Maghemite Functionalization for Antitumor Drug Vehiculization

Article pubs.acs.org/molecularpharmaceutics

Maghemite Functionalization for Antitumor Drug Vehiculization Katarzyna Rudzka,† Á ngel V. Delgado,*,† and Julián L. Viota‡ †

Department of Applied Physics, School of Science Campus Fuentenueva, University of Granada, 18071, Granada, Spain Department of Physics, Campus Las Lagunillas, University of Jaén, 23071, Jaén, Spain

‡

ABSTRACT: In this paper we describe the preparation and characterization of magnetic nanocomposites designed for applications in targeted drug delivery. Combining superparamagnetic behavior with proper surface functionalization in a single entity makes it possible to have altogether controlled location and drug loading, and release capabilities. The colloidal vehicles consist of maghemite (γ-Fe2O3) cores surrounded by a gold shell through an intermediate silica coating. The external Au layer confers the particles a high degree of biocompatibility and reactive sites for the transported drug binding. In addition, it permits to take advantage of the strong optical resonance, making it easy to visualize the particles or even control their payload release through temperature changes. The results of the analysis of relaxivity demonstrate that these nanostructures can be used as T2 contrast agents in magnetic resonance imaging (MRI), but the magnetic cores will be mainly useful in manipulating the particles using external magnetic fields. We describe how optical absorbance and electrokinetic data provide a followup of the progress of the nanostructure formation. Additionally, these techniques, together with confocal microscopy, are employed to demonstrate that the component nanoparticles are capable of loading significant amounts of the antitumor drug doxorubicin, very efficient in the chemotherapy of a wide range of tumors. Colon adenocarcinoma cells were used to test the in vitro release capabilities of the drug-loaded nanocomposites. KEYWORDS: doxorubicin, gold shell, maghemite, magnetic resonance imaging, nanostructures, silica coating, superparamagnetism, targeted drug delivery

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INTRODUCTION It is common knowledge that cancer is one of the most lethal world diseases, and fighting against it is a universal challenge for all fields of science involved. According to the 2008 World Cancer Report,1 the most complete global examination of the disease to date, over 12 million new cases are reported each year, with around 7 million deaths. To fight the battle against cancer, more and better diagnostic and treatment approaches should be developed. This includes improvements in conventional surgery, radiation therapy, and chemotherapy, as well as finding new medical and technological strategies and ideas in such fields as drug delivery, diagnostic techniques, and new treatments.2 Nanoscience and nanotechnology can help in achieving some of these objectives as they have proved promising in different biomedical applications.3−5 In fact, working with therapeutic nanoparticles offers us tremendous control of the behavior and distribution of the active molecules to which they serve as vehicles. This is not the only biomedical field of application of nanosized materials. Let us mention, for instance, the therapy known as magnetic hyperthermia, which allows increasing the temperature of a tumor area to a level (>40 °C) where tumor cells are killed.6−11 In these applications, the use of nanoparticles has the additional advantage that extravasation of particles and their accumulation in tumors are favored by the passive mechanism of enhanced permeability and retention (EPR).12−14 © 2012 American Chemical Society

In addition to hyperthermia, magnetic nanoparticles are presently being employed as contrast agents in magnetic resonance imaging (MRI). Superparamagnetic iron oxide nanoparticles (SPIONs), which possess a high transverse (or spin−spin) relaxivity, are T2 contrast agents, and as a consequence they produce darker images if accumulated in a certain area.15−18 Because of their magnetic properties, SPIONs have been widely studied as delivery vehicles for therapeutic agents, using an external magnetic field with the aim of driving and locating the particles at the desired site.19−24 In such systems, drugs or genes are bound to the magnetic nanoparticles and administered by injection near the target area. When the therapeutic compound is located in the appropriate position, a magnetic field is externally applied to the site so as to concentrate magnetic nanoparticles with attached drug at the target place. Thus, the use of magnetic nanostructures as drug delivery systems can be advantageous in cancer therapy in different ways.25,26 Their magnetic behavior not only permits their location and accumulation in the target tumor but also makes possible monitoring their distribution by MRI. This in turn allows optimizing the drug dosing while reducing side effects. In addition, the magnetically guided particles can Received: February 21, 2012 Accepted: June 13, 2012 Published: June 13, 2012 2017

dx.doi.org/10.1021/mp3002705 | Mol. Pharmaceutics 2012, 9, 2017−2028

Molecular Pharmaceutics

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compensate for the limited penetration of free drugs through the tumor cell membrane.27 Because of such a wide range of potential and actual applications, efforts have been applied to the improvement of the medical performance of these nanoparticles in aspects like biocompatibility, drug loading and release capabilities, target specificity, and so on. One of the modifications that have been suggested is the preparation of SPIONs with a gold shell termination. The resulting magnetic nanostructures would have the additional advantage of particular optical properties, specifically increased absorbance in the visible spectrum, opening the possibility of using them in photothermal therapy. In summary these particles could be first directed and accumulated in tumors by application of external magnetic fields. Second, their MRI contrast properties can provide real time imaging of their biodistribution; finally the gold shell could provide photothermal activity while the drug is released. Moreover, gold is by itself under investigation for drug delivery purposes.28−34 Mukherjee35 reported that gold nanoparticles might also inhibit angiogenesis. Recently, You36 noted that gold nanospheres, employed as nanocarriers, are not cytotoxic for gold concentrations varying from 0.04 to160 μg/ mL, which is congruent with other literature sources indicating that, as a rule, Au-based nanoparticles are satisfactorily tolerated.37,38 In this work, we report on the use of composite particles consisting of a maghemite core coated by silica and terminated by a gold layer, as nanocarriers for a therapeutic drug, doxorubicin (DOX). The behavior of the magnetic silica substrate in MRI will first be analyzed and compared to commercially available contrast agents. A good representative example of DOX-based nanocarrier in the market is Doxil (commercial name of doxorubicin in a long-circulating PEGcoated liposome).39 Another proposal is a compound of liposomes under the trade name of Myocet, which is used as part of a combinatory therapy of several kinds of cancer.40 Excellent results have been reported for DOX in PEG liposomes against metastatic breast carcinoma,39,41,42 unresectable hepatocellular carcinoma,43 cutaneous T-cell lymphoma,44 sarcoma,45 squamous cell cancer of the head and neck,46 and ovarian cancer.47 Magnetic nanovehicles for doxorubicin have also been described. These can be based on covalent bonding of DOX to magnetite coated by amine-PEG,48 magnetite-PEGsilica nanostructures,49 or citrate-stabilized magnetite,50 to mention a few recent examples. As to the mechanism of action, it is known that DOX binds to DNA, preventing the genetic code from being read and used for manufacturing proteins. Doxorubicin also hinders DNA recoiling and copying. This stops cell replication and slows down the growth of the tumor.51 Electrokinetic determinations will be used as a qualitative probe of the surface composition of the particles obtained. On the other hand, UV−vis absorbance maxima of DOX are very well-defined, which facilitates control of its attachment/binding to nanospheres through spectroscopic evaluation, and hence electrophoresis together with spectrophotometric measurements will also be used for the evaluation of the amount of DOX loaded on the magnetic composites. The drug loading and its release in tumor cell nuclei will also be qualitatively ascertained by means of confocal microscopy by taking advantage of the fluorescence properties of DOX.

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EXPERIMENTAL SECTION

Materials. Ethanol 96% solution was purchased from Guinama, Spain. Ammonia (NH4OH) 32% solution was reagent grade from Scharlau, Germany. Iron(III) chloride hexahydrate; iron(II) chloride; ammonium hydroxide; hydrochloric acid (HCl); sodium chloride; iron(III) nitrate; nitric acid (HNO3); TEOS (tetraethoxysilane), 98% solution; sodium hydroxide; chloroauric acid or hydrogen tetrachloroaurate (HAuCl4); trisodium citrate dihydrate (Na3C6H5O7·2H2O); poly(diallyldimethylammonium chloride) (PDADMAC) (C8H16ClN), (low molecular weight) 20 wt % in water; poly(sodium 4-styrenesulfonate, PSS) (typical molecular weight, Mw = 70,000) 30 wt % solution in water; L-ascorbic a ci d ( C 6 H 8 O 6 ), 99 % solutio n; and d oxorubicin (C27H29NO·HCl) were all reagent grade from either SigmaAldrich (USA) or Panreac (Spain). All the chemicals and solvents were used without further purification. Water used in the preparation of the suspensions was deionized and filtered in a Milli-Q Academic (Millipore, Spain) system. Methods. Synthesis of Maghemite Particles. Maghemite nanoparticles were obtained from the oxidation of magnetite particles synthesized according to Massart’s coprecipitation method.52 All the synthesis procedures were carried out in a fume hood. First, a solution of iron chlorides was prepared by dissolving 3.97 g of FeCl2 in 10 mL of 2 M HCl and 10.8 g of FeCl3 in 40 mL of Milli-Q water. Both solutions were simultaneously and rapidly added to 500 mL of 7.7 M NH4OH contained in a 1 L beaker, while stirring vigorously. After the sedimentation of the produced black precipitate with a magnet, the supernatant was separated and the moist precipitate was heated up to about 90 °C, while stirring on a hot plate. After about 5 min, magnetite nanoparticles were oxidized by adding to the solution 40 mL of 2 M HNO3 and 60 mL of 0.33 M FeNO3. The temperature of 90 °C and the stirring were maintained during 1 h. After this time the synthesis was terminated. The maghemite nanoparticle dispersion was repeatedly centrifuged and redispersed in water. Silica Coating on Maghemite Particles. A silica shell was produced on maghemite by using a modified sol−gel process described in ref 53, which originates from the well-known Stöber method.54 First, a solution containing 4.85 mL of 28% NH4OH, 28.8 mL of H2O, and 27.5 mL of 96% EtOH was prepared. The second step consisted of adding 6.22 mL of a suspension of maghemite particles in water with 4.83 g/L solids concentration to the ammonia solution referred to above while stirring. Finally a solution containing 2 mL of TEOS in 30 mL of ethanol was added to the maghemite/ammonia solution in small portions with a pipet. The condensation of SiO2 onto the magnetic particles took 4 h. In order to minimize particle aggregation during the process, the synthesis was carried out under constant agitation in a bath sonicator (Selecta, Spain). The final magnetic silica suspension was cleaned of impurities and unreacted compounds by repeated cycles of centrifugation and redispersion in water. Formation of a Gold Shell on Maghemite/Silica Spheres. The formation of the gold shell requires the previous coating of the substrates with a dense layer of Au nanoparticles which act as seeds for gold condensation. The preparation of Au nanoparticles was performed according to the standard sodium citrate reduction method.55 100 mL of a 5 × 10−4 M aqueous solution of HAuCl4 was prepared and driven to boil. Next, 5 mL of a 1% sodium citrate solution was added, and the mixture 2018



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Scheme 1. Schematic Illustration of the Formation of Magnetic Silica Spheres Coated with an Outer Shell of Gold53

and the supernatant was spectrophotometrically evaluated for DOX content. Physical Characterization of the Nanoparticles. Particle sizing was carried out based both on the microscopic observations (pictures of the particles were obtained using a high-resolution transmission electron microscope (Philips STEM CM20, Netherlands)), and on static and dynamic light scattering. This was performed, respectively, in a Mastersizer 2000 and a PCS 3700 (both from Malvern Instruments, U.K.). Electrophoretic mobility measurements were carried out in a Zetasizer Nano-ZS (also from Malvern Instruments, U.K.). The suspensions were prepared as follows. In the case of suspensions with different pH values they were prepared by simply adding dropwise a small amount of the iron oxide mother suspensions to 50 mL of 5 × 10−3 M KNO3 until finally obtaining a slightly turbid solution adequate for this type of determination. The pH value of the suspensions was then adjusted by adding a suitable amount of KOH (0.01 or 0.1 M) or HNO3 (0.01 or 0.1 M). The suspensions were left to stay at least 3 h, and then the electrophoretic mobility was determined. The temperature was 25.0 ± 0.5 °C. For each suspension, 3 measuring runs were taken, with 3 cycles in each run. In the case of suspensions with different ionic strengths, these were prepared by suspending a small amount of the iron oxide mother suspensions in KNO3 solutions containing the specified amount of electrolyte, in the concentration range 10−4 M to 0.1 M. Suspensions when prepared were equilibrated for 3 h as before. In all cases, the zeta potential was obtained from mobility data using the O’Brien and White theory,57 and the maximum relative uncertainty was typically below 5%. The XRD analysis of the maghemite powder sample was carried out in a BRUKER D8 ADVANCE (USA) powder diffractometer using Cu Kα radiation. The parameters chosen for the measurement were 2Θ steps of 0.02°, 8 s of counting per step, and 2Θ range from 3° to 80° at room temperature. Magnetic characterization was performed using a QUANTUM DESIGN MPMS XL SQUID magnetometer from EVERCOOL (USA). In Vitro DOX Release Evaluation. For the in vitro evaluation of doxorubicin release, a well-characterized colorectal adenocarcinoma cell line (DLD-1) was maintained as an adherent monolayer in the culture medium RPMI-1640 (GIBCO-BRL) supplemented with 10% fetal bovine serum (FBS), 100 U/mL

was stirred vigorously while boiling. The synthesis was continued during five more minutes until obtaining a redwine color, which indicates gold particle formation. The final suspension was kept in a glass bottle in a refrigerator (5 °C) in the dark. The electrostatic repulsion between the negatively charged silica coating and the gold nanoparticles prevents the deposit of the latter on the former. It has been shown56 that the layer-bylayer technique can be used to produce a number of stacked polyelectrolyte layers terminated in the positively charged PDADMAC to produce a homogeneous surface capable of electrostatically binding gold. As described in refs 53 and 56, it suffices to successively adsorb PDADMAC-PSS-PDADMAC onto the maghemite/silica spheres for obtaining the desired positively charged substrate. Briefly, we took 30 mL of silicacoated maghemite particles (after being sonicated for at least 20 min) with a solids concentration of 0.1 wt % and added it dropwise into 30 mL of an aqueous solution of polyelectrolyte (0.05 g/mL) while it was mechanically stirred and sonicated for 12 h. After that time the solution was centrifuged and redispersed in 30 mL of pure water at least twice. The same procedure was followed for each of the polyelectrolyte layers. A 5 mL volume of the suspension of polyelectrolyte-coated spheres (with ionic strength adjusted to 0.2 M NaCl) was mixed with 5 mL of the gold nanoparticle solution (0.05%) and kept under stirring for 20 min. As before, cleaning was achieved by repeated centrifugation and redispersion in 10 mL of pure water. In order to produce the final condensed gold shell,53 the gold nanoparticles deposited as above were used as nuclei for the further growth on the nanocomposite particles (Scheme 1). To that aim 10 μL of 5 × 10−4 M HAuCl4 and subsequently 10 μL of 0.34 × 10−3 M ascorbic acid solutions were added five times to 10 mL of aqueous solution of gold-seeded magnetic silica spheres. No more than six of the described addition processes appeared necessary to perform an optimum gold coating. Doxorubicin (DOX) Loading. We followed the procedure described by You et al.36 Aliquots of aqueous DOX solutions (1 mM, 0.03 mL) were added to an aqueous solution of magnetic silica spheres with gold shell (0.1 wt %, 0.5 mL), and the mixtures were stirred at room temperature for 24 h. After that time the solutions were centrifuged (14000 rpm for 20 min) 2019



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Figure 1. TEM of magnetic maghemite particles (A) and size distribution histogram deduced from static light scattering (B).

the morphology of γ-Fe2O3 (Figure 1A). It can be seen that the maghemite nanoparticles are close to spherical and rather monodisperse in size. The average particle size estimated directly from TEM micrographs is 15 nm with a standard deviation of 5 nm, which, as will be discussed below, is similar to the size measured by means of light scattering determinations. In fact, as determined by the static light scattering method, the average diameter of the synthesized maghemite particles was 17 ± 4 nm. Figure 1B is an example of the distributions found. The magnetic nanoparticles could be easily dispersed in water and be stably preserved for several months, suggesting that the tendency to aggregation is considerably weak. The quality of these particles is excellent for performing studies on biomedical applications. The X-ray diffraction pattern of the synthesized iron oxide particles is displayed in Figure 2. This pattern matches that of

penicillin, and 100 μg/mL streptomycin (Sigma, USA) and incubated at 37 °C in 5% CO2. For the cell culture experiments, cells were grown in chamber slides (8 well Chamber Slide, Sigma-Aldrich) and treated with a known concentration of DOX-loaded nanoparticles for 1 h in RPMI-1640 medium supplemented with 10% FBS at 37 °C, under gentle agitation during 2 h in a humidified 5% CO2 atmosphere. After treatment, the cells were fixed in ice-cold paraformaldehyde (4%) for 10 min and nuclear counterstaining with DAPI (4′-6diamidino-2-phenylindole) was performed. Nanoparticle autofluorescence was visualized with a Leica Spectral confocal (Bensheim, Germany) laser microscope, and DAPI staining with a Leica Fluorescence Microscope, using a 63× oilimmersion objective. X-ray Photoelectron Spectroscopy (XPS). XPS measurements were performed on an X-ray photoelectron spectrometer Kratos Axis Ultra-DLD. For all samples a monochromatic Kα X-ray source was employed. The conditions for the wide survey scan were as follows: energy range = 1200−0 eV, pass energy = 160 eV, step size = 1 eV, dwell(s) time = 0.1 s, number of sweeps = 1. In case of the Fe 2p region as well as for the O 1s region, for the high-resolution spectra, measurements with an energy range of 50−20 eV and a step size of 0.1 eV were done. Magnetic Resonance Imaging. The relaxivity of bare maghemite and silica coated maghemite samples was determined at room temperature in a 7 T Bruker Pharmascan (USA) MRI instrument using both water and fetal calf serum (FCS) as dispersion media. Previously the amount of iron in each sample was determined by total X-ray reflection in a TXRF 8030C FEI spectrometer (Germany). Different dilutions of the original suspensions (iron concentrations 6575 ± 14 mg/ mL for maghemite suspensions, and 288.9 ± 0.5 mg/mL in the case of maghemite/silica particles) in both water and bovine serum were placed in capillaries 1 mm in diameter and located in the isocenter of the magnetic field. Conventional weighting sequences were followed in T1 (longitudinal relaxation time, controlled by the spin environment), T2 (transversal or spinrelaxation time, related to the loss of phase between spins in the plane perpendicular to the magnetic field direction), and T2* (including also field inhomogeneities in the transversal relaxation).

Figure 2. X-ray diffraction pattern of the synthesized maghemite.

standard maghemite, although some of the exhibited peaks correspond to both maghemite (γ-Fe2O3) and magnetite (Fe3O4). All XRD peaks are characteristic of the iron oxide spinel structure (Fe3O4 or γ-Fe2O3). Since maghemite (γFe2O3) is commonly formed by oxidation of magnetite and the crystallographic (in fact, also magnetic) properties of maghemite are very close to those of magnetite, it is very difficult to distinguish between magnetite and magnetite/ maghemite phases because X-ray diffraction patterns are not different enough in that respect. In our case, the six diffraction peaks for standard maghemite, which are (220), (311), (400),

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RESULTS AND DISCUSSION Physical Characterization of the Nanocomposites. High-resolution transmission electron microscopy revealed 2020



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Figure 3. The XPS wide spectrum (A) and the Fe 2p spectrum (B) obtained for the maghemite powder sample.

particles is clearly observable. Dynamic light scattering determinations in dilute suspensions of these particles yielded an average hydrodynamic diameter of 202 ± 14 nm (around the size of a doublet like that in Figure 4). As mentioned, the production of a gold shell terminating the particles requires the use of gold nanoparticles deposited on the silica surface, and acting as seeds for the formation of the shell. Figure 5A is an example of the Au nanoparticles synthesized.

(422), (511), (440), indicated that the obtained XRD pattern (peak positions and intensity) corresponds to those of the reference data for γ-Fe2O358−62 up to 58%, suggesting that the maghemite phase (γ-Fe2O3) is likely the most abundant. The (311) peak broadening indicates that the average crystallite size of the fabricated particles is only a few nanometers.63 In order to further elucidate the presence of maghemite in our iron oxide particles, their XPS analysis was also conducted. The results are shown in Figures 3A (wide general spectrum) and 3B (iron bands). According to published studies,64−66 the peaks centered at 711.5 and 725 eV correspond, respectively, to Fe 2p 1/2 and Fe 2p 3/2, and they confirm the presence of maghemite. Indeed, the absence of the 798 eV Fe 2p 3/2 band (classified as the Fe2+ state of magnetite), as well as the presence of the 717 eV satellite peak, allows us to distinguish clearly that the sample presents Fe3+ states, and thus our particles are predominantly maghemite.64−66 Additionally, the sample peak at 530 eV observed in the O 1s region (Figure 3A) confirms that conclusion.67 The following step in the process of preparation of our final nanocomposites involved the coating of superparamagnetic iron oxide nanoparticles with silica in order to isolate the magnetic core from the surroundings. Although the iron oxide surface has a strong affinity for amorphous silica, the coating process can be complicated due to aggregation of magnetic particles. Because of this, the magnetic core consists of more than one iron oxide nanoparticle in most of the cases. Figure 4 illustrates these results: the presence of several maghemite

Figure 5. TEM image (A) and dynamic light-scattering measurements (B) of gold NPs synthesized and used as seeds to form the gold shell.

From several pictures like this, and also from dynamic light scattering data (Figure 5B), it can be estimated that the diameter of the gold particles is around 10 nm. The 10 nm gold colloids attached to the surface of the magnetic silica spheres were used to template the growth of the solid gold shell. Figure 6 contains a sample of the resulting final

Figure 6. TEM image of the gold shell forming on the magnetic silica spheres by filling the voids between the small gold particles deposited onto the surface of the magnetic silica spheres.

Figure 4. TEM picture of silica-coated maghemite nanoparticles. Bar length: 200 nm. The circles mark the maghemite nuclei. 2021



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composites. It can be observed that, although gold aggregates are present on the surface, the gold shell is also obtained, with a thickness close to the diameter of the gold nanoparticles, indicating that approximately a single monolayer of nanoparticles was deposited. The halo surrounding the particles, observed in Figure 6, corresponds to the polyelectrolyte film (∼3 nm according to ref 68), acting as substrate for the deposition of gold. Hydrodynamic diameters resulted to be 300 ± 20 nm. Electrokinetics. Zeta potential titration allowed verifying the value of about 7.5 for the isoelectric point of maghemite (Figure 7), in agreement with previous findings showing that

Figure 8. Effects of pH in 5 mM KNO3 solutions (A), and of ionic strength at natural pH (B) on the electrophoretic mobility of maghemite (γ-Fe2O3), and maghemite/silica (SiO2@γ-Fe2O3) before and after adsorption of PDADMAC and PSS/PDADMAC polyelectrolyte layers.

Figure 7. Zeta potential of γ-Fe2O3 and SiO2-coated γ-Fe2O3 dispersions as a function of pH.

ferrofluids are usually stable only under highly acidic or basic conditions. The presence of the silica shell significantly modifies the ζ−pH trend, approaching that of pure silica. Note that the isoelectric point of the mixed particles is close to 3.5, far away from that of maghemite and closer in fact to that of silica. The initial potential instability of bare maghemite at natural pH, which is associated with its low surface potential and small diameter, changes by the presence of the outer silica layer. Interestingly, magnetic silica spheres may form stable dispersions in the pH conditions typical of biological fluids and can have a predictable surface chemistry, thanks to the wellknown reactivity of silica toward coupling agents. The silica coating of magnetic nanoparticles results in an increased chemical stability of these suspensions, thus influencing their performance for bioapplications, especially when used for photodynamic therapy purposes. The outer silica layer also improves the polyelectrolyte compatibility. As mentioned, in most cases the magnetic core is composed of more than one iron oxide nanoparticle, because of the aggregation of iron oxide nanoparticles prior to or during the coating process. The molecular self-assembly (layer by layer, or LbL) technique was the first stage in a two-step process for the formation of the gold shell. Each successive precursor polyelectrolyte film (cationic PDADMAC was deposited first, followed later by anionic PSS adsorption) demonstrated a very significant effect on the surface charge of the magnetic silica spheres and their subsequent modifications. Figure 8A shows the dramatic changes experienced by the electrophoretic mobility of magnetic silica after the polyelectrolyte deposits. Note that by depositing one polyelectrolyte layer it is possible to control the surface charge totally, as it can be made to vary from highly positive in the case of the PDADMAC layer to very negative for the PSS layer located on the positive polyelec-

trolyte. Data corresponding to the third coating (PDADMAC on top of PSS) are almost identical to those obtained after the first PDADMAC deposit and will not be shown. These three layers of polyelectrolytes will generate a uniform and positively charged substrate for the negative gold coating.53 It is once more confirmed that the LbL technique is an excellent tool for exploiting the electrostatic attraction between the charged species to be deposited. Figure 8B shows the trend of variation of the electrophoretic mobility with ionic strength. Note that the important effect of the polyelectrolyte coatings on the mobility of magnetic silica is found for the whole range of ionic strength tested. Very significantly, the result previously reported by Ohshima69 that the mobility remains finite at very high ionic strength in the case of soft, polyelectrolyte-coated particles is confirmed in our case. Electrophoretic mobility determinations are also a good probe for the deposition of gold seeds on the PDADMACcoated magnetic silica spheres and the negatively charged gold nanoparticles. Data in Figure 9 demonstrate that after 3 goldcoating steps the electrophoretic mobility of the nanostructures obtained is practically indistinguishable from that of the particles coated with just the gold seeds, this being a good indication of the efficiency of the gold shell formation. Optical Study. The optical properties of gold particles enable the spectrophotometric control of the process of functionalizing the maghemite/silica nanospheres with gold in order to promote the subsequent drug conjugation. This is possible due to the strong, visible absorption band of gold53 and, as discussed later, of the therapeutic drug, doxorubicin. As shown in Figure 10, the aqueous solution of gold nanoparticles 2022



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plasmon resonance in the visible or near-infrared range but also have the advantage of magnetic responsiveness, as discussed below. Analysis of Magnetic Properties. Superparamagnetic properties are observed at sizes smaller than 15 nm for maghemite,74 and particles can then be considered SPIONs. It is essential to use SPIONs in biomedical applications since the magnetic remanence of larger particles within an organism can be even destructive when the magnetic field is removed.75 Our magnetic measurements (Figure 11) indicate that both samples (uncoated and coated magnetic iron oxide samples) are superparamagnetic at room temperature, that is, thermal fluctuations become dominant over spontaneous magnetization and the net magnetization will be zero at zero field.76 The action of an external field will produce net orientation of the magnetic moments of the particles, which behave as paramagnets. The saturation magnetization value obtained at 50 kOe for uncoated particles is 30.7 emu g−1, which is much lower than the value of its bulk counterpart, 74 emu g−1, for maghemite.77 Other literature values reported are 52 emu g−1 for 15 nm particles, and 31 emu g−1 for sizes around 7 nm.78 In fact, the saturation magnetization of single-domain superparamagnetic nanoparticles is size-dependent,79 which can be explained by surface effects.80 The surface spins of magnetic particles lack complete coordination and become disordered, thus being less susceptible to changes in the strength of the external field.81,82 This phenomenon becomes more significant for the nanosized particles due to their large surface-to-volume ratio. So, it is reasonable for the assynthesized nanoparticles to have a smaller saturation value even under fields as high as 50 kOe. In addition, it has been reported that the crystallinity could also affect the magnetic properties.83 Therefore, the amorphous impurities at grain boundaries, undetectable by XRD, might be another reason for the diminution in the effective magnetic moment.84 On the other hand, the magnetization value observed at 50 kOe for the coated magnetic nanoparticles was 2.8 emu g−1, significantly smaller than that for the naked ones. This is clearly due to the thickness of the silica layer (about 50 nm, Figure 4), although maghemite nanoparticles preserve their supermagnetic behavior despite being embedded in silica shells (Figure 11). Magnetic Resonance Imaging. It is easy to understand that the clinical objective of MRI is to optimize the contrast and sharpness in observation of body structures. This is associated with differences in relaxation times coming from different tissues and, for given magnetic field strength, can be improved

Figure 9. Comparison of the effect of pH on the electrophoretic mobility of magnetic silica spheres with infiltrated gold seeds, and of maghemite/silica/gold shell nanostructures.

Figure 10. Optical absorbance spectra of aqueous solutions of 10 nm Au NPs (1), and magnetic silica nanospheres, both bare (2) and coated with five gold layers (3).

used for the deposition has a peak absorption wavelength at 525 nm, associated with the surface plasmon. The same wavelength has been reported by Brust et al.70 and Mayya et al.71 Upon gold shell formation the adsorption band becomes wider and shifts to longer wavelengths. This has been justified in refs 72 and 73 as due to the coupling of the surface plasmons of neighbor particles. In summary, it can be verified that magnetic silica spheres with an outer shell of gold not only possess a highly tunable

Figure 11. Left: room-temperature magnetization curves of silica-coated and uncoated maghemite particles. Right: a picture illustrating the action of a permanent magnet on a maghemite suspension. 2023



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through generation of T2 and T2* contrast. These two relaxations are hence most useful for applications of nanoparticles in medical imaging: because T2 time is longer the larger the water content, tissues with larger water concentrations will appear brighter in T2-weighted images.85,86 One existing way for increasing contrast in this sort of image is to use paramagnetic gadolinium chelates, which reduce both T2 and T2*, and thus contribute to darkening the area where they are present. An even better alternative is superparamagnetic nanoparticles, as those employed in this work. Figure 12 shows

with their gold shell as drug carriers for the transport and delivery of doxorubicin (DOX). The complexes of DOX with the different kinds of particles prepared in this work were readily formed by simply mixing them with DOX for 24 h at room temperature. As a first step in verifying the required functionality, an attempt of DOX loading onto the synthesized Au NPs (seeds) was first investigated. The results can be found in Figure 13.

Figure 12. Spin−spin relaxation time of suspensions of maghemite and maghemite/silica for different values of total iron concentration in water.

Figure 13. Comparison of the optical absorbance of free DOX in solution, Au nanoparticles and DOX on Au NPs. DOX concentration in all cases: 10−4 M.

the reduction in T2 achieved with maghemite/silica and (mainly) maghemite nanoparticles, and a similar effect was measured on T1 and T2*. The quantity characterizing the efficiency in contrast enhancement is relaxivity, R, or slope of the relation 1/T vs iron concentration. This kind of plot is shown in Figure 12 for T2. Note that a linear relationship is obtained between the reciprocal relaxation time and the concentration of iron in the sample. Values of the relaxivity in this and the other cases studied are shown in Table 1. These values are quite small as compared to the few data

The differences in absorbance spectra of gold and DOXloaded gold nanoparticles are clear. In fact, the color of the Au NP suspension changed from reddish to dark purple upon adsorption of DOX, while DOX@Au NPs displayed a UV−vis absorption peak at ∼490 nm, characteristic of DOX. After 24 h mixing, the absorbance peak intensity of DOX in the UV− visible region was significantly reduced compared to that obtained immediately after mixing DOX and Au NPs, when DOX was unbound. In summary, the absorbance data in Figure 13 confirm that the surface plasmon of gold is strongly quenched by DOX, which can be considered as a clear proof of adsorption. This result is in agreement with those presented recently by You et al.36 and Mirza and Shamshad,88 who showed that DOX molecules were able to cap gold nanoparticles. The adsorption of DOX to the polyelectrolyte/silica/ maghemite nanocomposites is demonstrated by data in Figure 14A, where the absorbance spectrum of a 10−4 M drug solution is compared to that of the supernatant produced after 24 h contact between the same solution and the nanoparticle suspension (0.1 wt % solids contents). Experiments for gold/ silica/maghemite nanoparticles were also performed with 2 × 10−4 M DOX concentrations (Figure 14B), with similar results. From the absorbance differences it is possible to obtain the DOX loading efficiency of magnetic particles coated with gold shells, as shown in Figure 15. Note that the drug adsorption increases with the equilibrium concentration of doxorubicin in solution up to 10−4 M, where saturation is reached at approximately 40 μmol/g. The adsorption isotherm model which best fits the data is the Langmuir one. The adsorption density Γ can be calculated as

Table 1. Values for Relaxivities in Fetal Calf Serum and Water Solutions R1

R2

R2*

Relaxivity (mM−1 s−1) in Fetal Calf Serum γ-Fe2O3 SiO2/γ-Fe2O3 γ-Fe2O3 SiO2/γ-Fe2O3

0.0112 4.13 0.0004 1.73 Relaxivity (mM−1 s−1) in Water 0.0101 3.16 0.0002 1.825

7.71 2.50 3.96 2.51

previously published on the relaxivity of maghemite NPs (most commercial formulations are based on magnetite): for example, Park et al.87 found R2 as high as 177.5 mM−1 s−1 for Dextrancoated maghemite nanocrystals, 358.9 mM−1 s−1 for carboxymethyl Dextran-coated maghemite, and 124.5 mM−1 s−1 for PEG-phospholipid coating, all in 3 T fields. These differences can be understood considering that the particles in the above work were specifically designed as MRI contrast agents, whereas our particles are rather drug delivery vehicles in which MR properties are an additional, or collateral, use. Drug Adsorption Study. The last stage of the present work was to investigate the feasibility of magnetic silica spheres

Γ = Γmax 2024

kceq 1 + kceq

(1)



Article

Hence, the conjugated drug may be later preferentially released in the more acidic environment (pH ∼ 5−6.5) of the endosome of cancer cells rather than in blood (pH ∼ 7.4). This is important, considering that it provides an easy release mechanism, in spite of the limitations (in particular, higher chance of premature release) of physically loaded drug as compared to chemically bound molecules.89,90 Because of the positive charge of DOX molecules in a wide pH range, their adsorption onto the negatively charged gold/ maghemite complexes can be further qualitatively followed by electrophoresis. As observed in Figure 16, increase of the

Figure 16. Electrophoretic mobility of maghemite/silica/gold nanostructures after 24 h contact with doxorubicin solutions of the concentrations indicated.

Figure 14. (A) Absorbance spectra of a 0.1 mM DOX solution, a 0.1 wt % suspension of nanospheres (polyelectrolyte/silica/maghemite nanoparticles) in water, and the supernatant of the same suspension after 24 h contact with the DOX solution. (B) Comparison of the absorbance spectra of 0.2 mM DOX solution and of the supernatant of 0.1 wt % suspension of gold-coated nanospheres (Au-nanospheres) after 24 h contact with the solution.

(positive) electrophoretic mobility of the nanostructures with DOX concentration in solution confirms incorporation of the drug, and agreement with the direct adsorption determination also manifests in the mobility plateau starting at 0.3 mmol/L initial concentration. In Vitro Drug Release. The presence of the partial planar structure of the tetracyclic ring in the doxorubicin molecule makes it fluorescent at 533 nm upon excitation at 488 nm. This makes it possible to confirm the presence of the drug on the particles, and eventually its release in the selected tumor cells. The results obtained with confocal microscopy confirm that DOX is released in the nuclei of cancer cells, as can be noticed in Figure 17: the red fluorescence characteristic of doxorubicin is observed when either free drug or drug loaded on the nanoparticles is in the culture medium. The fact that the fluororescence is limited to the nuclei in both cases confirms that the nanocomposites gain access to the nuclei and are not stuck in the cytoplasm, after two hours contact.

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CONCLUSIONS Gold functionalized maghemite-silica nanospheres were synthesized and investigated as drug delivery systems for doxorubicin. Maghemite nanoparticles were first prepared and coated by a silica shell, making them more biocompatible and providing them with a more reactive surface. Both bare and silica-coated maghemite behaved as superparamagnetic particles. A layer-by-layer coating of polyelectrolytes prepared the particles for receiving a final layer of gold on which DOX was adsorbed. Spectrophotometry, electrophoresis, and fluorescence experiments were used to demonstrate that the prepared particles are able to load the drug after contact with its solutions. The maximum payload appears to be 40 μmol/g. The efficiency of the nanoparticles in releasing the DOX inside cells

Figure 15. DOX adsorption on magnetic silica spheres with gold shell for varying equilibrium concentrations of doxorubicin in solution. The line is the best-fit to the Langmuir isotherm.

where Γmax is the plateau observed in the adsorption density at high eqiulibrium concentration of the adsorbate, ceq, and k is the Langmuir equilibrium constant. The best-fit parameters in our case (Figure 15) were k = 37 ± 22 (L/mmol), Γmax = 46 ± 8 μmol/g. The mechanism of DOX binding to nanospheres is based on electrostatic interactions between the positive charge of dissolved DOX (through protonation of the amino group of the drug molecule), and the negative surface charge of gold. 2025



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was tested in vitro by confocal microscopy using colon adenocarcinoma cells grown in the presence of drug-loaded nanocomposites.

AUTHOR INFORMATION

Corresponding Author

*University of Granada, Department of Applied Physics, Facultad de Ciencias, Fuentenueva s/n, 18071, Granada, Spain. E-mail: [email protected]. Phone: +34 958 243 209. Fax: +34 958 243 214. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by Project Project PE-2008 FQM 3993 (Junta de Andaluciá and FEDER funds) is greatly appreciated. One of us (K.R.) also acknowledges MICINN, Spain, for a grant associated with Project SAF 2009-06367-E. Special thanks goes to Prof. S. Cerdán and Dr. D. Calle, Universidad Autónoma de Madrid, Spain, for their kind assistance in performing and interpreting MRI results. Also, the help of Drs. J. Salmerón, J. A. Munoz-Gamez, and A. Carazo in carrying out the in vitro experiments with cancer cells is deeply acknowledged.

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Figure 17. Confocal images of colorectal adinocarcinoma cells (DLD1) untreated (top) treated with free DOX solution (0.3 mM concentration, middle row) and treated with magnetic nanocomposites loaded in the same drug solution (bottom). The column named “Nuclei (DAPI)” indicates the positions of the nuclei of the cancer cells; the column “Doxorubicin” is the fluororescence due to the drug released in the nuclei; the rightmost column is the superposition of the other two.

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ABBREVIATIONS USED

DOX, doxorubicin; PDADMAC, poly(diallyldimethylammonium chloride); PSS, poly(sodium 4styrenesulfonate); SPION, superparamagnetic iron oxide nanoparticle; TEOS, tetraethoxysilane 2026



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