Article pubs.acs.org/Langmuir
Stimuli-Responsive Magnetic Nanomicelles as Multifunctional Heat and Cargo Delivery Vehicles Dong-Hyun Kim,†,∥ Elina A. Vitol,†,‡ Jing Liu,§ Shankar Balasubramanian,‡ David J. Gosztola,‡ Ezra E. Cohen,§ Valentyn Novosad,*,† and Elena A. Rozhkova*,‡ †
Materials Science Division and ‡The Center for Nanoscale Materials, Argonne National Laboratory, Argonne, Illinois 60439, United States § Department of Medicine, University of Chicago, 5841 S. Maryland Avenue, MC 2115 Chicago, Illinois 60637, United States S Supporting Information *
ABSTRACT: Hybrid nanoarchitectures are among the most promising nanotechnology-enabled materials for biomedical applications. Interfacing of nanoparticles with active materials gives rise to the structures with unique multiple functionality. Superparamagnetic iron oxide nanoparticles particles SPION are widely employed in the biology and in developing of advanced medical technologies. Polymeric micelles offer the advantage of multifunctional carriers which can serve as delivery vehicles carrying nanoparticles, hydrophobic chemotherapeutics and other functional materials and molecules. Stimuli-responsive polymers are especially attractive since their properties can be modulated in a controlled manner. Here we report on multifunctional thermo-responsive poly(N-isopropylacrylamide-co-acrylamide)-block-poly(ε-caprolactone) random block copolymer micelles as magnetic hyperthermia-mediated payload release and imaging agents. The combination of copolymers, nanoparticles and doxorubicin drug was tailored the way that the loaded micelles were cable to respond to magnetic heating at physiologically-relevant temperatures. A surface functionalization of the micelles with the integrin β4 antibody and consequent interfacing of the resulting nanobio hybrid with squamous head and neck carcinoma cells which is known to specifically over-express the A9 antigen resulted in concentration of the micelles on the surface of cells. No inherent cytotoxicity was detected for the magnetic micelles without external stimuli application. Furthermore, SPION-loaded micelles demonstrate significant MRI contrast enhancement abilities.
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INTRODUCTION Hybrid nanoarchitectures are among the most promising nanotechnology-enabled materials for biomedical applications.1−3 Interfacing nanoparticles with active materials gives rise to structures with unique multiple functionality. Superparamagnetic Fe3O4 iron oxide particles are widely employed in studies of cell rheology, magnetic-field-guided drug delivery, and magnetofection4 and as energy transducers that affect the cell fate through magnetically induced hyperthermia5 and the direct activation of ion channels in the cell membrane.6−8 Wet chemistry synthesis of iron oxide particles in organic solvents, typically employing the thermal decomposition of the ironcontaining precursor,9 yields particles with a tightly controlled size distribution, giving it an advantage over aqueous-based synthesis. However, the nanoparticles dispersed in organics cannot be directly used in conjunction with living matter. One of the convenient routes to making such particles biocompatible and to improve their stability in solution is to coat them with a noncytotoxic polymer10 such as poly(ethylene glycol)11 or dextran.12,13 As a step forward, polymeric micelles offer the advantage of multifunctional capsules that can serve as nanocontainers carrying not only the nanoparticles but also © XXXX American Chemical Society
drugs, dyes, plasmid DNA, miRNA, and other functional materials.14 Stimuli-responsive polymers are especially attractive because their properties can be modulated in a controlled manner.15−17 For polymeric micelles, externally triggered drug release is one of the key applications realized by employing stimuli-responsive polymers.18 Temperature- and pH-dependent modulations of polymeric structures represent the external routes of polymer stimulation resulting in phase transitions.19−21 At the same time, loading the micelles with nanoparticles allows for applying an internal stimulus, such as magnetically induced heating in the case of superparamagnetic particles22 or optically induced heating through plasmonic nanoparticles.23,24 In this work, we report on multifunctional thermoresponsive poly(N-isopropylacrylamide-co-acrylamide)-block-poly(ε-caprolactone) (P(NIPAAm-co-AAm)-b-PCL) random block copolySpecial Issue: Interfacial Nanoarchitectonics Received: November 7, 2012 Revised: January 25, 2013
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the micelles was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern, Herrenberg, Germany). Synthesis of SPION. Magnetite nanoparticles with 11 nm diameter were synthesized by a seed-mediated method.26 Briefly, Fe(acac)3 (2 mmol) was mixed with 1,2-hexadecanediol (10 mmol), oleic acid (6 mmol), and oleylamine (6 mmol) in benzyl ether (20 mL) under positive nitrogen pressure. The mixture was held at 200 °C for 2 h under nitrogen and then refluxed at 300 °C for 1 h. After being cooled to room temperature, the solution was precipitated with ethanol and resuspended in hexane. The resulting 6 nm particles were used as seeds for the synthesis of 11 nm particles. Synthesis of P(NIPAAm-co-AAm)-b-PCL. P(NIPAAm-co-AAm) was prepared by radical polymerization according to previous reports.27,28 NIPAAm (3 g), AAm (0.3063 g), chain-transfer agent ME (60 μL), and initiator ACPA (9 mg) were dissolved in 10 mL of methanol. The solution was degassed by bubbling with nitrogen for 30 min. After continuous agitation at 70 °C for 24 h under positive nitrogen pressure, the resulting product was precipitated out by the addition of 20 mL of diethyl ether. The intermediate copolymer was purified by repeated precipitation in diethyl ether and then dried in vacuum. An amorphous white solid was obtained. FT-IR (KBr pellet, cm−1): 1645 (amide carbonyl), 1550 (bending frequency of amide N− H). MS (MALDI TOF): 11 545 [M]+. P(NIPAAm-co-AAm)-b-PCL was synthesized from P(NIPAAm-coAAm) and CL via ring-opening polymerization. P(NIPAAm-co-AAm) (0.73 g), CL (146 μL), and Sn(Oct)2 (1 mg) as a catalyst were dissolved in 20 mL of toluene. The solution was degassed by bubbling with nitrogen for 30 min. After continuous agitation at 115 °C for 24 h under positive nitrogen pressure, the product was precipitated with diethyl ether and then purified by repeated precipitation. The resulting polymer was dried in vacuum. The polymer was soluble in water, methanol, chloroform, and THF and insoluble in hexanes. An amorphous white solid was obtained. 1H NMR (500 MHz, CDCl3, δ): 3.98 (d in Figure S2, −CH(CH3)2 in P(NIPAAm)), 1.19 (a, −CH(CH3)2 in P(NIPAAm)), 1.20−2.48 (b and c, P(NIPAAm) main chain CH− and −CH2−), 2.58 (h, −OCOCH2(CH2)4− in PCL), 2.65 (j, −OCOCH2CH2CH2CH2CH2− in PCL), 1.41 (k, −OCOCH2CH2CH2CH2CH2− in PCL), 4.05 (h, −OCOCH2CH2CH2CH2CH2− in PCL) (Figure S2). FT-IR (KBr pellet, cm−1): 1720 cm−1 (CO ester in PCL). MS (MALDI TOF): 13 828 [M]+. PDI = 1.3 (Mw/Mn). Preparation of Magnetic Micelles. SPION micelles were formed by a solvent-evaporation method.14 A set of samples with various weight ratios (1:0, 1:0.2, 1:0.5, 1:1, 1:2, 1:3, 1:4, and 1:5) of P(NIPAAm-co-AAm)-b-PCL and SPIONs were prepared. The polymer−MNP mixture was dissolved in 10 mL of chloroform. After the complete evaporation of chloroform under a gentle flow of nitrogen, the dried film was dissolved in THF (1 mL) in a glass vial. The solution was added dropwise to Milli-Q water (10 mL) under vigorous ultrasonication (40 kHz, 130 W, Branson 2510, Branson, Danbury, CT) for 5 min. The solution was open to air, allowing for the slow evaporation of the organic solvent and the formation of micelles. The final volume was adjusted to 10 mL with Milli-Q water. The micelle solution was filtered through a nylon syringe filter (pore size 0.2 μm, Whatman, Clifton, NJ) to reduce the size polydispersity. Raman Spectroscopy. Raman spectra of aqueous samples were collected in the temperature range of 25−55 °C using a Renishaw inVia Raman microscope. Samples were excited using the 514 nm line of an Ar+ laser focused through a 50×, 0.5 N.A. objective. The backscattered signal was collected with the same objective, passed through a notch filter to reject the excitation wavelength, and then dispersed on a cooled CCD camera using a 1800 lines/mm grating. The sample was placed inside a TMS600 heating stage (Linkam Scientific Ltd., Tadworth, England), which allows for precise temperature control. Drug Loading and in Vitro Drug Release. A solution of DOX in chloroform (1 mg/mL) containing TEA (molar ratio 1:1) was loaded into SPION micelles. Magnetic micelles were formed by a solventevaporation method as described above. The DOX-loaded SPION micelle (DOX-SPION micelles) solution was divided into two parts.
mer micelles as magnetic hyperthermia-mediated drug release and imaging agents. PNIPAAm is a well-known thermosensitive polymer that exhibits lower critical solution temperature (LCST) phase-transition behavior at around 32 °C. The latter can be tailored by incorporating either hydrophilic or hydrophobic copolymer into the PNIPAAm structure, resulting in an increase or decrease in the LCST, respectively.25 Here, PNIPAAm was copolymerized with acrylamide (Am) in order to adjust the LCST to 43 °C, which translates to the stability of the polymer structure at the physiological temperature of 37 °C, thus making it suitable for applications involving living cells. Loading polymeric micelles with magnetic particles allows, on the one hand, for remote magnetic modulation of the polymer hydration state resulting in controlled payload release from the micelle core. On the other hand, such micelles can be used as carriers of locally concentrated nanoparticles serving as contrast enhancement agents for magnetic resonance imaging (MRI). The article is organized as follows. First, we discuss the procedure for the formation of micelles with encapsulated superparamagnetic inorganic magnetic nanoparticles (SPIONs) and an anthracycline anticancer antibiotic, doxorubicin (DOX). Second, the results of a Raman spectroscopic study of the changes in micelle structure as a function of temperature will be discussed. Third, we investigate two different approaches for inducing drug release by either heating the micelles in a water bath or heating them in the external ac magnetic field, with the latter approach enabled by the presence of superparamagnetic iron oxide particles in micelles. The surface functionalization of the micelles with the integrin β4 antibody and interaction of the resulting nanobiohybrid with various cell lines are discussed in the next section. Finally, we demonstrate that the SPIONloaded micelles can be used as contrast enhancement agents for MRI.
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MATERIALS AND METHODS
Fe(acac)3, 1,2-hexadecanediol, oleic acid, oleylamine, benzyl ether, tetrahydrofuran (THF), chloroform, hexane, methanol, N-isopropylacrylamide (NIPAAm), acrylamide (AAm), 2-mercaptoethanol (ME), 4,4′-azobis(4-cyanopentanoic acid) (ACPA), ε-caprolactone (CL), tin(II) 2-ethylhexanoate (Sn(Oct)2), pyrene, doxorubicin (DOX) hydrochloride, triethylamine (TEA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemical Co. (Saint Louis, MO). N-Hydroxysulfosuccinimide (sulfo-NHS) was obtained from Pierce (Rockford, IL). 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC) was obtained from Sigma. Integrin β4 (A9) antibody and its fluorescein conjugate (A9-FITC) were from the Santa Cruz Biotechnology, Inc. Near-IR lipophilic dye 1,1′-dioctadecyl3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD), was from Invitrogen. Dialysis cassettes (2000 MW cutoff) was obtained from Pierce (Rockford, IL). General Methods. The crystal structure, size, and magnetic properties of the synthesized samples were characterized by X-ray diffractometry (Philips X’Pert Pro diffractometer), vibrating sample magnetometry (model 7400, Lake Shore, Westerville, OH), and transmission electron microscopy with a cryo sample holder (−176 °C) (CM 30, Philips, Mahwah, NJ) operating at 120 kV. The molecular weight of the copolymers was measured with a Voyager DEPRO MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA). The 1H NMR spectra were recorded on a Bruker DMX-500 spectrometer in CDCl3, with tetramethylsilane (TMS) as an internal standard. Fourier transform infrared FT-IR spectra of the samples pressed into potassium bromide pellets were recorded with a Vertex 70 Bruker FTIR spectrophotometer. The morphology of self-assembled magnetic micelles was investigated by atomic force microscopy (Nano V, Veeco, Santa Barbara, CA). The mean particle size distribution of B
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Figure 1. (a) Topographic AFM image of polymer micelles loaded with magnetic nanoparticles. (b) TEM image of several micelles and (c) a closeup view of a single micelle clearly showing the encapsulated iron oxide NP. The first part was frozen at −80 °C and then lyophilized using a freeze-dry system (FreeZone 1 L, Labconco, Kansas, MO) for 36 h at −40 °C. The freeze-dried micelles were dissolved in DMSO to determine the drug-loading efficiency. The measured drug-loading efficiency of as-assembled MNP micelles was ∼66%. The remaining portion of DOX-SPION micelles was employed for the drug release test. The micelles were precipitated magnetically, dispersed in phosphate-buffered saline (PBS), and dialyzed in Milli-Q water for 24 h. The dialyzed DOX-SPION micelle solution (4 mL) was placed into a preswollen membrane bag (Spectra/Por MWCO 3500, Spectrum, Los Angeles, CA) and then immersed in 40 mL of PBS. The temperature of the medium was controlled to 37 or 43 °C with a water bath. Aliquots of PBS were aspirated periodically and replaced with a fresh solution to keep the volume of the solution constant. The amount of DOX released from the micelles into PBS was measured using UV absorbance at 480 nm (UV−vis spectrophotometer, Lambda 950, Perkin-Elmer, Waltham, MA). Inductive Heating Experiments. An external ac magnetic field with a fixed frequency of 330 kHz was generated using a 3.5 kW power supply (Ameritherm HOTSHOT 3.5, Scottsville, NY) equipped with a heating station and a custom-made coil. The temperature of the sample was monitored remotely with an FDA-certified infrared camera (ICI 7640 P-series infrared camera, Beaumont, TX). The heating effect was controlled by varying the micelle concentration, magnetic field amplitude, and exposure time. Surface Functionalization of the Magnetic Micelles. Six microliters of 88 mM sulfo-NHS and 4 μL of 50 mM EDAC in a 10 mM PBS buffer (pH 6.3) were mixed with 300 μL of 2.5 mg/mL magnetic micelles. The reaction mixture was incubated for 1 h at room temperature with continuous gentle shaking in the dark, and 1 μL of 2mercaptoethanol was added to quench the excess EDAC. The A9FITC (100 μg, excitation 494 nm, emission max 521 nm) antibody in 50 μL of 100 mM phosphate buffer at pH 7.6 was mixed with the preactivated micelles (hydroxysulfosuccinimide ester) and incubated for 6 h at room temperature with continuous gentle mixing in the dark. The reaction mixture was placed into 2000 MW cutoff dialysis bags and dialyzed against 2 L of Milli-Q water for 4 h. The dialyzed product was mixed with 25 μL of 25 mM glycine in 10 mM PBS buffer at pH 7.4 and incubated for 15 min to quench the remaining active sites on the particle surfaces. The final FITC-A9-modified magnetic micelles were spin-washed three times with 500 μL of 0.1 M PBS at pH 7.4 to remove unbound protein and then redispersed in 500 μL of 0.1 M PBS at pH 7.4 and stored at 4 °C. FT-IR (cm−1): 1650 (amide I) and 1550 (amide II) cm−1. MRI Relaxivity Measurements. T1 and T2 relaxivities of the Fe3O4-loaded micelles and Fe3O4 nanoparticles were measured on a 9.4 T MRI scanner (BioSpec, Bruker, Billerica, MA). Samples with different concentrations of magnetic carriers were suspended in an agarose gel (1%) matrix. The atomic Fe concentrations of the stock solutions were determined using inductively coupled plasma optical emission spectroscopy (Optima 3300 (ICP-OES), Perkin-Elmer, Waltham, MA). The samples were dissolved in concentrated hydrochloric acid. For relaxation time measurements, a multiecho fast spin−echo sequence (TR = 5000 ms) was used to collect a series of data points simultaneously at different echo times (TE of 15 in 15
ms increments). The relaxation time was calculated by fitting the decay curve using the nonlinear monoexponential algorithm of M(TE) = M0exp(−TE/T2). Assessment of the Magnetic Micelles Acute Cytotoxicity and Particle−Cell Interaction. The intrinsic cytotoxicity (with no magnetic field applied) of the magnetic micelles was assessed for five cancer cell lines from the American Type Culture Collection: A375 melanoma, MDA.MB.231 breast cancer, HeyA8 ovarian cancer, oral squamous cell carcinoma SQ20B, and Cal-27 squamous cell carcinoma (ATCC CRL-2095). In addition, noncancerous normal human keratinocytes NHEK were used. Cells were cultured in 96-well plates to reach a concentration of ∼500 cells/well. The magnetic micelles (5 × 106 micells/mL) were added to the cells and incubated for 48 h under standard conditions, and then their viability was evaluated using the CellTiter-Blue (Promega) in accordance with the manufacturer’s protocol. Complete culture media appropriate for each cell line were used as a negative control. For a visualization of the interaction between cells and magnetic micelles, the A9-FITC-biofunctionalized micelles were loaded with near-IR fluorescent, lipophilic dye DiD (exitation 644 nm, emission 665 nm) in a similar way as described above for DOX loading. Cells were incubated with surface biofunctionalized magnetic micelles for 1 h under standard culture conditions, washed with culture medium three times to remove all unbound micelles, and then supplemented with fresh medium. Magnetic micelles with no surface functionalization were used as a control.
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RESULTS AND DISCUSSION Synthesis and Characterization of Magnetic Micelles. Iron oxide nanoparticles with an 11 nm diameter were synthesized by seed-mediated thermal decomposition. The selected size of the nanoparticles is known to be optimal for magnetically induced hyperthermia,29 which in our case translates to the favorable conditions for heating the temperature-responsive polymer. Amphiphilic random block copolymer P(NIPAAm-co-AAm)-b-PCL), with Mn = 13 828, was synthesized via radical polymerization followed by ring-opening polymerization. The lower critical solution temperature (LCST) of P(NIPAAm-co-AAm), representing the coil-toglobule transition in the configuration of a hydrated polymer chain, was tailored to ∼45 °C by the copolymerization of NIPAAm with the hydrophilic acrylamide (AAm) monomer in a molar ratio of 86:14 (PNIPAAm/AAm). Then, the resulting P(NIPAAm-co-AAm) (Mn = 11 545) copolymer was further copolymerized with polycaprolactone (PCL). The amphiphilic properties of this copolymer allow for the formation of micellar structures in aqueous solution with a hydrophobic PCL core and a hydrophilic P(NIPAAm-co-AAm) outer layer. Adding a payload of interest (e.g., nanoparticles, quantum dots, drugs, dyes, etc.) to the copolymer solution results in its subsequent encapsulation in micelle cores, as long as the payload is hydrophobic in nature. The LCST of the micelles was C
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Figure 2. (a) Temperature-dependent Raman spectra of 10 wt % micelles in water. (b) Ratio between the area of the C−H (AC−H) and O−H stretching vibrational bands (AO−H), which is proportional to the level of micelle hydration.
around the phase-transition temperature of 43 °C (Figure 2b), which is in agreement with the cloud point measured by UV− vis spectroscopy. At this temperature, water dissociates from around the N−H and CO bonds of the polymer, resulting in the dehydration of micelles. As the temperature increases further, the magnitude of AC−H/AO−H decreases because the water molecules diffuse within the relaxed PCL chains in the core, caused by approaching a melting point (Figure 2b). Temperature-Controlled Drug Release from Magnetic Micelles. Heating Micelles in a Water Bath. The temperature-responsive behavior of SPIONs-loaded micelles can be directly utilized for controlled drug release. Micelles with a 1:1 weight ratio of polymer to SPION were selected in order to study noncovalent binding and thermostimulated drug release using the antitumor anthracycline antibiotic doxorubicin (DOX) as a model payload. A vial with micelle solution was placed in a water bath, and the temperature was cycled between 37 and 43 °C two times and then increased to 55 °C and finally cooled to the initial 37 °C. The corresponding amounts of the released DOX with respect to the initial amount of the loaded drug are shown in Figure 3. Only an insignificant amount of DOX was released from stable magnetic micelles at a normal physiological temperature (37 °C). However, an accelerated
measured to be 43 °C (Figure S3). Importantly, the PCL softening starts at ∼40 °C with the melting transition occurring at 57 °C.30 It is therefore reasonable to expect that the micelles will be stable up to this temperature whereas their structure can be altered starting at ∼40 °C because of the increased permeability and the temperature-sensitive movement of the outer shell. This has direct implications for facilitating the drug release. Importantly, the micelles are expected to be stable at a physiological temperature of 37 °C, which allows us to employ them in conjunction with living cells. The size of the SPION-loaded micelles was measured to be around 70 nm in the swollen state and ∼52 nm when dried on a mica substrate for atomic force microscopy (AFM) measurements. A high nanoparticle loading capacity of micelle is required for efficient heating of the composite micelles by an applied ac magnetic field. Transmission electron microscopy (TEM) morphology studies of SPION-loaded micelles revealed that for the weight ratios from 1:1 to 1:3 between amphiphilic P(NIPAAm-co-AAm)-b-PCL and magnetic nanoparticles, stable core−shell structures with diameters
