of magnetic materials, such as iron oxides (Fe2O3 and Fe3O4), metal alloy (Fe, Co, and Ni), and iron cobalt alloy, have been widely studied for magnetic drug delivery2. Among these materials, magnetite (Fe3O4, single domains of about 5-20 2 Environment ACS Paragon Plus

Page 3 of 39


nm), a common magnetic iron oxide, is a very promising candidate for its biocompatibility and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 haemoglobin) and thus eliminated by the normal iron recycling pathways3,4. The main advantages of MNPs, compared to their bulk counterparts, are not only their high specific surface areas, low sedimentation rate and reduced magnetic dipoledipole interaction, but especially their magnetic behaviour. In fact, MNPs having dimensions below 20 nm, exhibit superparamagnetic behaviour (superparamagnetic iron oxide nanoparticles, SPIONs). They magnetize strongly when an external magnetic field is applied but no residual magnetic force exist between the particles upon removal of the magnetic filed5. This behaviour makes them suitable to use experimentally for numerous in vitro applications, such as cell separation experiments6,7, and in vivo applications such as magnetic resonance imaging (MRI) contrast enhancement8,9, hyperthermia and drug delivery2,10 and targeting11. Moreover, although MNP are highly biocompatible, their high superficial area and hydrophobic surfaces may results in aggregation, and cause also their uptake by the body reticuloendothelial system. Therefore, it is necessary coating MNPs with a suitable polymer to improve their stability and their circulation time. MNP coatings to date explored comprises several polymer materials, including both synthetic and natural polymers5. 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 alcohol13 and poly(ethyl-2-cyanoacrylate)14,15. While, natural polymer include gelatine16, dextran, chitosan13 and pullulan13,17. The polymer coating not only stabilize the nanoparticles but also provide them active functional groups for controllable bioconjugation of targeting ligands18. The targeting by drug delivery systems containing iron oxide such as polymeric vesicles18 , magnetic nanoparticles20,21, magnetic liposomes22-24 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 magnetic property, 3 Environment ACS Paragon Plus


Page 4 of 39

and could target the system to a specific site by the action of the external magnetic field. However, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the polymeric shell could entrap drug molecules in order 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-IB-p(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. FLUMNCs were prepared 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. 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 METHOD α,β-Poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was prepared and purified according to the previously reported procedure27,28. 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. 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, 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 (SPIONs) (10 ± 1 nm) in toluene 4 Environment ACS Paragon Plus

Page 5 of 39


(magnetization emu/g, at room temperature under 4500 Oe), polyvinyl pyrrolidone (PVP), glyceryl 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

monostearate, sodium dodecyl sulphate (SDS), flutamide (FLU) hydroxyflutamide (FLU-OH), dihydrotestosterone (DHT), L-Ascorbic acid sodium salt, bovine serum albumin, ferrozine (3-(2pyridyl)-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 purchase 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)graft-poly(butyl methacrylate) (PHEA-IB-p(BMA) copolymer) Derivatization of PHEA with 2-bromoisobutyryl bromide (BIB) to obtain PHEA-BIB multifunctional macroinitiator was carried out using the previously described protocol29. 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 elsewhere29, was 30 mol-%. The homopolymerization of butyl methacrylate, using PHEA-BIB as the macroinitiator, was carried out according to a previously reported procedure30, 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 drop-wise 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 5 Environment ACS Paragon Plus


Page 6 of 39 30 1

and spectroscopic data were in agreement with the previous results . H NMR (300 MHz, DMSO1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 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 & Kunkel Ika–Labortechnik) for 20 min at 24,000 rpm. 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, Missouri, U.S.A.). Analogous procedure was adopted for the preparation of FLU-loaded magnetic (FLU-MNCs) and FLU-loaded non-magnetic (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 6 Environment ACS Paragon Plus

Page 7 of 39


temperature overnight. The samples were analyzed using a JEM-2100 LaB6 transmission electron 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

microscope operating at an accelerating voltage of 200 kV, equipped with a Multi Scan CCD camera.

Total iron determination: Iron content in the nanocarriers was determined spectrophotometrically using a method elsewhere reported based on the formation of the highly coloured complexes ironthiocyanate ion16. Firstly, 5 mg of nanoparticles were 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 oxidise 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 coloured 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 K - 350 K temperature range with applied field up to 6.5 T and recorded as a function of temperature and magnetic field using. Measurements were performed both on the lyophilized sample and on the water dispersion (1 % w/w of magnetite). All 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 magnetisation was measured after zero field cooling (ZFC) and field cooling (FC) procedures with an applied magnetic field of µ0H = 5mT in the temperature range 5300 K. ZFC curves were obtained after cooling samples until starting temperature (5 K) in the absence of external magnetic field. On the contrary, FC curves were obtained by cooling samples from 300 K to 5 K and applying an external magnetic field of 5 mT.



Dynamic Light Scattering (DLS) measurement and Z-potential analysis: DLS studies and Z1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 90°. Aqueous dispersion of each nanoparticles sample prepared in bi-distilled water at a final copolymer concentration of 0.2 mg/mL, were analysed as prepared and also after filtration through a 5 µm cellulose membrane filter. The intensity-average hydrodynamic diameter and polydispersity index (PDI) were obtained by cumulants analysis of the correlation function. The zeta potential (mV) was calculated from the electrophoretic mobility using the Smoluchowsky relationship and assuming that K·a >>1 (where K and a are the Debye-Hückel parameter and particle radius, respectively).

FT-IR Analysis FT-IR spectra of MNCs copolymer, FLU-MNCs and solid iron oxide nanoparticles were recorded in KBr pellets in the frequency range of 4000–400 cm−1 by using a Perkin-Elmer 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, 4.6×250mm column with a pre-column 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 and FLU-NC samples and FLU alone (1 mg, as positive control) were suspended in bi-distilled water (20 mL) and 8 Environment ACS Paragon Plus

Page 8 of 39

Page 9 of 39


transferred inside of a Spectra/Por dialysis membrane (MWCO 12,000-14,000 Da). This dialysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 analysed 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 microparticles. 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 drug loaded magnetic nanocarriers Human metastatic prostate adenocarcinoma (LNCaP) cells were cultured in RPMI 1640 medium supplemented with 5% heat-inactivated foetal bovine serum (FBS), 1mM sodium pyruvate, 1mM 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 steroids concentration 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 1x105 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 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 co-treated 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-(39 Environment ACS Paragon Plus


carboxymethoxyphenyl)-2-(4-sulphopheyl)2H-tetrazolium] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

was

Page 10 of 39

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 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, 1mM 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 5x105 cells/mL for 96 h. After 96 h cell growth the medium was replaced with fresh medium (500 µL) and cell incubated with further 500 µL of DMEM containing different amount 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 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 (100µL) 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 10 Environment ACS Paragon Plus

Page 11 of 39


solutions containing FeCl3 in the range concentration between 0.05 and 5 µg/mL in 10 mM HCl, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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 triplicates.

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 ethyl2-cyanoacrylate 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, were treated with the same amount of FLU-MNPs as 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

9,800 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 on line with a computerized workstation monitoring at 250 nm. The used column was a reversed-phase Gemini C18 Phenomenex (5 µm, 4.6 × 250mm column with a pre-column 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 firstly 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 oxidise 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 coloured 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 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

Polymeric Nanocarriers for Magnetic Targeted ... - ACS Publications

Recommend Documents