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Magnetically Triggered Multidrug Release by Hybrid Mesoporous Silica Nanoparticles Alejandro Baeza,‡ Eduardo Guisasola,†,§ Eduardo Ruiz-Hernández,‡ and María Vallet-Regí†,‡,* †

Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain ‡ Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, CIBER-BBN, Spain § Grupo de Dispositivos Magnéticos, Instituto de Sistemas Optoelectrónicos y Microtecnología (ISOM), Universidad Politécnica de Madrid, 28040 Madrid, Spain S Supporting Information *

ABSTRACT: The treatment of complex diseases such as cancer pathologies requires the simultaneously administration of several drugs in order to improve the effectiveness of the therapy and overwhelm the defensive mechanisms of tumor cells, responsible of the apparition of multidrug resistance (MDR). In this manuscript, a novel nanodevice able to perform remotely controlled release of small molecules and proteins in response to an alternating magnetic field has been presented. This device is based on mesoporous silica nanoparticles with iron oxide nanocrystals encapsulated inside the silica matrix and decorated on the surface with a thermoresponsive copolymer of poly(ethyleneimine)-b-poly(N-isopropylacrylamide) (PEI/ NIPAM). The polymer structure has been designed with a double purpose, to act as temperature-responsive gatekeeper for the drugs trapped inside the silica matrix and, on the other hand, to retain proteins into the polymer shell by electrostatic or hydrogen bonds interactions. The nanocarrier traps the different cargos at low temperatures (20 °C) and releases the retained molecules when the temperature exceeds 35−40 °C following different kinetics. The ability to remotely trigger the release of different therapeutic agents in a controlled manner in response to a nontoxic and highly penetrating external stimulus as alternating magnetic field, along with the synergic effect associated to hyperthermia and chemotherapy, and the possibility to use this nanocarrier as contrast agent in magnetic resonance imagining (MRI) convert this nanodevice in an excellent promising candidate for further studies for oncology therapy. KEYWORDS: controlled drug release, hyperthermia, magnetic mesoporous nanoparticles, stimuli-responsive

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INTRODUCTION Nanoparticles as drug delivery devices can be made using different materials including polymers, lipids, ceramics, and even virus capsides.1−6 The development of nanocarriers able to transport a unique drug combination to specific target cells or tissues can significantly improve the efficacy of the treatment of many pathologies, specially cancer diseases.7 A successful nanocarrier may be engineered to evade the reticuloendothelial system, must present high biocompatibility, protection of active therapeutic drugs, colloidal stability, and improved pharmacokinetics. One of the main problems associated to cancer diseases corresponds to the acquired multidrug resistance (MDR) upon repeated chemotherapy cycles.8 It is hypothesized that high doses of different therapeutic agents, such as small molecules, antibodies, proteins, enzymes, and oligonucleotides, among others, may overwhelm these resistance mechanisms.9,10 Biotechnology provides a great number of peptide and protein drugs for clinical applications.11 However, their use is hampered by the high vulnerability of these molecules in physiological conditions due to degradation by enzymes and proteases. An interesting strategy is based on the © 2012 American Chemical Society

encapsulation of the macromolecules into a suitable matrix in order to achieve the controlled protein release, protecting the cargo until reaching the target tissue.12−14 Mesoporous silica nanoparticles (MSNs) show very interesting properties for the application in the development of drug delivery devices, such as stable mesoporous structure, high surface areas (ca. 1000 m2/g), large pore volume (ca. 1 cm3/g), regular and tunable mesopore diameters (2−50 nm), and pore channel systems homogeneously organized in hexagonal (2D) or cubic (3D) mesostructures. The biocompatibility of these materials has been recently proved.15,16 Moreover, Lin et al.17 have demonstrated the good hemocompatibility of MSNs, which has great significance for intravenous administration of these drug nanocarriers. The drug delivery ability of this mesoporous materials has also been modified via reversible capping of the pore surface, providing these materials with the ability to control the release in response to different external Received: October 5, 2011 Revised: January 4, 2012 Published: January 11, 2012 517

dx.doi.org/10.1021/cm203000u | Chem. Mater. 2012, 24, 517−524

Chemistry of Materials

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stimuli such as pH,18−20 temperature,21,22 light,23,24 magnetic fields,25,26 redox reactions,27,28 enzymes,29,30 and even antibodies.31 However, less effort has been made to produce smart multidrug delivery nanocarriers able to release two or more agents that present different chemical natures.32−34 In this text, we report on the synthesis of a new thermoresponsive hybrid polymer/magnetic mesoporous silica nanocarrier able to release different cargos, proteins, and small molecules, housed in the polymer branches and inside the mesoporous matrix, respectively, in a controlled manner in response to an external alternating magnetic field. Superparamagnetic iron oxide nanocrystals have been trapped inside the mesoporous silica matrix, in order to provide the heating capability under alternating magnetic fields, which is also suitable for hyperthermia treatment of cancer.35−37 As a reversible gating system, the thermoresponsive polymer based on the well-known properties of poly(N-isopropylacrylamide) (PNIPAM) has been attached on the mesoporous surface. This polymer exhibits phase transition at a lower critical solution temperature (LCST) of approximately 32 °C in water.38−40 Below this value, the polymer is hydrated and blocks the release of the drugs trapped inside the pores, whereas it releases water and suffers from shrinkage if the temperature is increased, which involves pore opening and subsequent release of the housed drugs. Different strategies for producing mesoporous silica−PNIPAM hybrid nanoparticles have been recently published,41−46 but as far as we know, all of them are focused on the release of a small molecule encapsulated inside the mesoporous matrix in response to temperature changes. In our work, branched polyethyleneimine (PEI) chains (Mn ≈ 10 KDa) have been grafted to the end of each of the PNIPAM chains, and N,N′-methylenebis(acrylamide) (MBA) has been used as cross-linker in order to increase the electrostatic interactions with the housed proteins and to create a more extensively branched polymeric network, respectively, hampering as much as possible the premature protein release. The polymer shell is responsible for the partial retention of the macromolecules when the temperature is below the LCST, while provoking the pore opening and a significant increase in the protein release rate at higher temperatures, due to polarity inversion (hydrophilic to hydrophobic transition) and tridimensional polymeric network changes. The nanodevice as produced combines different interesting properties; on one hand it can release different therapeutic agents in response to a nontoxic and highly penetrating in living tissues external stimulus, as magnetic field. At the same time, the ability to increase the temperature of the surroundings by the trapped superparamagnetic iron oxide nanocrystals under alternating magnetic fields can improve the therapeutic effect of the released cytotoxic drugs by a synergic effect.47 Finally, the presence of the magnetic nanocrystals could allow that the nanocarriers be magnetically targeted to the desired place using a permanent magnet48,49 or could be used as contrast agents in magnetic resonance imagining (MRI).50 We envision that this multifunctional nanodevice can be a promising candidate for further studies for oncology therapy.

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ammonium persulfate (APS), trypsin inhibitor protein (type II−S: soybean, 21500 Da), catalase from bovine liver (powder, 2000−5000 units/mg protein), and fluorescein sodium salt were also purchased from Aldrich Inc. These compounds were used without further purification. Deionized water was purified by passage through a MilliQ Advantage A-10 Purification System (Millipore Corporation) to a final resistivity of 18.2 M·cm. All other chemicals (65 wt % nitric acid, 25 wt % ammonia, absolute ethanol, acetone, etc.) were of the best quality commercially available and used as received. Characterization Techniques. Powder X-ray diffraction (XRD) experiments were performed with a Philips X’Pert diffractometer equipped with Cu Kα radiation (wavelength 1.5406 Å). XRD patterns were collected in the 2θ range between 0.6° and 8 with a step size of 0.02° and counting time of 5 s per step. Fourier transform infrared spectroscopy (FTIR) was measured in a Thermo Nicolet nexus equipped with a Goldengate attenuated total reflectance device. The textural properties of the materials were determined by nitrogen sorption porosimetry by using a Micromeritics ASAP 2020. To perform the N2 measurements, the samples were previously degassed under vacuum for 24 h at room temperature. Thermogravimetry (TGA) was performed in a Perkin-Elmer Pyris Diamond TG/DTA analyzer, with 10 °C/min heating ramps, from room temperature to 800 °C. A vibrating sample magnetometer (VSM, ISOM, UPM, Madrid) with a maximum applied continuous field of 5000 Oe was used, at room temperature, to study the magnetic properties. The hydrodynamic size of magnetic nanoparticles in the ferrofluid and the mesoporous silica particles was measured by means of a Zetasizer Nano ZS (Malvern Instruments) equipped with a 633 nm “red” laser. Fluorescent spectrometry was used to determine fluorescein release (λexc 490, λem 514 nm) by means of a Biotek Synergy 4 device. Transmission electron microscopy (TEM) was carried out with a JEOL JEM 2000 FX instrument operated at 200 kV, coupled with energy-dispersive X-ray spectroscopy (EDX) and equipped with a CCD camera (KeenView Camera). Sample preparation was performed by dispersing in distilled water and subsequent deposition onto carbon-coated copper grids. Scanning electron microscopy (SEM) analyses were made on a JEOL 6400-LINK AN10000 microscope (Electron Microscopy Center, UCM). The samples underwent Au metallization previous to observation. Calculation Procedures. The surface area was determined using the Brunauer−Emmett−Teller (BET) method, and the pore volume, Vpore (cm3/g−1), was estimated from the amount of N2 adsorbed at a relative pressure around 0.99. The pore size distribution between 0.5 and 40 nm was calculated from the desorption branch of the isotherm by means of the Barrett−Joyner−Halenda (BJH) method. The mesopore size, Øpore (nm), was determined from the maximum of the pore size distribution curve. Synthesis of Maghemite, γ-Fe2O3. The superparamagnetic maghemite nanocrystals were obtained by coprecipitation of Fe(II) and Fe(III) chloride. A total of 31.33 g of FeCl2·4H2O (0.156 mol) was dissolved in 3.5 L of H2O (mQ), pouring a second solution of FeCl3·6H2O (86.05 g, 0.312 mol) in 170 mL of 1.5 M HCl under strong stirring, yielding nanometric magnetite (Fe3O4). To the previous solution was added 300 mL of ammonia (25%) under strong stirring during 15 min, followed by two days of decantation on a magnet. The black flocculate was dispersed to a 2 M HNO3 solution and stirred for 2−3 min. After decantation, the particles were oxidized to maghemite by pouring a solution of 81.62 g of Fe(NO3)3·9H2O in 600 mL of H2O (mQ) at 100 °C, keeping that temperature for 30 min. Another decantation (2−3 h) was carried out, and the product was dispersed to a 2 M HNO3 solution and stirred during 15 min. Over the last decantation, the ferrofluid was washed with acetone several times and finally dispersed in water to a concentration of 21.08 mg·mL−1 (as measured by titration with K2Cr2O7). The so-obtained ferrofluid was composed of magnetic nanocrystals with an average diameter of 16 nm in acidic pH, as measured by dynamic light scattering. Synthesis of Magnetic Mesoporous Silica Nanoparticles (MMSNs). To a 1 L round-bottom flask were added 1 g of CTAB as a structure-directing agent, 480 mL of H2O (HPLC-grade), 3.5 mL of NaOH (2 M), and the corresponding amount of maghemite (21.08 g/

EXPERIMENTAL SECTION

Reagents. The following compounds were purchased from SigmaAldrich Inc.: Iron chlorides, ammonium nitrate, cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), and 3(trimethoxysilyl)propyl methacrylate (MPS). For polymerization and release experiments, NIPAM, PEI (branched, Mn ∼ 10 000), MBA, 518



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L) (250, 500, and 1000 mg, respectively). The suspension was heated to 80 °C and stirred at 1000 rpm. When the reaction mixture reached 80 °C, 5 mL of TEOS were added dropwise, and the suspension was stirred during 2 h. The reaction mixture was filtered and washed with 50 mL of H2O and then three more times with 50 mL of EtOH. The surfactant template was removed by ion exchange using an extracting solution with 1.59 g of NH4NO3, 573 mL of EtOH (99.5%), and 27 mL of pure water. The mixture was heated to 65 °C and stirred for 15 min. Then, the brown solid obtained was washed with 50 mL of EtOH. The extraction was repeated three more times. The samples were named as MMSN25, MMSN50, and MMSN100, respectively. Synthesis of MMSN-MPS. A total of 0.25 g of MMSN100 was added to 10 mL of dry toluene containing 0.6 mL of MPS. After being stirred during 12 h at 110 °C, the mixture was filtered, washed with toluene and diethylether, and air-dried. Synthesis of MMSN-NIPAM. A total of 250 mg of MMSN-MPS was suspended in 125 mL of H2O (Milli-Q). A total of 2.125 g of NIPAM and 330 mg of MBA were added to this suspension. A second solution of 440 mg of PEI in 125 mL of EtOH (99.5%) was added to the previous suspension. The reaction mixture was frozen, and the flask was deoxygenated by three vacuum/N2 cycles. Then, 5 mg of APS were added, and the solution was stirred overnight under N2 at 30 °C. The reaction mixture was filtered (nylon, 0.22 μm) and washed several times with H2O (Milli-Q) and EtOH (99.5%). The orange powder obtained was dried under vacuum during 1.5 h at room temperature. Loading and Release of Fluorescein and Soybean Trypsin Inhibitor type II−S (STI). A total of 50 mg of MMSN-NIPAM was added to 5 mL of fluorescein sodium salt aqueous solution (15 mg/ mL), and the suspension was stirred in an orbital shaker at 100 rpm and 45 °C overnight. Then, the mixture was rapidly cooled below 15 °C and washed with cold water. For the protein loading, the asobtained solid was added to 3 mL of STI protein solution (10 mg/ mL) in phosphate buffered saline (PBS 0.1 M, pH 7.2) at 15 °C and was gently stirred overnight. After that time, the sample was filtered and washed with cold water. Finally, the products were dried under vacuum at 25 °C. In vitro release of fluorescein and STI protein from 50 mg of the corresponding loaded particles was carried out in 10 mL of PBS solution. Temperature changes were produced in an Infors-HT Ecotron incubator shaker. The alternating magnetic field at 24 kA·m−1 and 100 kHz was applied within a thermostatic chamber at 20 °C by means of a Celes AC function generator. Variations of temperature were monitored by Luxtron fluoroptic probes inside and outside the liquid. The amount of fluorescein released was measured by fluorescent spectrometry (λexc 490, λem 514 nm). The amount of protein released was measured using a Waters 2695 HPLC. The employed column was a Symmetry300 C4 column 4.6 × 150 mm containing 5 μm of C4 silica beads, using injected volumes of 100 μL. The mobile phase consisted of acetonitrile with 0.1% trifluoroacetic acid (A) and water with 0.08% trifluoroacetic acid (B) at 303 K. The gradient was 20−95% B in 40 min at a flow rate of 0.5 mL/min. Under these conditions, the retention time of the protein was 4.4−4.5 min and fluorescein was 19.0−19.1 min. Detection was performed by UV at 190−380 nm, and chromatograms were recorded on a Millenium integrator.

obtain particles within this size distribution and high content of trapped iron oxide nanocrystals, a modification of the previously published method by Zink et al. has been employed.54 In our new approach, TEOS was added to an aqueous solution of nonfunctionalized iron oxide particles, CTAB and NaOH as base, at 80 °C, because the size distribution of the obtained mesoporous silica particles is highly dependent on the reaction temperature and the nature of the employed base.55 In order to achieve the higher degree of iron oxide encapsulation and obtain a suitable material that could combine controlled release with hyperthermia treatment, different amounts of iron particles (25, 50, and 100% w/w respect to CTAB) have been added to the synthesis mixture to provide MMSN25, MMSN50, and MMSN100, respectively. Once the surfactant is removed, all of the samples show a narrow average size distribution, centered on 50−100 nm, measured by dynamic light scattering (Figure S1, Supporting Information). The percentage of encapsulated maghemite estimated by VSM measurements is higher in the sample MMSN100 (Figure

Figure 1. VSM curve of (a) MMSN25, (b) MMSN50, and (c) MMSN100 and percentage of encapsulated maghemite.

1). Despite the fact that the small-angle XRD pattern of this material shows that the characteristic profile of MCM-41 materials is affected by the encapsulation process (see Figure S2, Supporting Information), the nitrogen sorption isotherm (Figure 2a) displays a characteristic adsorption step in the 0.1− 0.3 relative pressure range and can be classified as type IV according to IUPAC. The absence of the hysteresis loop is assigned to mesoporous matrices with cylindrical pores open at both ends. The surface area of the particles is 747 m2·g−1, with pore size distribution centered at 2.7 nm (see inset of Figure 2). These results indicate that MMSN100 presents high loading capacity surface area and has been chosen as the best candidate for further studies. Once MMSN100 was selected as the best magnetic mesoporous material, the nanoparticle surface was functionalized with MPS, necessary for the further polymer attachment

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RESULTS AND DISCUSSION Maghemite nanoparticles were synthesized and encapsulated into MCM-41 type mesoporous silica materials. In previous works,25,51 we managed to encapsulate a certain amount of magnetic nanocrystals within mesoporous particles by the addition of TEOS at room temperature to a solution containing CTAB, iron oxide nanoparticles, and NH4OH as base. However, the size distribution of the obtained particles was centered on 200 nm and between 0.3 and 3 μm in each case, which could be slightly large for drug delivery applications, where the best value is around 50−100 nm.52,53 In order to 519



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Figure 3. TG curves of samples of (a) MMSN100, (b) MMSN-MPS, and (c) MMSNNIPAM.

Figure 2. N2 adsorption isotherms of samples: (a) MMSN100, (b) MMSN-MPS, (c) MMSN-NIPAM, and (d) fluorescein and soybean trypsin inhibitor loaded MMSN-NIPAM.

FT-IR was employed to verify the successful functionalization with MPS and the polymer attachment (Figure 4). For

(Scheme 1). The reaction was performed by the addition of MPS to a solution of MMSN100 in dry toluene at 110 °C. The amount of functional groups grafted was around 10% determined by TGA (Figure 3b). As mentioned in the Introduction, the aim of the work was to prepare a material able to release different chemical agents housed inside the pores and between the branches of the attached polymer, respectively. Polyethyleneimine coated nanoparticles have been widely used as DNA carriers due to the strong affinity of the negatively charged phosphate groups of the DNA with the polycationic shell.56−58 This polymer can also present a strong affinity for certain proteins through the formation of hydrogen bonds or electrostatic interactions and can be used as protein carrier.59,60 Moreover, the presence of a polycationic shell on the surface of a nanocarrier enhances the cellular uptake, improving the drug delivery efficacy.61 Copolymerization of poly-NIPAM-block-PEI and in situ attachment of the polymer on the mesoporous surface was performed by radical polymerization of NIPAM and MBA as cross-linker agent, using APS as initiator in the presence of PEI and MMSN-MPS particles. The presence of PEI served as promoter as well as a starting block62 in such a way that the PEI block is always at the end of the polymeric chain attached on the nanoparticle surface. The amount of attached polymer was 18−20% determined by TGA (Figure 3c). Textural parameters of MMSN100 (Figure 2b,c) are subsequently decreased after functionalization (30% reduction of surface area, 50% reduction of pore volume) and after the polymer attachment (60% reduction of surface area, 75% reduction of pore volume), which suggests an effective blockage of the access to the mesoporous channels.

Figure 4. FTIR spectra of (a) MMSN100, (b) MMSN-MPS, and (c) MMSN-NIPAM.

bare magnetic mesoporous silica nanoparticles, the characteristic absorption peaks of the tetrahedron silica structures at 1100 cm−1 (SiO stretching) and 800 cm−1 (SiOSi bending) were clearly observed. After the functionalization with MPS, the peaks corresponding to sp3 CH and CO (methacrylate) stretching vibrations appeared at 2980 cm−1 and 1700 cm−1, respectively. Finally, the polymer attachment on the silica surface was confirmed by the presence of the characteristic amide bands at 1650 cm−1 (CO stretching) and 1553 cm−1 (NH stretch) and the significant increase of the sp3 CH band. It is important to note that the superparamagnetic

Scheme 1. Synthesis of MMSN-NIPAM

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Figure 5. SEM and TEM images of samples MMSN100 (a, d), MMSN-MPS (b, e), and MMSN-NIPAM (c, f) (obtained by stained uranyl acetate and slight overfocus in order to enhance the polymer coating contrast).

heated to 40 °C, indicating that the LCST should be between 30 and 40 °C, which is in accordance with the typical values of PNIPAM brushes. The fluorescein release profile over time when the temperature is maintained at 45 °C (Figure 7a) follows the typical pattern associated to uncapped mesoporous

behavior of MMSN-NIPAM is maintained after the polymerization attachment since no hysteresis is shown in the magnetization curve (Figure S3, Supporting Information), showing a percentage of encapsulated maghemite of 38−40%. According to the TEM and SEM images (Figure 5), the functionalization with MPS and subsequent polymer attachment barely alters the morphology and size of the particles. Staining of the polymer by uranyl acetate (Figure 5f) allows a clear visualization of the organic shell structure. In order to establish the LCST of the polymer grafted onto the nanoparticle surface, MMSN-NIPAM was soaked with fluorescein (15 mg/mL) in a phosphate buffered solution (PBS 0.1 M, pH 7.2) at 45 °C during 12 h. After that, the material was filtered and vigorously washed with cold water (10−15 °C) in order to remove the adsorbed fluorescein molecules on the surface. The amount of loaded fluorescein was 5−8%, determined by TGA. The as loaded particles were suspended in a phosphate solution and stirred in an orbital shaker (100 rpm) at the corresponding temperature during 5 h. After that time, the suspension was centrifuged and the fluorescein amount of the supernatant media was measured by fluorescence spectroscopy. As can be observed in Figure 6, the maximum amount of fluorescein released was obtained when the sample is

Figure 7. Fluorescein release profile over time at different temperatures: (a) 45 °C (⧫) and (b) 20 °C (■).

matrices. On the other hand, if the temperature is set at 20 °C, the fluorescein release is greatly hindered (Figure 7b). Once the ability of this system to release drugs trapped inside the mesoporous channels in response to temperature changes was demonstrated, the next step was to determine if this material is able to release two different molecules, one of them housed inside the pores and the other one retained in the polymer branches, by the application of an external stimulus. Moreover, the presence of superparamagnetic crystals encapsulated into the mesoporous silica nanoparticles can be used as a heating source by the application of an alternating magnetic field. In order to investigate the stimuli-response behavior of this material, the sample MMSN-NIPAM was loaded with fluorescein following the procedure described above, and then it was soaked in a solution of cold water (10−15 °C) in the presence of a protein model, soybean trypsin inhibitor type II− S (STI) during 12 h. After that, the particles were filtered and

Figure 6. Determination of LCST of MMSN-NIPAM particles. 521



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Figure 8. Time dependent protein release at 20 °C, 45 °C, and under alternating magnetic field exposition, as determined by HPLC.

Figure 9. Time dependent fluorescein release at 20 °C, 45 °C, and under alternating magnetic field exposition, as determined by fluorescence spectroscopy.

during 6 h. The effect of the temperature and magnetic field on the protein and fluorescein release is shown in Figures 8 and 9, respectively. Regarding the protein release (Figure 8), during the first hour the release rate is very similar in the three samples, which can be due to the weakly attached protein located in the outer surface of the polymer shell. After that time, the sample placed at 20 °C shows a slow release, reaching a final value of almost 50% of the released protein by the sample heated at 45 °C. The profile of the protein release using magnetic fields was similar to the heated sample, which is consistent with the hypothesis that the polymer collapses and changes to a more hydrophobic surface, causing the increase in the release rate of the attached protein. Surprisingly, the fluorescein release under magnetic field is faster than that achieved at 45 °C (Figure 9). A similar effect using magnetic silica-based microspheres loaded with ibuprofen has been recently described.63 The higher fluorescein release achieved with magnetic field, as ibuprofen in the previously cited work, can be due to the rapid rotation of the magnetic nanocrystals trapped in the silica matrix by the application of high-frequency

washed with cold water to remove the proteins weakly attached on the polymer surface. It is important to note that during the protein loading step, about 5−10% of the previously trapped fluorescein was prematurely released. This quantity was determined by measuring the fluorescence in the employed loading media and the washing solutions. At the end of the process, the total amount of fluorescein and protein retained was 15−20% determined by TGA. The as-prepared sample was divided into three batches; two of them were soaked in a phosphate buffered solution (PBS 0.1 M, pH 7.2) and placed in a oven at 20 and 45 °C, respectively, in order to test the response to temperature changes above and below the LCST of the polymer shell. The third batch was exposed to an alternating magnetic field of 24 kA·m−1 and 100 kHz inside a thermostatic chamber at 20 °C. This kind of particles is able to heat the environment after several minutes under the influence of the magnetic field, as previously demonstrated by our group.25,51 In this case, the maximum temperature reached was 32−33 °C after 30 min of exposure. Then, this temperature was maintained by continuous application of the magnetic field 522



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control the release of the macromolecules attached into the polymer branches. This dual release along with the synergistic effect of hyperthermia treatment, all triggered by a nontoxic and highly penetrating in human tissues external stimulus such as magnetic field, could greatly improve the effectiveness of the current antitumoral therapy. Further work is underway to achieve a precise control in the trigger temperature, comprised in the upper limit of hyperthermia range (42−45 °C), as well as to obtain stronger attachment of the loaded proteins and also to extend the application to different therapeutic macromolecules such as DNA and siRNA, among others.

magnetic field. This fact generates heat energy, and subsequently the pore structure is enlarged causing the faster drug release. It is also important to note that the fluorescein release at 45 °C presents a significant increase around 15−20 h after the application of the thermal stimulus, corresponding to the time when more than 90% of the protein has been released. This may show that the attached protein on the polymer branches also blocks the fluorescein diffusion. Consequently, in this case the sample at 20 °C only reaches a 5% of fluorescein release after 24 h that is slightly less than the release achieved when only fluorescein is loaded in the material. The application of a high-energy magnetic field could provoke that the iron oxide nanocrystals encapsulated into the silica matrix reach higher temperatures than the surroundings. These hot spots could cause alterations or denaturalization in the proteins trapped into the polymer branches. In order to test the protein stability during the heating process under the application of alternative magnetic fields, an enzyme (catalase) was loaded in the polymer coated magnetic mesoporous nanoparticles (MMNP-NIPAM) following a similar method to the previously described. The enzymatic activity of the released protein was measured after 6 h of heating process in the oven and under magnetic field, respectively. The total protein released was determined by bicinchoninic acid (BCA) colorimetric protein assay.64 The catalase activity was measured by UV−vis spectrophotometry following the decomposition of a hydrogen peroxide solution at 240 nm65 (see Supporting Information for the detailed procedure). According to the literature, catalase is stable up to temperatures of 55−60 °C;66 therefore, its enzymatic activity would suffer a sharp decline if the temperature in the polymer shell, where the protein is housed, exceeds this value during the application of the magnetic field. However, as can be observed in Figure 10, the catalase activity loss was only around 10%, which indicates that the protein is placed far enough to be

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ASSOCIATED CONTENT

S Supporting Information *

Description of total protein released and enzymatic activity assay, size distribution and XRD pattern of MMSN100, and VSM curve of MMSN-NIPAM (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Phone 34 91 3941870. Fax 34 91 3941786.

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ACKNOWLEDGMENTS This work has been financially supported by the Spanish CICYT through project MAT-2008-00736 and by the Spanish National CAM through project S2009/MAT-172. We also thank the X-ray Diffraction CAI and the Electron Microscopy CAI of Universidad Complutense de Madrid. E.G. thanks the financial support of Programa Internacional de Captación de Talento (PICATA) of the Campus de Excelencia Internacional de Moncloa.

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REFERENCES

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Figure 10. Total catalase release (blue) and enzymatic activity (red) after magnetic field exposition and conventional temperature increase.

affected by the iron oxide hot spots. In the case of the thermal heating, only less than 5% of the enzyme was affected during the process.

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CONCLUSIONS In this work, a proof of concept of a new smart nanodevice able to release two different cargos, proteins and small molecules, in response to external stimuli, such as temperature or alternating magnetic field, has been presented. The results demonstrate that the phase state of the polymer can act as gate-keeper, opening or closing the pores of the silica matrix, and may also 523



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