Nonhomogeneous Silica Promotes the Biologically Induced Delivery

Article pubs.acs.org/JPCC

Nonhomogeneous Silica Promotes the Biologically Induced Delivery of Metal Ions from Silica-Coated Magnetic Nanoparticles Ana B. Dávila-Ibáñez,† Rosalía Mariño-Fernández,† Melodie Maceira-Campos,† Andrés García-Lorenzo,‡ Vicenta Martínez-Zorzano,‡ and Verónica Salgueiriño*,† †

Departamento de Física Aplicada, Universidade de Vigo, Vigo 36310, Spain Departamento de Bioquímica, Genética e Inmunologı ́a, Universidade de Vigo, Vigo 36310, Spain

‡

S Supporting Information *

ABSTRACT: Herein we report the endocytosis of magnetic nanoparticles of two different transition metal ferrites, which are coated with silica. The variation in the cytotoxicity results, which correlate with the metal ions from the magnetic cores, stems from the inhomogeneity of the silica shell and consequent partial degradation of the nanoparticles once loaded into the endosomes of Caco-2 cells.

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INTRODUCTION Nanoparticles have become very useful for targeted therapies. This stems from the fact that they can go beyond biological barriers, enter and distribute within cells, with processes dependent on size and surface chemistry of the nanoparticles themselves but also on the cell cycle.1−6 Accordingly, having in mind the delivery of nanoparticles to particular biological targets, recent studies have focused on understanding the effects of nanoparticle physicochemical properties as they interact with cells and tissues.7,8 In this regard, the silica coating of nanoparticles has turned out to be promising due to the physical chemistry involved,9 which endows nanomaterials with the possibility of subsequent functionalization and manipulation for both diagnostic and therapeutic techniques.10−17 Most of the very small molecular drugs imply a risk of systemic toxicity, reaching the disease site only in a minor proportion.18 In this regard, nanoparticles (and magnetic in particular) can alter the pharmacokinetic profile of these drugs with the aim of reducing this toxicity out of the proposed target, and consequently improving the expected therapeutic activity.19−22 Additionally, magnetic nanoparticles offer potential for imaging, diagnosis, hyperthermia, and cell separation when considering the advantage of an external magnetic manipulation.23,24 However, the need for both increased biocompatibility and low cytotoxicity creates a design conflict for drug delivery vehicles based on magnetic nanoparticles, because the inclusion of specific transition metal ions in the magnetic material improves the magnetic properties, but their intrinsic toxicity hinders the bioapplications proposed. Additionally, nanoparticles designed to target tumors must have a © 2014 American Chemical Society

sufficiently large size to reduce clearance and improve retention and also the capability to biodegrade.8,25 A different perspective can call into question these conflicts, focusing the interest on nanoparticles of transition metal ferrites (MFe2O4, M: Fe, Co, Ni, Mn, Zn) coated with silica, attending to how the physicochemical properties of these inorganic nanoparticles can be modified to facilitate first a drug delivery and subsequently their own elimination. In this regard, after the endocytosis of these nanoparticles from the extracellular milieu, the variations in the pH values26 can induce, attending to the type of silica surrounding the magnetic cores, the acidic dissolution of the nanoparticles once loaded in the endosomes. In fact, nanometer-sized silica dissolves in certain aqueous conditions27,28 and previous reports have shown the possibility of a selective etching.29,30 This offers an outstanding opportunity for biological applications since the biological interaction occurring at the surface can also trigger a process from the interior of the nanoparticles.31−33 According to this, we can promote a particular degradation that triggers the delivery of the metal ions from the magnetic cores, which initially improved the magnetic properties, to increase then the cytotoxicity results. Davidson et al. have pointed out the molecular responses exhibited by cells that come into contact with toxic metals, since they can interfere with the activity of some enzymes.34 The amount of iron in cells is regulated by both uptake and export proteins since deficiency and excess of Received: September 17, 2014 Revised: November 10, 2014 Published: November 10, 2014 28266

dx.doi.org/10.1021/jp5094228 | J. Phys. Chem. C 2014, 118, 28266−28273

The Journal of Physical Chemistry C

Article

this metal can be toxic. Accordingly, if these nanoparticles are internalized and degraded in an acidic endosome where the metallic ions (it also works with nickel, manganese, cobalt, copper, and zinc, besides iron) are subsequently released, the divalent metal transporter (DMT 1) has the chance to transport them out of the endosome and into the cell. Since the chemical degradation of the nanoparticles takes place precisely at the endosomes where they are trapped, this process by which proteins can help to transport the ions released from the magnetic cores can be triggered.10 Herein we report the partial degradation of silica-coated transition metal ferrite nanoparticles after an endocytosis process. The cytotoxicity values correlate with the initial metal ionic payload, delivered after a corrosion process promoted by the inhomogeneity of the silica shell.

(3 mmol) was added to a mixture of 1,2-hexadecanediol (10 mmol), oleic acid (6 mmol), oleylamine (6 mmol) and octhyl ether (20 mL) under magnetic stirring and N2 flow. The mixture was heated up to 290 °C for 1 h and let to reflux (2 h). Again, a change of color from yellow to black was observed. The formed nanoparticles were separated by centrifugation adding ethanol, and the process was repeated at least three times. Finally, the Fe3O4 nanoparticles were dispersed in hexane solution using oleic acid.37 Fe3O4 Nanoparticles (15.1 (±4.1) nm in Diameter) in Aqueous Solution. Magnetite (partially oxidized to maghemite) nanoparticles were prepared according to Massart’s method, exploiting a coprecipitation process of ferrous and ferric ion solutions (1:2 molar ratio).38 Aqueous solution of FeCl3 (20 mL, 1M) and FeSO4 (5 mL, 2 M in HCl (2M)) were added to NH4OH (250 mL, 0.7M) under mechanical stirring for 30 min, in order to obtain a black precipitate. The sediment was redispersed in distilled water (50 mL) and subsequently three aliquots of tetramethylammonium hydroxide solution (10 mL, 1M) were added while stirring. Finally, the solution was brought up to a volume of 250 mL with water. The magnetite nanoparticles were further washed, prior to using the magnetic nanoparticles solution (4 mL), and diluted to 50 mL with distilled water, centrifuged, and redispersed in pure water (100 mL).39 Silica Coating. For incorporation in silica, the Fe3O4 and CoFe2O4 nanoparticles were dispersed in chloroform (the nanoparticles synthesized in aqueous solution were first mixed with different amounts of oleic acid for the phase transfer). For the silica coating to take place by a reverse microemulsion, 2.6 mL of NP-5 were dispersed in 18 mL of cyclohexane and stirred for 15 min.40 Then, independently, Fe3O4 or CoFe2O4 nanoparticles dispersed in chloroform (100−200 μL, 0.05M) were added, jointly with subsequent volumes of TEOS (20− 150 μL in 2 mL of cyclohexane, for the thinner or thicker silica shell) and 850 μL of ammonia hydroxide solution were added. After every addition, the reaction mixture was allowed to stir for 15 min (1800 g) and then stored for 48 h. The silica-coated magnetite or cobalt ferrite nanoparticles were washed and redispersed in water.4 Cytotoxicity Assays. To evaluate cell viability in the presence of the different types of nanoparticles, a human colon adenocarcinoma cell line (Caco-2) was used. Cells were grown in 25 or 75 cm2 plastic flasks in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose, 10% fetal calf serum (FCS), 100 units/mL of penicillin, 100 μg/mL of streptomycin, 2 mM L-glutamine, and 1% nonessential amino acids at 37 °C, in a 5% CO2 humidified atmosphere. For the evaluation of the cytotoxicity results, a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was employed. This assay is based on the conversion of the yellow tetrazolium salt to purple formazan crystals by metabolic active cells. In these experiments, cells were seeded at a density of 10 000 cells per well into 96-well tissue culture plates and grown for 24 h before adding the tested nanoparticles. These nanoparticles, previously diluted in culture medium without fetal bovine serum, were added to the wells at final concentrations in the range 5−100 μg/mL, and the cells were incubated for 24 h. After the incubation period, 10 μL of the MTT reagent were added to each well, and the cells were incubated for an additional 4 h period to allow the cleavage of the MTT reagent by viable cell mitochondrial succinate dehydrogenase. Afterward, 100 μL of the solubiliza-

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EXPERIMENTAL SECTION Chemicals.4 Ammonium hydroxide solution, NH4OH (28− 30% NH3) and iron(III) chloride hexahydrate (FeCl3·6H2O, 97%) were obtained from Fluka. Oleic acid, iron acetylacetonate (Fe(acac)3), hydrochloric acid (37%), sodium hydroxide, dioctyl ether, iron pentacarbonyl (Fe(CO)5), 1,2-hexadecandiol, oleylamine, benzylether, tetramethylammonium hydroxide (TMAOH), cobalt(II) chloride hexahydrate (CoCl2·6H2O, 98%), iron(III) nitrate nonahydrate (Fe(NO3)3·9H2O, 98%), iron(II) sulfate heptahydrate (FeSO4·7H2O, 99%), nitric acid (HNO3 65%), poly(5)-oxyethylene-4-nonylphenyl-ether (NP5), tetraethylortho-silicate (TEOS), hexane, and cyclohexane were supplied from Sigma-Aldrich. Chloroform was supplied from Merck. All chemicals were used as received. Distilled water was deionized using a Millipore system (with conductivity lower than 18 mS cm−1). The Caco-2 human colon adenocarcinoma cell line was obtained from the European Collection of Cell Cultures (ECACC no. 86010202). CoFe2O4 Nanoparticles (21.5 (±10.7) nm in Diameter) in Aqueous Solution.35 CoCl2·6H2O (5 mL, 2 M in HCl 7.4%) and FeCl3·6H2O (40 mL, 0.5 M in Milli Q water) solutions were heated at 50 °C and added under vigorous stirring to a boiling solution of NaOH (200 mL, 1M). After 30 min, boiling was stopped and the solution was cooled to room temperature. Afterward, five water-cleaning stages by magnetic sedimentation were made. The resultant ferrofluid was treated with an oxidative reaction to passivate the surface. For that, nanoparticles were dispersed in 30 mL containing HNO3 (2M) and Fe(NO3)3·9H2O (0.35M) and heated to 100 °C for 45 min under continuous stirring. The final product was magnetically sedimented overnight and finally dispersed in 100 mL Milli Q water. Fe3O4 Nanoparticles (6.6 (±1.2) nm and 10.4 (±2.0) nm in Diameter) in Organic Solution. These magnetite nanoparticles were synthesized as follows: Fe(CO)5 (0.4 mL, 3.04 mmol) was injected under N2 flow into a mixture containing 20 mL of octyl ether and 3.44 mL of oleic acid (10.84 mmol) at 100 °C, previously deoxygenated under N2 for 20 min. The resulting mixture was slowly heated (280−290 °C) and refluxed for 2 h under open atmosphere, observing a change of color from yellow to black. The solution was led to cool down to room temperature under continuous magnetic stirring and subsequently treated with excess of ethanol. The formed nanoparticles were separated by centrifugation, and the process was repeated at least three times. Finally, the Fe3O4 nanoparticles were dispersed in hexane solution.36 The 10.4 nm magnetite nanoparticles were synthesized as follows: Fe(acac)3 28267



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Figure 1. Summary of samples of transition metal ferrite nanoparticles employed, representative TEM image of the Fe3O4@SiO2 nanoparticles from sample 6 and MvsH hysteresis loops of nanoparticles from samples 2, 4, and 7.

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RESULTS AND DISCUSSION Figure 1 includes a summary of the magnetic nanoparticles employed using cobalt ferrite (CoFe2O4) and magnetite (Fe3O4) as examples of transition metal ferrites. The cobalt ferrite nanoparticles (sample 1) were synthesized by a standard coprecipitation process35 and further coated with silica (samples 2 and 3) after being transferred into a reverse microemulsion system.40 The magnetite nanoparticles were synthesized employing two different methods: the Massart method based on the coprecipitation of two salts of iron38 and on the thermal decomposition of an iron precursor.36,37 These methods produce 15.1 nm in the first case (sample 4), and 6.6 nm particles, that were grown to 10.4 nm (average diameter), in the second case. These 10.4 nm magnetite nanoparticles grown in an organic solvent were transferred to aqueous solution (sample 5), using tetramethylammonium hydroxide (TMAOH).39 The initial 6.6 nm magnetite nanoparticles produced were coated with silica, employing again the reverse microemulsion method (samples 6 and 7). Thus, three samples based on CoFe2O4 and four based on Fe3O4 were made available, as summarized. Figure 1 also includes a representative TEM image of silica-coated transition metal ferrite nanoparticles, while Figure S1 in the Supporting Information (SI) includes TEM images with general overviews of all the samples employed. Both transition metal ferrite nanoparticles have been widely used as examples of nanoparticle magnetism, with emphasis on studies of the nanoparticle intrinsic spin structure as influenced by surface and finite-size effects.41 The different samples herein employed consider the variation of important magnetic parameters, as reflected by the MvsH hysteresis loops (Figure

tion solution were added, and the plates were incubated overnight. The absorbance of the solubilized formazan product was then spectrophotometrically quantified in an ELISA microplate reader at 570 nm with the reference at 630 nm. All experiments were performed in triplicate. The cell viability is expressed as a percentage according to the formula: % Cell viability= [(A c+n+m − A n+m)/Ac+m] × 100, where A c+n+m corresponds to the absorbance of the cells with nanoparticles present in the medium, An+m corresponds to the absorbance of the nanoparticles in the medium (no cells present), and Ac+m corresponds to the absorbance of cells in the medium (no nanoparticles). The cell viabilities were therefore measured as a function of the relative cell survival percentages when compared to nanoparticle-free cell control, and the data reported represent an average of three independent experiments in all cases (with three different measurements registered in every case).4 Characterization. TEM measurements were performed on a JEOL JEM 1010 instrument operating at an acceleration voltage of 100 kV. Samples for TEM were prepared by placing a drop of the dispersions on a Cu grid letting the liquid evaporate at room temperature in the case of nanoparticles. For imaging the nanoparticles inside cells, we have used a specific protocol for cell culture developed by the microscopy unit (http://webs. uvigo.es/cactiweb/s_microe/pmme01.htm). To study the magnetic properties using SQUID magnetometry, the magnetic nanoparticles were dried. ICP analysis was performed using a PerkinElmer Optima 4300 DV. 28268



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Figure 2. TEM images of Caco-2 cells from the assays using nanoparticles from samples 3 (top), 4 (center), and 6 (bottom).

percentages of cell viability of the Caco-2 cells as a function of the added concentration (in μg/mL) of the three cobalt ferritebased samples (1, 2, and 3) and the four magnetite-based samples (4, 5, 6, and 7), respectively. An overall observation of the cytotoxicity results points toward higher cell viability for the noncoated nanoparticles (samples 1, 4, and 5). On the contrary, all the samples coated with silica show an accentuated decrease in the cell viability dependent on the nanoparticle concentration (samples 2, 3, 6, and 7). In order to assess the intracellular localization of the nanoparticles, a cryo-TEM analysis of the Caco-2 cells upon exposure was also performed, addressing the cellular uptake. Figure 2 includes representative TEM images of the Caco-2 cells that have interacted with nanoparticles from samples 3, 4, and 6. While nanoparticles from samples 3 and 6 have internalized the cells and have accumulated into endosomes, nanoparticles from sample 4 appear accumulated outside the cells (Figure 2-center). Indeed, no silica-free particles were taken up in the cell and in contrast, TEM imaging showed that all the silica-coated nanoparticles (independently of the magnetic core nature or size) were internalized. The fact that the noncoated (samples 1 and 4) and the TMAOHfunctionalized nanoparticles (sample 5) do not internalize the cells explains the very low values of cytotoxicity encountered in all cases and even at the highest doses used, because they are unable to cross the cell membrane. To explain, however, the differences in the cell viability results among the noncoated nanoparticles even staying outside the cells, it is necessary to underline the differences in the nanoparticle surface chemistry.

1) from samples 2, 4, and 7. Sample 2 includes nanoparticles with the larger magnetic core and magnetocrystalline anisotropy (CoFe2O4) and accordingly there is a multidomain ground state that leads to a broad hysteresis loop (increased HC) at low temperature since it takes important field energy to make domain walls move. This situation persists, although in a much smaller way, at room temperature. Samples 4 and 7 have small (15.1 nm) and even smaller (6.6 nm) magnetic cores (with additionally lower magnetocrystalline anisotropy) and offer consequently nearly (at low temperature) and completely closed loops at room temperature (HC = 0), characteristic of a superparamagnetic behavior. This means that the magnetic moment of the nanoparticles as a whole is free to fluctuate in response to thermal energy, while the individual atomic moments maintain their ordered state relative to each other. Different values of saturation magnetization (MS) can be extracted at room temperature due to the different materials forming part (the two ferrimagnetic transition metal ferrites and the diamagnetic silica, which reduces the initial MS values in the coated magnetic nanoparticles). Accordingly, all the different magnetic responses discussed above depending on size and material can therefore be exploited in the different biorelated applications.24,42 To benchmark the relative toxicities of the magnetic nanoparticles (noncoated and coated with silica), the cellular viability of Caco-2 cells was assessed by colorimetric assay.43 The cells were treated for 24 h with known concentrations of nanoparticles from the seven samples described, over a 5−100 μg/mL dose range. Figures S2 and S3 (see SI) show the 28269



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condensation reactions of soluble silanol species, conducted in aqueous medium under basic conditions at room temperature, but confined in the microemulsion cavity. The fact of performing this process in the confined volume given by the reverse microemulsion just amplifies this chemical gradient and favors therefore the aforementioned partial etching.30 Sample 6 includes very small (6.6 nm) magnetic nanoparticles, which were located therefore in the voids of the micelles before the coating process, on which there was more available space for the silica shell to grow. This favors the swelling of the silica shell as being produced by successive molecular condensation reactions of soluble silanol species. Nanoparticles from sample 3 were obtained by coating the much larger (21.5 nm) CoFe2O4 cores with silica. In this case then, there is less room available in the microemulsion cavity for the molecular condensation reactions of soluble silanol species, resulting in more cross-linked silica. This means that the silica partial etching is more important in the nanoparticles from sample 6 than in the nanoparticles from sample 3, as actually shown in the TEM images in Figure 2. Summarizing this part, the silicacoated magnetic nanoparticles undergo a degradation process, promoted not only by the pH encountered when trapped in the endosomes of the cells, but also and mainly because of the nonhomogeneity of the silica, which favors its internal etching.27,30,44 Figure 3 reflects the cell viability as a function of silica-coated nanoparticle dosage from samples 2, 3, 6, and 7. We can draw a

In the case of samples 1 and 4, there are not stabilizers in the nanoparticle surface, situation that favors an aggregation process when added in the cell culture, rendering very difficult the endocytosis of the larger aggregates formed and any influence in the cell activity. In the case of sample 5, nanoparticles have TMAOH as the surfactant, with the negative hydroxide ions attached at their surface and the cationic species [(CH3)4N]+ around forming the electrostatic double layer.39 This circumstance increases the colloidal stability of the nanoparticles, although we have not observed them internalized in cells. The cytotoxicity values as increasing the positively charged nanoparticle dose (SI Figure S3) are likely related to the basic pH of the nanoparticle solution with a visible effect only at the highest dose. The silica-coated magnetic nanoparticles on the contrary, have interacted with and internalized the cells. All the silicacoated transition metal ferrite nanoparticles detected appeared inside cells and, paying more attention to the TEM images, the nanoparticles have actually changed their morphology once inside. The TEM image (top-right) in Figure 2 shows the nanoparticles (from sample 3) inside the cells and degraded to a certain extent starting from the interface between the silica and the magnetic core. The TEM (bottom-right) image in Figure 2, instead of the initial nanoparticles from sample 6, shows however swelled spherical units that result from the partial degradation of the magnetic core and the silica shell. Attending to the changes in the morphology of the nanoparticles pointed out it is also particularly important to take into account the chemistry of the silica surrounding the magnetic cores. Indeed, the physiological conditions encountered by the nanoparticles once crossing the cell membrane can initialize different chemical processes.44 During the cellular endocytosis, nanoparticles experience a decrease of pH, from that of the extracellular medium (7−7.5), early and late endosomes (6− 5.5), to the acidic environment of lysosomes (4.5−4.8). In the lysosomes, nanoparticles can be exposed to the combined effect of acidic pH and enzymes (lysosomal hydrolases that can hydrolyze proteins, DNA, RNA, polysaccharides, and lipids).26 Although we would expect the silica of the coated nanoparticles to have a similar framework structure, Wong et al. have revisited the Stöber method reporting the inhomogeneity of the silica formed as a source for preferential etching.30 The complete hydrolysis of TEOS and subsequent condensation of silicic acid give a theoretically uniform network of tetrahedral SiO4 units with shared vertices. However, through controlled experiments, they were able to demonstrate the inhomogeneity of the silica precipitated by etching interior concentric shells of silica nanoparticles with hot water. According to their hypothesis, the outer silica shell that persisted must be porous and permeable to allow the solvent to diffuse in, and the etched part to dissolve out. Since no protecting agent was employed, they postulated the inhomogeneous nature of the silica formed by the Stöber method due to a possible sequential hydrolysis, to explain the preferentially dissolved inner silica. That is, the hydrolysis of TEOS brings about a gradient of chemical stability since becomes slower as advancing on the process and as decreasing the concentration of TEOS in solution. Analogously, the inner silica of the silica-coated transition metal ferrite nanoparticles becomes etched because it was formed faster and with a more porous structure, while the outmost layer persists since it was derived from silicic acid and derivatives and has reached an important degree of cross-linking. In fact, the silica shell of these nanoparticles is produced by successive molecular

Figure 3. Comparison of cell viability for the silica-coated transition metal ferrite nanoparticles.

better picture of these cytotoxicity values considering the different factors playing a role, since the nanoparticle−cell interactions are governed by a spectrum of physicochemical properties that depend on different parameters.45 Silica cytotoxicity and cellular uptake have been demonstrated to be dependent on nanoparticle size; considering a range between 14 and 335 nm, with important cytotoxicity values at the smaller sizes,46 and revealing an optimum size of about 50 nm for cell uptake.47 The total diameter is not a differentiating parameter in cell viability in our case, since we are considering a rather small range (samples 2 (40.5 nm), 3 (52.9 nm), 6 (28.3 nm) and 7 (44.2 nm)) of sizes. Additionally, although silica, which dissolves to form monosilicic acid or oligosilicic acid, has been shown to have no intrinsic toxicity,47 it can actually have some negative effect once inside the Caco-2 cells, which would be similar and proportional to the 28270



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the magnetic core and the silica shell were observed. Given the fact that the products of the silica degradation do not affect (or to a very minimal extent) the cell viability, we correlate therefore the cytotoxicity results to the release of metallic ions into the cytosol, which connotes an important impact in the cells.34 As mentioned, Davidson et al. have summarized some of the molecular responses exhibited by cells that come into contact with metallic ions, since they can interfere, for example, with the essential metals of some enzymes. Since the chemical degradation of the nanoparticles takes place precisely at the endosomes, we can point out the capability of the divalent metal transporter (DMT 1), to transport ferric iron (Fe3+) and Co2+ ions into the cell.34 Additionally, we have to take into account the reactive oxygen species (ROS), which are highly reactive molecules with unpaired electrons formed during oxidative metabolism, such as O2− ions, OH radicals or hydrogen peroxide (H2O2) molecules. They are continuously generated and eliminated in biological systems and play important roles in the regulation of cell proliferation or apoptosis processes since they can cause oxidative damage to cellular macromolecules, including DNA, proteins, and lipids. Exposure of cells to As, Cd, Co, Cr, Cu, or Ni (and even excess of Fe49) can generate excessive ROS in cells (or depletion of cellular antioxidant capacity) by Fenton-type reaction, HaberWeiss reaction, or by reacting directly with cellular molecules. The high ROS levels can damage cells by peroxidizing lipids, disrupting DNA, modulating gene transcription, and altering proteins, consequently resulting in a decline in physiological function and cell apoptosis/death. Lastly, we can relate the more important cytotoxicity values to the release of Co2+ ions from the CoFe2O4 magnetic cores. Although cobalt is also an essential element as part of vitamin B12, it becomes toxic at high concentrations. The common modes of action include induction of oxidative stress and damage to cellular components, particularly DNA, interference with DNA repair systems, resulting in genomic instability and deregulation of signaling pathways (and oncogenes or tumor suppressors in case of carcinogenicity). Co2+ ions are able to impair p53-DNA binding capacity and inhibit cell cycle arrest, due to the disruption of p53 native conformation.50 One more aspect to consider is related to the fact that some nanomaterials can induce autophagy; a process by which the cell degrades intracellular pathogens and damaged organelles and proteins.7 Both autophagy and endocytosis (endolysosomal) ways can influence the disposition of nanomaterials, leading therefore to important values of cytotoxicity.

concentration of nanoparticles in all the experiments we have carried out. However, according to a previous report,48 the range of concentration of nanoparticles employed is rather low for the silica to cause a significant impact in the cytotoxicity values registered. The cell viability curves from samples 6 and 7 are very similar in terms of tendency and quantitative values of cytotoxicity over the whole range of dosage, which means that the only parameter differentiating the two samples (the silica shell thickness) was not playing a role. However, the comparison of the two samples containing the cobalt ferrite as the magnetic core reflects that although they have a similar dosagedependent tendency, the smaller one (sample 2) shows higher percentages of cell viability, which means that in this case, the differentiating parameter (the silica shell thickness) was indeed playing a role. To obtain thinner silica shells around magnetic cores of the same size, the process for the silica precipitation in the reverse microemulsion is carried out using lower concentrations of TEOS and consequently at slower reaction rate, which also generates less porous silica. Since this thinner but more compact shell is consequently more difficult to etch, it will slow down the degradation of the magnetic core and the release of metallic ions. These differences, although also present in the samples with the 6.6 nm magnetite nanoparticles (samples 6 and 7), are not that accentuated to cause variations in the cell viability. According to the right top and bottom images in Figure 2, we can state that the acidic medium the nanoparticles have encountered once internalized in the cells degrades the silica shell and the magnetic core. Although detected by ICP, it is not possible to differentiate the amount of metallic ions from the magnetic material in the cells which still form part of the partially degraded magnetic cores or which were released. Consequently, we have performed a comparison of average size distributions. Figure 4 includes the size distribution analyses

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CONCLUSIONS In summary, we demonstrate the complementary action played by the silica shell and the magnetic core of the silica-coated transition metal ferrite nanoparticles to mediate the biological delivery and cell viability. While silica helps to internalize the nanoparticles in the cells and differentiate their degradation once located in the endosomes and exposed to a moderate acidic pH, the transition metal ferrite material determines the type of metallic ions released and consequently the final cytotoxicity results.

Figure 4. Comparison of size distribution analyses (Gaussian fit) of the CoFe2O4 magnetic cores from sample 3 before and after cell exposure.

(Gaussian fit) of the CoFe2O4 magnetic cores from sample 3, before and after cell exposure. The plot of size distributions reflects the significant reduction in size (from 21.5 ± 10.70 to 12.8 ± 4.74 nm average diameter) of the CoFe2O4 magnetic cores, due the very important degradation process they have undergone. We cannot perform the same analysis for the Fe3O4-based samples since instead of core−shell nanostructures, swelled spherical units resulting from the degradation of

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1. Representative TEM images of the nanoparticles from the different samples employed. Figures S2 and S3. 28271



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Percentages of cell viability as a function of concentration of nanoparticles employed (CoFe2O4 (sample 1), CoFe2O4@SiO2 (samples 2 and 3)), Fe3O4 (samples 4 and 5), and Fe3O4@SiO2 (samples 6 and 7)). 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]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Xunta de Galicia (Regional Government, Spain) has supported this work under projects EM2014/035 and InBioMed (Investigadores Mozos).

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REFERENCES

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Nonhomogeneous Silica Promotes the Biologically Induced Delivery

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