www.acsanm.org Article Magnetic Mesoporous Silica Nanorods Loaded with Ceria and Functionalized with Fluorophores for Multimodal Imaging Jan Grzelak, Jaume Gázquez, Alba Grayston, Mariana Teles, Fernando Herranz, Nerea Roher, Anna Rosell, Anna Roig,* and Martí Gich* Cite This: ACS Appl. Nano Mater. 2022, 5, 2113−2125 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Multifunctional magnetic nanocomposites based on mesoporous silica have a wide range of potential applications in catalysis, biomedicine, or sensing. Such particles combine responsiveness to external magnetic fields with other functionalities endowed by the agents loaded inside the pores or conjugated to the particle surface. Different applications might benefit from specific particle morphologies. In the case of biomedical applications, mesoporous silica nanospheres have been extensively studied while nanorods, with a more challenging preparation, have attracted much less attention despite the positive impact on the therapeutic performance shown by seminal studies. Here, we report on a sol−gel synthesis of mesoporous rodlike silica particles of two distinct lengths (1.4 and 0.9 μm) and aspect ratios (4.7 and 2.2) using Pluronic P123 as a structure-directing template and rendering ∼1 g of rods per batch. Iron oxide nanoparticles have been synthesized within the pores yielding maghemite (γ-Fe2O3) nanocrystals of elongated shape (∼7 nm × 5 nm) with a [110] preferential orientation along the rod axis and a superparamagnetic character. The performance of the rods as T2-weighted MRI contrast agents has also been confirmed. In a subsequent step, the mesoporous silica rods were loaded with a cerium compound and their surface was functionalized with fluorophores (fluorescamine and Cyanine5) emitting at λ = 525 and 730 nm, respectively, thus highlighting the possibility of multiple imaging modalities. The biocompatibility of the rods was evaluated in vitro in a zebrafish (Danio rerio) liver cell line (ZFL), with results showing that neither long nor short rods with magnetic particles caused cytotoxicity in ZFL cells for concentrations up to 50 μg/ml. We advocate that such nanocomposites can find applications in medical imaging and therapy, where the influence of shape on performance can be also assessed. KEYWORDS: mesoporous silica rods, superparamagnetic nanoparticles, magnetic resonance imaging, fluorescence imaging, multimodal nanoparticles, anisotropic nanoparticles ■ INTRODUCTION Nanocomposites with unique properties and synergistic actions with magnetic particles benefit from a response to external magnetic fields, and can be combined with additional from different components have a great potential to be used in functionalities by hosting other chemical species. This can be very useful for applications such as site-specific cargo fields such as biomedicine,1,2 catalysis,3,4 and energy.5,6 delivery,18,19 magnetic resonance imaging (MRI),20−22 mag- Mesoporous silica nanocomposites are mechanically and netically switchable catalytic reactions,23−25 sensing combined thermally stable, present large surface areas and pore volumes with magnetic separation,26−29 recycling of magnetic car- with narrow pore size distributions, and their particle and pore riers,30,31 or the development of catalytic nanomotors.32 In sizes can be controlled.7−14 These characteristics endorse particular, mesoporous silica particles incorporating different mesoporous silica as an attractive platform to design moieties can be of interest in multimodal contrast agents for nanocomposites by loading the pores with different chemical medical imaging, combining the advantages of each imaging species. Such materials can encapsulate large amounts of technique and improving the diagnostics of diseases.33 A nanoscopic cargo while maintaining their robustness and pore significant number of studies have been devoted to the ordering. Among a variety of silica mesophases, Santa Barbara biomedical applications of spherical mesoporous silica Amorphous-15 (SBA-15) is one of the most extensively studied because of its highly ordered and hexagonally arranged cylindrical pores with tunable size between ∼4 and 30 Received: November 12, 2021 nm.7,15−17 Accepted: January 26, 2022 The performance of mesoporous silica nanocomposites can Published: February 10, 2022 be adjusted by controlling the size and porosity of silica, the amount and chemical character of the cargo(s), and the surface chemistry of the composites. For instance, nanocomposites © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsanm.1c03837 2113 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article particles,34−38 but much less research considered other specific static conditions for 24 h. After that, the reaction bottle was shapes (only ∼10% concern rods). Indeed, preparing silica in transferred to an oven at 80 °C and kept there for 24 h. In a well-defined asymmetric morphologies is extremely challeng- subsequent step, silica was filtered and dried at 55 °C overnight. The ing, as it requires stringent control of the synthesis conditions. surfactant was removed by washing the product in ethanol (24−40 cycles of a 500 mL soxhlet). Then, the material was calcined in air at But it appears that these experimental efforts would pay off. 550 °C for 5 h. One batch synthesis typically yielded approximately 1 Indeed, pioneering studies focusing on rodlike particles have g. shown increased performance in relevant aspects, such as cell Synthesis of Silica Rods with a Low Aspect Ratio (Short uptake, toxicity, degradation, or drug release,39−47 and it has Rods, SR). Pluronic P123 (2.4 g) was dissolved in 80 mL of 2 M been suggested that the efficient uptake of anisotropic hydrochloric acid solution in a 250 mL bottle (Simax). After nanoparticles could mimic the advantage of rod-shaped obtaining a homogeneous solution, the temperature was increased to bacteria for internalization in nonphagocytic cells.48 40 °C and the solution was stirred at 700 rpm using a magnetic Following these ideas, in the present study, we report on the stirring bar (dimensions: 25 mm × 6 mm). After 3 h, 5.1 g of TEOS was added dropwise. Stirring was stopped after 4 min and the reaction synthesis of mesoporous silica rods intended for multimodal was kept under static conditions for 3 h. Then, the reaction bottle was imaging and therapeutic action using biocompatible materials transferred to an oven at 80 °C and kept there for 24 h. Then, silica from the design phase. We have developed two different highly was filtered and dried at 55 °C overnight. The surfactant was removed reproducible synthetic protocols to prepare rods of two distinct by washing the product in ethanol (24−40 cycles of a 500 mL aspect ratios with narrow size distributions. The silica pores soxhlet). Finally, the material was calcined in air at 550 °C for 5 h. were loaded with magnetic γ-iron oxide (maghemite) particles One batch synthesis typically yielded approximately 1.2 g. following a solvent-free impregnation method.9 Silica powders Synthesis of Iron Oxide Nanoparticles Inside Silica Pores. were mixed with molten iron nitrate, which could flow into the The impregnation of mesoporous silica rods consisted of mixing the pores by capillarity. The impregnation was followed by a silica powder with iron (III) nitrate nonahydrate. To induce capillary action, it was made sure that the volume of the iron precursor thermal treatment to form iron oxide nanocrystals of around 7 VFe(NO3)3·9H2O = (mFe(NO3)3·9H2O)/(ρFe(NO3)3·9H2O) was not larger than nm in length inside the pores. The possibility of material multimodality is demonstrated by additionally impregnating the volume of the pores (calculated from BET isotherm; 0.65 cm3/g for LR, 0.79 cm3/g for SR). In a typical impregnation experiment, the the rods with cerium (IV) oxide, a material with excellent powders were mixed at a ratio of 1:1 wt/wt. Iron (III) nitrate antioxidant capacity recently reported as a potential nonahydrate was ground in a mortar, weighed, and added to a vial therapeutic agent in liver inflammation,49,50 and modifying containing the required amount of MSRs. The two powders were the silica surface with two fluorophores emitting in two distinct mixed thoroughly with a spatula and the vial was put in a silicone oil spectral ranges (525 and ∼730 nm). Additionally, the bath at 60 °C. After 30 min, the powders were mixed again. This step biocompatibility of the rods was evaluated using the ZFL cell was performed twice. After another 30 min of heating, the powder was line as an in vitro model. We believe that such versatile cooled down. Then, the impregnated silica was heated in a tubular materials could potentially be used in different fields in the furnace (ST 1002540, HOBERSAL) at 425 °C for 3 h under a flow of near future, especially as multimodal agents in biomedicine. argon with 5% of hydrogen (v/v; 100 cm3/min). Rods were labeled as ■ Fe2O3@LR and Fe2O3@SR. Synthesis of Ceria and Iron Oxide Nanoparticles Inside MATERIALS AND METHODS Silica Pores. The MSR powder was mixed with cerium (III) nitrate Materials. Mesoporous silica rods (MSR) were synthesized using hexahydrate (3:1 wt/wt). The cerium precursor was ground in a the following reagents. Hydrochloric acid (37%), poly(ethylene mortar, weighed, and added to a vial containing MSRs. The two glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) powders were mixed thoroughly with a spatula, and the vial was put in (Pluronic P123), tetraethyl orthosilicate (TEOS), iron (III) nitrate a silicone oil bath at 80 °C. After 30 min, the powders were mixed nonahydrate, (3-aminopropyl)triethoxysilane (APTES), agarose, again. This step was performed twice. After another 30 min of heating, sodium hydroxide, sodium citrate, fluorescamine, and triethylamine the powder was cooled down. Then, iron (III) nitrate nonahydrate were purchased from Sigma-Aldrich and used as received. Ethanol was ground in a mortar was added (miron precursor = mcerium precursor). The purchased from PanReac. Iron (III) nitrate nonahydrate and cerium powders were mixed thoroughly, and the vial was put in a silicone oil (III) nitrate hexahydrate were purchased from Acros Organics and bath at 60 °C. After 30 min, the powders were mixed again. This step used as received. Cyanine5 NHS ester (Cy5-NHS) was purchased was performed twice. After another 30 min of heating, the powders from Lumiprobe and used as received. The purity of all reagents was were cooled down. The impregnated MSR were heated in a furnace 98% or higher. (ST 1002540, HOBERSAL) at 600 °C for 3 h under a flow of argon Zebrafish liver (ZFL) cells were cultured at 28 °C in 5% CO2 in with 5% of hydrogen (v/v; 100 cm3/min). Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose, Surface Functionalization with Amine Groups. Amine groups supplemented with 0.01 mg/mL insulin, 50 ng/mL EGF, 5% (v/v) were introduced to the silica rod surface by aminosilanization with antibiotic/antimycotic solution, 10% (v/v) heat inactivated fetal APTES, in a protocol that was adapted from the literature53,54 with bovine serum (FBS), and 0.5% (v/v) heat inactivated trout serum modifications. A solution of ethanol in water (70:30 v/v) was (TS) as described in the literature.51 The MTT (3-(4,5-dimethylth- prepared and heated to 70 °C. MSRs (20 mg) were dispersed in 20 iazol-2-yl)-2,5-diphenyltetrazolium bromide) substrate and dimethyl mL of this solution by ultrasonication. APTES (120 μL) was added to sulfoxide (DMSO) were purchased from Sigma-Aldrich. the dispersion, and the mixture was kept at 70 °C under reflux for 1 h. Synthesis of Silica Rods with a High Aspect Ratio (Long The particles were then washed three times in ethanol and centrifuged Rods, LR). In the first step of the study, mesoporous silica rods of two at 16 773g. Then, the particles were dispersed in a small amount of aspect ratios were synthesized. In a typical synthesis, adapted from the ethanol and dried at 60 °C. This step was followed by heating at 120 literature,9,10,52 2 g of Pluronic P123 was dissolved in 95 mL of 1.7 M °C for 2 h in air. hydrochloric acid solution contained in a reaction bottle of 250 mL MSR Functionalization with Fluorescamine and Cyanine5. capacity (Simax). After obtaining a homogeneous solution, the In a typical synthesis, a stock solution was prepared by dissolving 1.4 temperature was increased to 40 °C and the solution was stirred at mg of fluorescamine in 4 mL of acetone. Amino-functionalized MSRs 700 rpm with a magnetic stirring bar (dimensions: 25 mm × 6 mm). (5 mg) were dispersed in 2.5 mL of acetone and sonicated in an After 3 h, 4.2 g of tetraethyl orthosilicate (TEOS) were added ultrasound bath. Then, 2.5 mL of the fluorescamine stock solution dropwise. Stirring was stopped after 5 min, the reaction bottle was was added to the dispersion. The mixture was left to react overnight at hermetically closed with a plastic cap, and the reaction was kept under room temperature, in the dark, under magnetic stirring. Then, the 2114 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article particles were purified by washing three times with ethanol and Magnetic measurements were performed in a superconducting centrifugation (16 773g, 8 min). Finally, the precipitate was dispersed quantum interference magnetometer (SQUID, Quantum Design, in a small amount of acetone and dried in vacuum. The product was Inc.) The magnetization at room temperature was measured up to a stored at 4 °C in the dark. maximum applied field of 50 kOe. The temperature dependence of A Cyanine5 solution was prepared by dissolving 1 mg of Cyanine5 the magnetization (M-T) under a dc field of 50 Oe was recorded on NHS ester in 5 mL of acetone. Amino-functionalized MSRs (10 mg) heating at 2 K/min after zero-field-cooling (ZFC) and field-cooling were dispersed in the solution by sonication in an ultrasound bath. (FC) conditions in the 10−300 K temperature range. Then, 80 μL of trimethylamine was added. The mixture was left to The iron content in the samples was determined by inductively react overnight at room temperature, in the dark, under magnetic coupled plasma - optical emission spectrometry (ICP-OES) at the stirring. Then, the particles were purified by washing three times with chemical analysis service at Universitat Autònoma de Barcelona. ethanol and centrifugation (16 773g, 8 min). Finally, the precipitate Approximately 1 mg of sample (weighed on an MX5 microbalance, was dispersed in a small amount of acetone and dried in vacuum. The Mettler Toledo) was digested with a mixture of concentrated HCl and product was stored at 4 °C in the dark. HNO3 (2:1 v/v) in a microwave oven. The resulting digestions were Characterization of Silica Rods. The morphology of MSRs was diluted with 1% HCl (v/v) and introduced into an ICP-OES examined in a JEOL 1210 transmission electron microscope (TEM) spectrometer (PerkinElmer, model Optima 4300 DV) for the operating at 120 kV accelerating voltage. Samples for TEM analysis measurement. were prepared by drop-casting ethanol dispersions of the particles (1 The MRI studies on phantoms were performed at the Institute of mg/mL) on a holey carbon film supported on a copper grid. Medical Chemistry (IQM-CSIC, Madrid). The MRI equipment used The size distributions were obtained using ImageJ software55 from in this study was an Agilent/Varian scanner (Agilent, Santa Clara, a statistical number of the particles (n = 300) from several frames CA) equipped with a DD2 console and an actively shielded 205/120 acquired from different regions of the sample. gradient insert coil with 130 mT m−1 maximum gradient strength, a A field emitting scanning electron microscope (SEM, FEI Quanta TX/RX volume quadrature coil, and a 1H circularly polarized 200 FEG, Thermo Fisher Scientific, Oregon) was used to study the transmit−receive volume coil of 35 mm inner diameter and 30 mm morphologies of silica particles. The images were acquired in high active length built by Neos Biotec (Pamplona, Spain). The scans were vacuum and with an accelerating voltage of 10 kV. The samples were performed at 7 T. prepared by covering a carbon tape attached to a metal SEM holder ζ potential values of silica rods before and after surface with a small amount of silica powder. The unattached excess of functionalization were measured by Zetasizer Nano ZS (Malvern). powder was then removed. MSRs were dispersed in distilled water at a concentration of 0.5 mg/ Nitrogen adsorption−desorption isotherms were measured at 77 K mL and placed in a ζ potential cuvette for the measurements. using Micromeritics ASAP 2000 apparatus in standard operating Fourier transform infrared spectra were measured by an FT-IR mode. Before analysis, all samples were degassed for 20 h at 180 °C spectrometer (FT-IR JASCO 4700LE) in transmittance mode in a under vacuum (p < 2 × 10−3 Torr). Pore size distribution was range from 4000 to 400 cm−1, using a circular pellet made of sample calculated from the Barrett−Joyner−Halenda (BJH) model from the powder mixed with KBr. desorption branch. The Brunauer−Emmett−Teller (BET) specific The XPS analysis was performed at the Advanced Microscopy surface area was obtained in the 0.05−0.30p/p0 range. Laboratory (LMA) unit at the Universidad de Zaragoza. The Small-angle X-ray scattering (SAXS) measurements were per- measurements were performed with Kratos AXIS Supra XPS formed using Bruker D8-Discover in 2θ range from 0.15 to 5° in steps spectrometer using a monochromatic X-ray source (Al Kα 120 W, of 0.02° with Cu Kα radiation (λ = 1.5406 Å). A lead plate was placed 8 mA/15 kV) under a pressure of 10−9 Torr. The size of the area perpendicular to the sample plane, slightly above it, and held with a analyzed was 2 mm × 1 mm. The samples in powder form were specific support designed to minimize the background at the detector. placed together on a holder with a conducting double-sided tape to be Thermogravimetric analysis (TGA) was performed by measuring analyzed under the same conditions. Because the samples under around 2 mg of silica rods sample in a SETSYS Evolution TGA investigation were magnetic, the magnetic lenses of the spectrometer (Setaram) from room temperature to 650 °C at a heating rate of 10 could not be used and the measurements were performed in °C/min and under dynamic dry airflow. electrostatic mode. It was necessary to employ a charge neutralizer The colloidal stability of the rods can only be qualitatively and optimize its parameters to the samples because a strong effect of evaluated by visual inspection because measuring the hydrodynamic differential charge was observed could not be neutralized with the size by dynamic light scattering is not possible for these systems since default parameters of the equipment. For each sample, a general scan the software used to determine the size assumes spherical shapes. The was performed, followed by high-resolution acquisitions in the rods remain colloidally dispersed in an aqueous solution for about 30 following regions of interest: C 1s, N 1s, O 1s, and Si 2p. min before starting to precipitate. They can be easily redispersed by a The fluorescence spectra were recorded with an LS 45 gentle sonication and the addition of a small amount of mannitol spectrofluorometer (PerkinElmer). The samples were dispersed in slows down the sedimentation. acetone at a concentration of 1 mg/mL and placed in a quartz cuvette. A Siemens D-5000 diffractometer in Bragg−Brentano geometry The emission spectra of fluorescamine-functionalized samples were with conventional X-ray source (Cu Kα radiation, λ = 1.5406 Å) was recorded for an excitation wavelength λ = 390 nm. used for X-ray diffraction measurements, which were analyzed by The UV−vis spectra were collected on an Infinite M200PRO Rietveld refinement using the Materials Using Diffraction (MAUD) Microplate Reader (TECAN) working in absorbance mode. software.56 Dispersions of MSRs in acetone at 1 mg/mL were prepared in 96- The scanning transmission electron microscopy/electron energy well plates (Thermo Fisher). loss spectroscopy (STEM/EELS) measurements were performed at Fluorescence molecular imaging (FMI) studies were performed at the ICTS National Center of Electron Microscopy at Universidad the Pre-clinical imaging Platform of Vall d’Hebron Institut de Recerca Complutense de Madrid. Samples were characterized using a JEOL (VHIR, Barcelona). The fluorescence of MSRs was characterized JEM ARM200cF operated at 200 kV equipped with a CEOS using an IVIS Lumina LT Series III imaging system (PerkinElmer, aberration corrector and with a Gatan Quantum energy filter Waltham, MA). All images were acquired at the following λex/λem spectrometer for EELS measurements. The STEM microscope was ranges: 625−655/695−770 nm, centered at 640/732 nm, respec- operated in high angle annular dark-field (HAADF) imaging mode, tively. A series of concentrations of Cyanine5-functionalized short also referred to as Z-contrast because the brightness associated with silica rods with and without Fe2O3 nanoparticles (respectively, labeled each atomic column of the images scales with its atomic number.57 Fe2O3@SR-Cy5 and SR-Cy5) dispersed in a D-mannitol aqueous The samples were prepared, as described above, by dispersing the solution (3 mg/mL) were prepared in saline (1.5, 1, 0.75, 0.5, 0.25, samples in ethanol and depositing a droplet of the suspension on a and 0.1 mg/mL), and duplicates of 100 μL per well were imaged in a holey carbon film supported on a copper grid. 96-well plate. Background wells containing saline (0 mg/mL) were 2115 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article Figure 1. SEM images of (a) long and (b) short rods. TEM images of (c) long and (d) short rods. used for background measurements. The fluorescence was measured double nitrile gloves were used for working with silica. The interior of as total radiant efficiencyTRE ([photons/s]/[μW/cm2]). For the fume hood was then carefully cleaned with ethanol and all quantification, circular regions of interest (ROIs) were manually material contaminated with silica was disposed of in an appropriate drawn surrounding the fluorescence signal and TRE was measured solid residue container. ■ using the Living Image software (PerkinElmer, Waltham, MA) and corrected by the TRE from the corresponding ROI in the background control well. RESULTS AND DISCUSSION ZFL Cell Viability Studies. Cytotoxic effects of MSRs on ZFL Mesoporous silica rods (MSRs) of SBA-15 type were were assessed using the MTT assay. The studies were performed at synthesized using a sol−gel method (Figure S1) with Pluronic the Institute of Biotechnology and Biomedicine (IBB, UAB). After 2 h in P123 surfactant as a structure-directing template.7,52 This minimal medium (0−0.5% FBS; 2% antibiotic/antimycotic), cultures template was later removed by washing with ethanol and were incubated with MSRs (Fe2O3@LR, Fe2O3@SR, each MSR type and condition in triplicate). 10% of culture medium volume was calcination. removed and replaced with the same volume of either water or stock As it has been previously reported in the literature,52 the solution of MSRs, to obtain MSR concentrations of 0, 5, 20, 50, and silica morphology depends greatly on the synthesis conditions, 200 μg/mL. The cells were incubated with MSRs for 6 h at 28 °C. and we have observed different particle shapes for different The cells were then washed with PBS, and the MTT substrate was temperatures and stirring rates (Figure S2 and Table S1). added to 10% of the total volume and further incubated at 28 °C for Once establishing the optimal temperature and stirring rate for 30 min. The solution was removed, the cells were solubilized in obtaining rodlike particles, these parameters were kept DMSO, and the lysate absorbance was read on a Victor 3 plate reader constant, and other parameters, namely, the acid concen- (PerkinElmer) at 550 nm. The experiment was repeated four times for each type of MSRs. Data were normalized using GraphPad Prism tration, the surfactant concentration, and the reaction mixture (version 7.01 for Windows, GraphPad Software, San Diego, California volume, were varied. The SEM and TEM images of Figure 1 USA, www.graphpad.com) such that the control readings were set at show the morphologies of the two types of silica rods with 100%. Two-way ANOVA was performed with Dunnett’s multiple distinct aspect ratios (AR) resulting from the optimization of comparison test, comparing treatment and control means. two synthetic protocols. These are denominated as long rods Safety. Silica powders used in this work were airborne. Data (LR, AR = 4.7, Figure 1a,c) and bacteria-like short rods (SR, available for different silica materials indicate that such materials can AR = 2.2, Figure 1b,d). The SEM images show a large number accumulate in the lungs when inhaled, leading to adverse effects on of particles with a high degree of size uniformity (Figure 1a,b). health, such as silicosis.58 For this reason, special protective measures were taken while working with silica in powder form. Sample In the TEM images, the rodlike shape of the particles is clearly manipulation was performed exclusively in a fume hood. FFP3 masks evidenced (Figure 1c,d). TEM images were used to character- (3M Aura 9300/Handanhy HY9330/Labbox) were used by every ize the length and width of MSRs, and the measured mean person present in the laboratory at the time of manipulation, and values are shown in Table 1. 2116 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article Table 1. Morphologies of Silica Rods incorporated inside the pores by capillary forces. The conversion of the salt to iron oxide was performed by thermal sample ID length (μm) width (μm) aspect ratio treatment of 3 h at 425 °C in an Ar/H2 atmosphere. The LR 1.4 ± 0.3 0.3 ± 0.1 4.7 ± 1.3 samples obtained are referred to as Fe2O3@LR and Fe2O3@ SR 0.9 ± 0.1 0.4 ± 0.1 2.2 ± 0.7 SR, respectively, and were used in further experiments. The first crystallization attempts done at the same temperature in Studying the results using a factorial design,59 we found that an air atmosphere resulted in the undesired formation of a within the studied range of variable values, the acid mixture of maghemite and the weakly magnetic hematite, concentration was the critical parameter determining the AR which is detrimental to its use as an MRI contrast agent (see of the rods, as a higher HCl concentration promoted the Figure S6). formation of shorter rods (see Table S2). For the optimized annealing treatment, only maghemite The rods present an array of hexagonally ordered, well- peaks were observed by XRD in both materials (Figure 3a). defined mesopores of approximately 5 nm, as observed by The presence of a single phase was confirmed by Rietveld TEM (Figure 2a−c). The pores can be seen as bright domains, refinement analysis (Figure S7). Electron microscopy images separated by darker walls. The size of the pores spacing show a large number of nanoparticles aligned inside the observed in TEM (approximately 10 nm) was compared with channels of both LR and SR (Figure 3c). The particles can be the lattice parameter obtained from small-angle X-ray seen as slightly larger than the pores, also observed in an earlier scattering (SAXS, Figure 2d). The scattering reflections at study.9 To confirm that the particles locally expand the low angles are the ones expected from hexagonal pore ordering channels while growing in confinement and are not adsorbed (p6mm space group) previously reported for SBA-15.7 The on the outer surface of rods, the TEM stage was tilted over a calculated lattice parameter (a = 10.1 nm) was in agreement wide range of angles around the long axis of the rod (Figure with the sizes measured by TEM. S8). As expected, under certain angles the channels were more The Pluronic P123 surfactant removal upon ethanol visible and the particles appeared clearly aligned along with cleaning and calcination was confirmed by thermogravimetry them. No particles were observed on the outer surface of the analysis (Figure S3) and the increased porosity was further silica rods and only a very small number of empty rods were confirmed by nitrogen adsorption−desorption isotherms identified. The nanoparticles (NPs) induced local deforma- (Figure S4a−c). Both types of rods were characteristic of tions of the pores of both types of rods as indicated by the type-IV isotherms with H1 type hysteresis,60 corresponding to increased pore width seen around the particles and the mesopores with a narrow pore size distribution. The total surface area, pore volume, and pore diameter values calculated tortuosity of the channels along the rod axis. from the isotherms are presented in Table S3. LR and SR had The magnetization vs magnetic field at room temperature of surface areas of up to 827 and 672 m2/g, respectively, and a the MSRs (Figure 3b) shows a typical superparamagnetic total specific pore volume of up to 1 cm3/g. Pore sizes behavior, characterized by the absence of hysteresis in fields obtained from BET isotherm were in good agreement with larger than the residual fields (in the order of 50 Oe)62 values observed in TEM (approximately 5 nm). The resulting from a trapped flux in the SQUID coils (see the inset calcination slightly decreased the surface area and pore volume in Figure 3b). Such behavior is characteristic for small as a result of the densification which silica undergoes at high magnetic nanoparticles (typically below 14 nm in the case of temperatures,61 but the mesopore structure remained intact, as maghemite), which contain a single magnetic domain.63 ZFC- seen by TEM (Figure S5). FC measurements (Figure S9) showed similar blocking In a subsequent step, magnetic nanoparticles were temperatures in both Fe2O3@LR and Fe2O3@SR systems synthesized inside the pores by a wet impregnation method. (i.e., TB ca. 40 K) and no separation between the ZFC and FC The molten salt (iron (III) nitrate nonahydrate) was above these temperatures, indicating a similar magnetic Figure 2. (a) TEM image showing the pores inside a silica rod. Silica walls are seen darker and the parallel channels as bright lines. (b) Cross section of a silica rod showing a hexagonal arrangement of the pores. A unit cell is marked in orange. (c) Magnification of the image showing the unit cell. (d) SAXS pattern of LR. Scattering reflections are indexed in a hexagonal unit cell of the p6mm space group. 2117 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article Figure 3. (a) XRD diffractograms of Fe2O3@LR and Fe2O3@SR. The peaks are indexed in γ-Fe2O3 unit cell. (b) Magnetic hysteresis loops of Fe2O3@LR and Fe2O3@SR at 300 K, where the magnetic moment is given per gram of sample. Inset: Image of magnetic silica rods attracted by a magnet. (c) TEM images of MSRs loaded with iron oxide nanoparticles. The leftmost image shows LR, and the central one SR. The rightmost higher-magnification image corresponds to LR, with iron oxide nanoparticles visible within the silica pores. character of both composites and no significant magnetic having a slightly elongated, ellipsoidal shape. To visualize interactions. individual nanoparticles, silica was etched with a concentrated Electron diffraction studies of maghemite-loaded rods solution of sodium hydroxide. Although the particles formed indicated that the growth of particles in confinement inside aggregates, the elongated shape (6.9 nm × 4.9 nm) was the channels induces a preferential direction. The diffraction confirmed in TEM measurements (Figure S12). The pattern (Figure S10) indicates that the crystals have different maghemite phase was identified using electron diffraction. orientations in the direction perpendicular to the rod axis but The presence of γ-Fe2O3 was also confirmed by STEM in share a common preferential [110] orientation along the rod combination with EELS (see Figure 4). The Fe L-edge map axis. In particular, all of the elongated diffraction spots of this from the spectrum image coincides with the map obtained pattern can be indexed by considering the superposition of using the pre-peak feature of the O K-edge spectrum, which is patterns from maghemite crystals along the [1̲11], [001], characteristic for iron oxide, confirming the presence of iron [11̲0], [11̲2̲], and [3̲32] zone axes. oxide NPs inside the pores and excluding the presence of The periodicity and loading of the channels were studied metallic iron. Moreover, the intensity ratio of iron L3/L2 white using SAXS (Figure S11). Before loading with iron oxide NPs, lines in Fe L2,3 spectra of approximately 5.5 indicates the three peaks were observed in the SAXS pattern and indexed as presence of γ-Fe2O3 rather than Fe3O4, which presents a lower (100), (110), and (220) reflections associated with p6mm two- L3/L2 ratio.64,65 dimensional space group, indicating a hexagonal mesoscopic The iron oxide content was assessed using ICP-OES. The periodical organization characteristic of SBA-15.7 After loading results are shown in Table 2. The iron oxide content calculated with iron oxide NPs, the intensity of the peaks decreased as from iron content analyzed experimentally by ICP-OES agrees expected, and only the (100) reflection was observed with a with the theoretical values calculated from the precursor significantly decreased intensity. This suggests that loading the amounts used for impregnation (i.e., ∼14 wt % Fe2O3). From pores caused a decrease in the periodicity of electron density these values, we expressed the saturation magnetization (Ms) contrast, confirming the filling of a substantial fraction of the per mass of Fe2O3. pores. Next, the performance of the magnetic composites as T2 The size and shape of NPs filling the pores were difficult to MRI contrast agents was studied. Agarose phantoms assess from the TEM images of the rods but they appear as containing Fe2O3@LR or Fe2O3@SR were prepared at a series 2118 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article Figure 4. EELS data of Fe2O3@LR. (a) Zeta contrast image of a single rod. The inset shows the region where a spectrum image 28 × 127 pixels was acquired, with an exposure time of 0.1 s per pixel and an energy dispersion of 0.25 eV per channel. (b) Simultaneous annular dark-field (ADF) image of the area highlighted in the zeta contrast image. (c, d) O K-edge and Fe L-edge maps. Integration windows of width 30 eV were used after background subtraction using a power-law fit. (e) O K-edge pre-peak map obtained by integrating over a 5 eV window centered at 530 eV, after background subtraction. This pre-edge feature stems from the hybridization of O-2p and Fe-3d states of γ-iron oxide particles, and it is absent in the O K-edge signal obtained from the silica. (f) Fe L23 ratio map obtained using the second derivative method. The L23 ratio (∼5.5) is higher than that expected for Fe3O4 calculated using the same method (∼5.1).64 It is known that the Fe L23 ratio in γ-Fe2O3 is higher than that in Fe3O4.65 (g, h) Fe L-edge and O K-edge spectra, respectively. The O pre-peak is indicated by an arrow in the latter. Table 2. Iron Oxide Content in Fe2O3@LR and Fe2O3@SR studied vortex and a faster decrease of the time that the nuclear Obtained from ICP-OES Compared with the Values spins move coherently in the x−y plane.66 Calculated from the Amounts of Precursors Used for We also investigated the loading of the pores of silica rods Impregnationa with cerium oxide (ceria). This compound offers great potential as a therapeutic agent serving as a scavenger of Fe2O3 wt % Fe2O3 wt % MS at 300 K sample ID (theoretical) (ICP-OES) (emu/g Fe2O3) reactive oxygen species (ROS) due to its redox proper- Fe2O3@LR 14.3 15.0 ± 1.3 43 ± 4 ties.50,67,68 We investigated the possibility of introducing both Fe2O3@SR 14.3 15.0 ± 0.3 42 ± 4 ceria and iron oxide nanoparticles inside the rods, forming a a bimodal load for potential theranostic applications. To From the ICP-OES analysis, the saturation magnetization is given per gram of Fe2O3. introduce ceria inside the pores, we followed a loading approach analogous to that used for magnetic nanoparticles. For this purpose, cerium (III) nitrate hexahydrate (Tm = 65 °C) was used as a precursor. Impregnation of MSRs at 80 °C of concentrations and their MRI T2 signal was recorded (Table followed by a thermal treatment at 600 °C resulted in the S4). A decrease of the signal was observed as the filling of the pores with ceria, as indicated by the XRD pattern concentrations of rods increased, for both long and short (Figure S13). rods, and the inverse T2 relaxation time presented a linear The impregnation of MSRs with the cerium precursor dependence on iron concentration in the composite (Figure followed by a second impregnation with the iron precursor 5). The signal decay of long rods was higher than that of short yielded rods appearing as filled in the TEM images (Figure rods, which is illustrated by their slightly higher transverse S14a). It was also observed that the sample was magnetic, with relaxivity value, r2, at 7 T (143 and 108 s−1 mM−1, the magnetization measurements indicating the presence of respectively). The Fe2O3@LR are slightly more efficient T2 superparamagnetic and paramagnetic contributions (Figure contrast agents because they accommodate a larger mass of S14c). However, electron diffraction (Figure S14b) and XRD Fe2O3 meaning a larger magnetic moment (emu) per particle. (Figure S13) did not reveal the presence of any crystalline This causes larger magnetic field inhomogeneities in the phase formed within the rods. 2119 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article optimized using low APTES concentrations (6 μL/mL) and short reaction times (1 h), to avoid erosion of the rods observed at high pH. The grafting efficiency was increased by performing the reaction at an elevated temperature (70 °C), using an ethanol/water mixture as a solvent, and applying a post-grafting thermal treatment at 120 °C. After the reaction, the ζ potential measurements showed a significant shift in surface charge from negative to positive values (Figure S15a). Moreover, TEM images (Figure S15b,c) showed that the structure of MSRs remained intact. Further evidence for change of surface character was provided from FT-IR measurements (Figure S15d). The bands corresponding to Si−O−Si symmetric stretching at 805 cm−1 and Si−O−Si asymmetric stretching at 1090 cm−1 were observed in samples both before and after functionalization, suggesting that the main structure of silica was not affected by the modification. Bands at 1610−1643 cm−1 observed in the functionalized silica correspond to −NH2 in-plane scissoring in the amine groups,70 while bands at 2851 and 2920 cm−1 indicate −CH2 symmetric and asymmetric stretching vibrations from the propyl branch of APTES.71 The positions of the bands are indicative of a Figure 5. (a) T2 MRI signals of agarose phantoms with increasing dense packing of the propyl chains.72 An asymmetric −CH3 concentrations of magnetic LR corresponding to 0, 0.175, 0.35, 0.525, mode at 2962 cm−1 may suggest that some ethoxide groups 0.7, 1.75, and 3.5 mM Fe. (b) Evaluation of r2 relaxivities of Fe2O3@ have not been fully hydrolyzed and that the APTES molecules LR and Fe2O3@SR (n = 1). are not fully bonded with each other.70,72 The presence of −NH2 groups on the MSR surface was To confirm the presence of both iron and cerium inside the additionally investigated by X-ray photoelectron spectroscopy pores and determine the oxidation state of cerium, we (XPS). A general scan was performed for both Fe2O3@LR- performed STEM analysis in combination with EELS (Figure NH2 and Fe2O3@SR-NH2, followed by high-resolution scans 6). Both iron and cerium maps (Figure 6c,d) indicate that of regions of interest: C 1s, N 1s, and O 1s. The elements these elements are co-localized inside the pores, following the present on the surface could be identified in a general scan channels. Moreover, the relative intensity of cerium M4 to M5 (Figure S15e). The spectra show peaks at energies character- peaks in the EEL spectrum (located at 901 and 883 eV, istic for nitrogen and carbon, introduced by APTES respectively), being M4 larger than M5, is characteristic of functionalization, as well as the silicon and oxygen from Ce4+.69 This indicates that ceria is in form of CeO2 and could silica.73,74 be potentially used for capturing ROS. The high-resolution spectra of the studied regions are shown In subsequent steps, we have explored the possibility of in Figure S16. Because of the high width of peaks in high- introducing additional functionalities to the system grafting resolution scans, it was not possible to distinguish chemical molecular species on the surface of the silica rods. First, MSRs species with clarity. However, the shape of peaks of both N 1s have been functionalized with APTES to introduce amine and C 1s was in good agreement with data found in the groups on the surface. The functionalization protocol was literature regarding APTES-functionalized surfaces.75,76 The Figure 6. EELS spectrum at the Ce M4,5 edge for Fe@Ce@LR. (a) Zeta contrast image of a single rod. The inset shows the region where a spectrum image of 19 × 68 pixels was acquired, with an exposure time of 0.1 s per pixel and an energy dispersion of 1 eV per channel. (b) Simultaneous ADF image of the area highlighted in the zeta contrast image. (c, d) Fe L and Ce M-edge maps. Integration windows of width 30 eV were used after background subtraction using a power-law fit. (e) Ce M-edge signal extracted from the SI. The relative intensity of Ce M4 and M5 peaks suggests a Ce4+ oxidation state.69 2120 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article Figure 7. (a) Structure of Cy5-NHS ester. (b) Reaction scheme of an NHS ester derivative with a primary amine forming an amide bond. (c) Absorbance of SR-Cy5 compared to Cy5 solution. Characteristic double peak of Cy5 is observed after functionalization of silica. (d) TRE vs control of SR-Cy5 and Fe2O3@SR-Cy5 plotted as a function of sample concentration showing a linear dependence (n = 1). (e) Fluorescence images of SR-Cy5 and Fe2O3@SR-Cy5 dispersions for the same MSR concentrations as in (d). deconvolution of N 1s spectra (Figure S17) suggested surface irradiation at 390 nm, and an intense emission peak was species including C−NH2 and C−NH3+.76 The amine group observed at 525 nm (Figure S18d). (C−NH2) was dominant in both samples. MSRs were then functionalized with Cyanine5 (also The introduction of amine groups to the surface of MSRs described as Cy5, where Cy stands for cyanine, and the allowed us to chemically attach other molecules to obtain number 5 refers to the number of carbon atoms between the fluorescent materials for optical imaging. Fluorescamine, or indolenine groups).78 This fluorophore emitting in the red fluram, is a nonfluorescent compound that reacts quickly with region is often used in biomedical research for cell and tissue primary amines (R-NH2), forming a fluorescent derivative that labeling, as in this spectral range biological objects show low emits green light upon irradiation with ultraviolet (UV) light absorption and many detectors exhibit maximum sensitivity.79 (Figure S18a).77 Fluorescamine was conjugated to the amine To attach the Cy5 dye to the MSR surface, N-hydroxysucci- groups on the surface of the APTES-functionalized MSRs. The nimide (NHS) ester derivative of Cy5 (Figure 7a) was used attachment of fluram to LR and SR (yielding samples labeled and reacted with amine groups of APTES-functionalized silica as LR-FL and SR-FL, respectively) could be confirmed by surface, forming an amide bond (Figure 7b).80 After the fluorescence spectroscopy, considering that free fluram does reaction of MSRs-NH2 with Cy5, a blue product was observed. not show fluorescence. UV−vis absorption spectra (Figure 7c) confirmed the presence After the reaction, the color of the MSR dispersion changed of Cy5 comparing the absorbance of free Cy5 with Cy5- from white to intense yellow (Figure S18b). Under irradiation functionalized SR (denominated SR-Cy5). A characteristic with UV light, green fluorescence was observed (Figure S18c). Cy5 band was observed at 652 nm, slightly shifted (∼10 nm) The fluorescence emission spectrum was then acquired under toward higher wavelengths compared to the absorbance curve 2121 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article of free Cy5. A similar effect has been reported in the literature thermal treatment. A single-step treatment in a reducing for Cy5 and other fluorophores bound to surfaces81,82 and can atmosphere (Ar/5% H2, 100 cm3/min) results in the formation be explained by the change in polarity and polarizability of the of small particles within the silica pores, which consist of γ-iron fluorophore environment compared to the free fluorophore.83 oxide, and we confirmed that virtually no magnetic particles are The fluorescence emission of SR-Cy5 and Cy5-functionalized formed outside the rods or on its surface. We have observed Fe2O3@SR (Fe2O3@SR-Cy5) was studied by FMI. Optical that both long and short rods filled with iron oxide imaging phantoms were made by preparing a dispersion of nanoparticles exhibit superparamagnetic character at room rods in an aqueous solution of D-mannitol (55 mg/mL) that temperature with a significant saturation magnetization (∼42 was then diluted in saline to obtain a range of concentrations emu/g Fe2O3) which highlights the potential of this material to (0−1.5 mg/mL). Mannitol solution was used instead of water be used in biomedical applications, such as MRI imaging. The to increase the stability of the dispersion of rods in this performance of the materials as T2-weighed MRI contrast medium. Emission was measured with the excitation filter agents has been confirmed to be slightly superior in the long centered at 640 nm. The particles exhibited high fluorescence, rods (r2 = 143 mM−1 s−1) than short ones (r2 = 108 mM−1 linearly increasing with the concentration (Figure 7d,e). The s−1). The potential of the material for combining multiple TRE was very similar for both SR-Cy5 and Fe2O3@SR-Cy5. functional moieties has also been confirmed by functionalizing Therefore, we have shown that the MSRs offer the potential of the surface with fluorophores of distinct emission wavelengths: combining two imaging functional moieties without a decrease fluorescamine, producing a material emitting in green (525 in their performance as fluorescence imaging agents. nm), and Cyanine5, endowing the rods with red light emission To assess the safety of MSRs in the context of their potential (∼730 nm). Cerium oxide was additionally introduced inside use in biomedical applications, toxicity tests were performed in the pores. It was observed that a system incorporating ceria vitro in zebrafish liver cell line (ZFL). Neither Fe2O3@LR nor and iron oxide in the pores presented a magnetic response but Fe2O3@SR caused cytotoxicity in ZFL cells for concentrations was not crystalline, indicating the presence of amorphous iron up to 50 μg/mL (Figure 8). At 200 μg/mL MSRs, the number oxide co-localized with Ce4+ species. These multimodal magnetic materials can find applications in future theranostic developments for which the functional versatility demonstrated in this work and the therapeutic performance associated with their anisotropic shape could be advantageous over spherical silica carriers. ■ * ASSOCIATED CONTENT sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsanm.1c03837. Synthesis scheme, TEM images, thermogravimetric analysis, porosity data, X-ray diffractograms, Rietveld refinements, ZFC-FC curves, electron diffraction, SAXS, MRI T2 relaxation times, ζ potential values, FT-IR spectra, XPS spectra, and fluorescence spectra (PDF) Figure 8. Zebrafish liver cell (ZFL) viability after 6 h of exposure to the different types of MSRs at four different concentrations; n = 12. ***P ≤ 0.001, ****P ≤ 0.0001. ■ AUTHOR INFORMATION Corresponding Authors Anna Roig − Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), 08193 Bellaterra, Catalonia, Spain; of viable cells decreased significantly only for Fe2O3@LR. Due orcid.org/0000-0001-6464-7573; Email: roig@icmab.es to the scarcity of studies concerning silica particles in ZFL Martí Gich − Institut de Ciència de Materials de Barcelona cells, the results of toxicity studies are difficult to compare with (ICMAB-CSIC), 08193 Bellaterra, Catalonia, Spain; the literature. However, these results suggest low toxicity of orcid.org/0000-0001-9958-0057; Email: marti.gich@ MSRs, especially short rods, at concentrations higher than cisc.es those that have been used for carbon nanotubes.84 ■ CONCLUSIONS We have reported on two protocols for synthesizing uniformly Authors Jan Grzelak − Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), 08193 Bellaterra, Catalonia, Spain sized MSR with distinct aspect ratios: long rods (LR, AR = Jaume Gázquez − Institut de Ciència de Materials de 4.7) and short rods (SR, AR = 2.2). The MSRs display an array Barcelona (ICMAB-CSIC), 08193 Bellaterra, Catalonia, of hexagonally ordered cylindrical pores running along the rod Spain axis with a high surface area (up to 827 m2 g−1 for LR and 672 Alba Grayston − Neurovascular Research Laboratory, Vall m2 g−1 for SR) and pore volume (up to 1 cm3 g−1 for LR and d’Hebron Research Institute (VHIR), 08035 Barcelona, 0.8 cm3 g−1 for SR) with a pore diameter of approximately 5 Catalonia, Spain nm. These channels enable the modification of the rods with Mariana Teles − Institute of Biotechnology and Biomedicine chemical species. We loaded the silica pores with iron oxide (IBB), Universitat Autònoma de Barcelona, 08193 nanoparticles using a wet impregnation method followed by Bellaterra, Catalonia, Spain 2122 https://doi.org/10.1021/acsanm.1c03837 ACS Appl. Nano Mater. 2022, 5, 2113−2125 ACS Applied Nano Materials www.acsanm.org Article Fernando Herranz − Instituto de Química Médica (IQM), and O2 Self-Sufficient Photodynamic Therapy. Nanoscale 2019, 11, Consejo Superior de Investigaciones Científicas (CSIC), 14654−14667. 28006 Madrid, Spain; orcid.org/0000-0002-3743-0050 (2) Zhang, M.; Wu, F.; Wang, W.; Shen, J.; Zhou, N.; Wu, C. Nerea Roher − Institute of Biotechnology and Biomedicine Multifunctional Nanocomposites for Targeted, Photothermal, and (IBB), Universitat Autònoma de Barcelona, 08193 Chemotherapy. Chem. Mater. 2019, 31, 1847−1859. Bellaterra, Catalonia, Spain (3) Qi, B.; Wu, C.; Liu, Y.; Liu, J.; Zhang, H. Self-Assembled Magnetic Pt Nanocomposites for the Catalytic Reduction of Anna Rosell − Neurovascular Research Laboratory, Vall Nitrophenol. ACS Appl. Nano Mater. 2019, 2, 4377−4385. d’Hebron Research Institute (VHIR), 08035 Barcelona, (4) Avery, K. N.; Schaak, J. E.; Schaak, R. E. M13 Bacteriophage as a Catalonia, Spain Biological Scaffold for Magnetically-Recoverable Metal Nanowire Complete contact information is available at: Catalysts: Combining Specific and Nonspecific Interactions to Design https://pubs.acs.org/10.1021/acsanm.1c03837 Multifunctional Nanocomposites. Chem. Mater. 2009, 21, 2176− 2178. Author Contributions (5) Patra, K. K.; Ghosalya, M. K.; Bajpai, H.; Raj, S.; Gopinath, C. S. Oxidative Disproportionation of MoS2/GO to MoS2/MoO3- x/ J.G. contributed to methodology, validation, formal analysis, RGO: Integrated and Plasmonic 2D-Multifunctional Nanocomposites investigation, visualization, writingoriginal draft & review for Solar Hydrogen Generation from Near-Infrared and Visible and editing. J.G., A.G., and M.T., carried out investigation, Regions. J. Phys. Chem. C 2019, 123, 21685−21693. formal analysis, and writingreview. F.H. contributed to (6) Abol-Fotouh, D.; Dörling, B.; Zapata-Arteaga, O.; Rodríguez- resources, investigation, and writingreview. N.R. and A.R. Martínez, X.; Gómez, A.; Reparaz, J. S.; Laromaine, A.; Roig, A.; contributed to resources, investigation, formal analysis, and Campoy-Quiles, M. Farming Thermoelectric Paper. Energy Environ. writingreview. A.R. and M.G. involved in conceptualization, Sci. 2019, 12, 716−726. resources, funding acquisition, supervision, writingoriginal (7) Zhao, D.; Feng, J.; Huo, Q.; Melosh, N.; Fredrickson, G. H.; draft & review and editing. Chmelka, B. F.; Stucky, G. D. Triblock Copolymer Syntheses of Notes Mesoporous Silica with Periodic 50 to 300 Angstrom Pores. Science 1998, 279, 548−552. The authors declare no competing financial interest. (8) Langley, P. J.; Hulliger, J. Nanoporous and Mesoporous Organic ■ ACKNOWLEDGMENTS The authors acknowledge financial support from the Spanish Structures: New Openings for Materials Research. Chem. Soc. Rev. 1999, 28, 279−291. (9) Li, J. G.; Fornasieri, G.; Bleuzen, A.; Gich, M.; Gloter, A.; Bouquet, F.; Impéror-Clerc, M. Alignment under Magnetic Field of Ministry of Science and Innovation through the RTI2018- Mixed Fe2O3/SiO2Colloidal Mesoporous Particles Induced by Shape 096273-B-I00 project, the ‘Severo Ochoa’ Programme for Anisotropy. Small 2016, 12, 5981−5988. Centers of Excellence in R&D (CEX2019-000917-S). The (10) Li, J.; Fornasieri, G.; Bleuzen, A.; Gich, M.; Impéror-Clerc, M. Generalitat de Catalunya, project 2017SGR765, is also Epsilon-Fe2O3 Nanocrystals inside Mesoporous Silicas with Tailored acknowledged. This research work was performed in the Morphologies of Rod, Platelet and Donut. ChemNanoMat 2018, 4, framework of the Nanomedicine CSIC HUB (ref 1168−1176. 202180E048).The authors participate in the Aerogels COST (11) Suzuki, K.; Ikari, K.; Imai, H. Synthesis of Silica Nanoparticles ACTION (CA 18125). J.G. received financial support through Having a Well-Ordered Mesostructure Using a Double Surfactant the “la Caixa” INPhINIT Fellowship Grant for Doctoral System. J. Am. Chem. Soc. 2004, 126, 462−463. Studies at Spanish Research Centers of Excellence (Grant (12) Trewyn, B. G.; Whitman, C. M.; Lin, V. S. Y. Morphological code: LCF/BQ/DI17/11620041), “la Caixa” Banking Foun- Control of Room-Temperature Ionic Liquid Templated Mesoporous dation (ID100010434), Barcelona, Spain. J.G. was enrolled in Silica Nanoparticles for Controlled Release of Antibacterial Agents. the doctoral program in Materials Science at the UAB. M.T. Nano Lett. 2004, 4, 2139−2143. (ref RYC2019-026841-I) has a post-doctoral fellowship (13) Song, Y.; Li, Y.; Xu, Q.; Liu, Z. Mesoporous Silica “Ramón y Cajal” supported by the “Ministerio de Ciencia e Nanoparticles for Stimuli- Responsive Controlled Drug Delivery: Advances, Challenges, and Outlook. Int. J. Nanomedicine 2017, 12, Innovación”, Spanish Government. A. G. has been supported 87−110. by the fellowship from Instituto de Salud Carlos III with (14) Liu, J.; Liu, T.; Pan, J.; Liu, S.; Lu, G. Q. M. Advances in FEDER funds (FI17/00073). A.Rosell and A. G. work takes Multicompartment Mesoporous Silica Micro / Nanoparticles for part within the RETICS-INVICTUS PLUS from Instituto de Theranostic Applications. Annu. Rev. Chem. Biomol. 2018, 9, 389− Salud Carlos III with FEDER funds (RD16/0019/0021). G. 411. Antorrena from the LMA at Universidad de Zaragoza is (15) Zhao, D.; Huo, Q.; Feng, J.; Chmelka, B. F.; Stucky, G. D. acknowledged for performing XPS measurements. ICTS- Nonionic Triblock and Star Diblock Copolymer and Oligomeric CNME at UCM is also acknowledged for offering access to Surfactant Syntheses of Highly Ordered, Hydrothermally Stable, STEM and expertise. The ICMAB Scientific services of Mesoporous Silica Structures. J. Am. Chem. 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