Maghemite Nanoparticles with Enhanced Magnetic Properties: One

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Biological and Medical Applications of Materials and Interfaces

Maghemite nanoparticles with enhanced magnetic properties: one-pot preparation and ultrastable dextran shell. Riccardo Di Corato, Alessandra Aloisi, Simona Rella, Jean-Marc Greneche, Giammarino Pugliese, Teresa Pellegrino, Cosimino Malitesta, and Rosaria Rinaldi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18411 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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ACS Applied Materials & Interfaces

Maghemite nanoparticles with enhanced magnetic properties: one-pot preparation and ultrastable dextran shell Riccardo Di Corato, Alessandra Aloisi, Simona Rella, Jean-Marc Greneche, Giammarino Pugliese, Teresa Pellegrino, Cosimino Malitesta, Rosaria Rinaldi* Dr. R. Di Corato, Prof. R. Rinaldi Dipartimento di Matematica e Fisica “Ennio De Giorgi”, Università del Salento, Via Arnesano, I-73100 Lecce, Italy Dr. A. Aloisi, Prof. R. Rinaldi CNR Institute for Microelectronics and Microsystems, SP Lecce-Monteroni, I-73100 Lecce, Italy Dr. S. Rella, Prof. C. Malitesta Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, Via Arnesano, I-73100 Lecce, Italy Dr. Jean-Marc Greneche Institut des Molécules et Matériaux du Mans (IMMM UMR CNRS 6283), Université du Maine, Avenue Olivier Messiaen, 72085 - Le Mans cedex 9, France G. Pugliese, Prof. T. Pellegrino Istituto Italiano di Tecnologia, via Morego 30, 16163 Genoa, Italy

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Prof. R. Rinaldi University of Salento – ISUFI, Via Monteroni, University Campus, 73100 Lecce, Italy

KEYWORDS: magnetic nanoparticles, magnetic characterization, XPS,

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Fe Mössbauer

spectrometry, water-transferring, dextran

ABSTRACT. In the field on nanomedicine, superparamagnetic nanoparticles are one of the most studied nanomaterials for theranostics. In this paper, a one-pot synthesis of magnetic nanoparticles is presented, with elevated control on particles size from 10 to 40 nm. The monitoring of vacuum level is here introduced as a crucial parameter for achieving a fine particle morphology. Magnetic properties of these nanoparticles are highly affected by disorders or mismatches in crystal structure. A prolonged oxidation step is applied to the obtained nanoparticles to transform the magnetic phases into a pure maghemite one, confirmed by a high resolution XPS analysis, by Mössbauer spectrometry and, indirectly, by increased performances in magnetization curves and in relaxation times. Afterward, the attained nanoparticles are transferred in water by a non-derivatized dextran coating. The thermogravimetric analysis confirms that the polysaccharide molecules replace the oleic acid on the surface by stabilizing the particles in aqueous phase and culture media. Preliminary in vitro test reveals as the dextran coated nanoparticles are not passively internalized from the cells. As proof of concept, a secondary layer of chitosan assures a positive charge to the nanoparticle surface, thus enhancing the cellular internalization.


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INTRODUCTION Superparamagnetic nanoparticles have been used in medicine primarily as contrast agents for magnetic resonance imaging (MRI), with the formulations having been approved by the FDA ferumoxides (trade name Feridex in the United States, Endorem in Europe), ferucarbotran (trade name Resovist) or recently ferumoxtran (trade name Combidex in United States, Sinerem in Europe).1-2 Some of the above cited formulations have been stopped for further development or withdrawn from the market.3 This class of nanoparticles has been also investigated for application in various fields of nanomedicine, for example as magnetic carriers for drug delivery, as contrast agents for magnetic particle imaging (MPI), and as effectors in magnetic hyperthermia.4-6 In addition, many biocompatibility studies have shown that these particles are safe and have a very low impact on cellular functions. All the nanoparticles cited above were prepared by improving a method developed in the early 80s, using two iron salts in an alkaline environment. In parallel, several studies were conducted on other synthetic routes.2 In particular, thermal decomposition at high temperatures ensures an elevated control of the characteristics of nanoparticles (the size, shape and purity of the crystalline phase). In these preparations, however, it is difficult to obtain good reproducibility because of possible variables that are present in the reaction steps, especially due to the presence of impurities (e.g. low boiling point species) in the synthesis reagents.7 Herein, we focus on an upgrade of a synthesis method for superparamagnetic nanoparticles that was first reported in 2004 by Colvin’s group. The preparation was based on the decomposition of a non-usual iron precursor, iron(III) oxide-hydroxide (FeO(OH)).8 In principle,


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the synthesis is centered on the pyrolysis of the iron carboxylate salt with the presence of oleic acid, dispersed in 1-octadecene as a solvent. It is a one-pot method and in the original paper the size of the nanoparticle was tuned by the ratio between the iron precursor concentration and the oleic acid concentration. The main advantages of this synthesis reside in the possibility of obtaining different sizes of magnetic nanoparticles and in the single step reaction. In fact, many synthetic strategies consider the preparation of small magnetic seeds and the subsequent growth of a magnetic shell with the same crystalline phase. The latter could cause some mismatch at the interface between the seed and the shell, creating a multi-domain crystal that would degrade the magnetic properties.9 EXPERIMENTAL SECTION Chemicals. All solvents used were of analytical grade and used with no modifications. Iron(III) oxide hydroxide, oleic acid (90%), 1-octadecene (90%), poly(maleic anhydride-alt-1octadecene), low molecular weight chitosan (mol wt 50-190KDa), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), glutamine, penicilline/streptomicine, and epoxy resin kit were purchased from Sigma-Aldrich. Dextran (70KDa) was purchased from Carl Roth. Synthesis of iron oxide nanoparticles. To synthesize quasi-spherical iron oxide nanoparticles, iron(III) oxide hydroxide (from 1.33 to 2.75 mmol, please refer to Figure 1a) was mixed with 8 mmol of oleic acid in 5 mL of 1-octadecene, in a 25 mL three-necks round flask in presence of stirring (by magnetic bar, at 1000 rpm). After degassing for 1 h at 100 °C (the vacuum was monitored by means of a digital gauge), pure nitrogen was continuously fluxed into the flask and the solution was rapidly heated to 230 °C and kept for 30 minutes under nitrogen flux. The temperature was then raised to 320 °C by a heat rate of 9 °C/min and kept for 60 minutes. After


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cooling to 130 °C, the crude reaction was exposed to air for 60 minutes and the temperature was raised again to 280 °C and kept overnight. Finally, the particle suspension was cooled to room temperature and washed twice, by centrifugation at 2000 g, with a mixture of ethanol and acetone. The collected nanoparticles were easily redispersed in chloroform. In some cases, a further purification step was applied to remove persistent excess of surfactant. In detail, the nanoparticles were first redispersed in hexane and mixed with acetic acid and ethanol (2:10:1, v/v). The resultant emulsion was centrifuged and washed with acetone (to remove the excess of acetic acid) and the black pellet was redispersed in chloroform. In order to obtain a welldispersed sample, the particle suspension was ultrasonicated by a Bandelin Sonopuls HD3100, equipped with a 3mm probe. Water-transfer procedures. Here we report an example of a dextran coating for nanoparticles with a diameter of 17 nm. First, 0.48 g of dextran powder was dissolved in 20 mL of dimethyl sulfoxide (DMSO) in a round flask by warming the milky solution up to 60 °C. When the solution was clear, the flask was transferred to an ultrasonic bath, pre-heated to 80 °C, and 10 mL of the particle suspension at an iron concentration of 10 mM was injected at a flow rate of 0.25 mL/minute. The suspension was further ultrasonicated for 2 h after the injection. The clear brown suspension was transferred to a rotary evaporator to remove any traces of chloroform residue. Then, the particle suspension was transferred to a 50ml conical tube and an excess of ethanol and acetone (1:1, v/v) was added. The nanoparticles were collected by centrifugation and redispersed in ultrapure water. To remove any excess dextran and DMSO residue, the water suspension was further purified by centrifugal concentrator tubes (MWCO 100KDa). Having considered the moles of dextran (6.857·10-6) and the nanoparticles (1·10-9), we used 6857 molecules of dextran per nanoparticle (for the calculation of nanoparticles molarity we used an


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unpublished homemade program that considers the crystalline cell of the nanoparticles and the corresponding number of iron atoms). The surface area was 900 nm2 therefore 7.62 molecules of dextran per nm2 were used for coating a 17nm-particle. These values could be used as reference for each particles size. For a cellular experiment, a supplementary coating based on chitosan was applied. In detail, 50 µL of dextran coated-nanoparticles (50 mM in iron) were added to 500 µL of 2% chitosan aqueous solution (dissolved in 1% acetic acid) and mixed vigorously on an orbital shaker for 120 minutes. The suspension was then diluted 4 times with water and centrifuged at 13000 rpm for 30 minutes. The collected nanoparticles were finally redispersed in ultrapure water. In order to investigate the effective nature of the dextran coating (by thermogravimetric analysis, see below), the iron oxide nanoparticles were also transferred in water by using poly(maleic anhydride-alt-1-octadecene), as described in ref.10 with no modifications. Transmission Electron Microscopy (TEM). Low-resolution TEM images were acquired with a Jeol Jem 1011 microscope operating at an accelerating voltage of 100 kV. For biological samples, the cellular monolayer was first infiltrated with epoxy resin and slices of 70 µm were later prepared for analysis with the microscope. MRI. Magnetic resonance imaging was performed on a Bruker ICON scanner. The scanner was interfaced to ParaVision software for preclinical MRI research. The nanoparticles were dispersed in 1.5% gelatin and stored at 4°C. All samples were imaged by i) T2 multi-slice multiecho sequence with a TR of 3000 ms, multiple TE from 12 to 90 ms and a flip angle of 180 degree; ii) T1 echo planar sequence with a TR of 4000 ms, TE of 20 ms, multiple TI from 17 to 1150 ms and a flip angle of 45 degrees.


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Magnetic characterization. Characterization of the water-transferred samples was carried out using a Vibrating Sample Magnetometer (VSM) from Cryogenic Limited. Magnetization curves were measured at 300 K from – 0.1 to 0.1 T for hysteresis loop and up to 3 T for magnetization saturation. XPS. XPS measurements were recorded with an AXIS ULTRA DLD (Kratos Analytical) photoelectron spectrometer using a monochromatic AlKα source (1486.6 eV) operated at 225 W (15 kV, 15 mA). Base pressure in the analysis chamber was 5.733·10-7 Pa. Powder samples pressed on conductive adhesive tape were assembled in a dry box, transferred to the XPS lab in an inert atmosphere and introduced quickly to the spectrometer. Survey scan spectra were recorded using a pass energy of 160 eV and a 1 eV step. High resolution spectra were acquired using a pass energy of 20 eV and a 0.1 eV step. The hybrid lens mode was used for all measurements. In each case, the area of analysis was about 700 µm x 300 µm. During the data acquisition, a system of neutralization of the charge was used. The spectra were processed with CasaXPS Release 2.3.16 software. The binding energy (BE) scale was referenced to the adventitious C 1s peak (285 eV). For the analysis of high resolution spectra all peaks were fitted using Shirley background and GL(30) lineshape (a combination of Gaussian 70% and Lorentzian 30%). For quantitative analysis, the relative sensitivity factors present in the library of CasaXPS for the areas of the signals were used. Mössbauer spectrometry.

57

Fe Mössbauer spectrometry experiments were performed at 77 K

using a conventional constant acceleration transmission spectrometer with a 57Co diffused into a Rh matrix. The ferrofluid samples were cooled down into an adapted sample holder to be placed in a bath cryostat to get a frozen solution and an homogenous Fe thickness to prevent from thickness effects. The Mössbauer spectra were fitted by means of the MOSFIT program


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(unpublished homemade Mosfit program Le Mans Université) involving the superimposition of quadrupolar doublets and Zeeman magnetic sextets with lorentzian lines. The spectrometer was calibrated using a α-Fe foil while the values of isomer shift are quoted relative to that of α-Fe at 300 K. TGA. Thermogravimetric analysis (TGA) was carried out with a TGA Q500-TA Instrument. During TGA, samples are heated from 30 °C to 600 °C at a heating rate of 5 °C min−1 under nitrogen atmosphere set at a flow rate of 50 mL min−1. Dynamic Light Scattering. Dynamic light scattering (DLS) and zeta potential measurements were performed using a ZetaSizer Nano ZS90 (Malvern Instruments) equipped with a 4.0 mW He-Ne laser operating at 633 nm and an APD photodiode detector. Elemental analysis. The iron concentrations were measured by elemental analysis by using an inductively coupled plasma atomic emission spectrometer (ICP-OES Varian 720-ES). Before the analysis, samples were digested in a concentrated HCl/HNO3 3:1 (v/v) solution. Cell Culture. The rat glioma cells (C6) and neuronal Schwann cells (RSC96) were grown continuously as a monolayer at 37 °C and in a 5% CO2 atmosphere in DMEM medium supplemented with L-glutamine (2 mL), penicillin (100 units/mL), streptomycin (100 µg/mL), and 10% heat-inactivated fetal bovine serum (FBS). The nanoparticles were dispersed in complete medium and incubated with cells for 24 hours, with a concentration of iron (by elemental analysis) ranging from 0 to 10 mM. Cytotoxicity was evaluated by MTT assay. RESULTS AND DISCUSSION


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Starting from the above cited synthesis, our intent was to extend the dimensional range in order to obtain nanoparticles with an average size greater than 30 nm by varying the ratio between surfactants and precursor. Moreover, we decided to introduce a long, monitored degassing step at 100 °C at the beginning of the synthesis to remove any trace of humidity contained in solvents, surfactants or precursors. The control of the vacuum performance is not often reported in syntheses papers but, in this particular case, we found that a vacuum-level of 6.5-9.5 Pa is necessary to obtain a valuable reproducible protocol (Figure S1). By introducing this high-vacuum step, the previously reported trend of the surfactant / precursor ratio has been modified. Herein, we kept the volumes of the surfactant and solvent constant and tuned the amount of iron precursors, ranging from 1.33 to 2.75 mmol (Figure 1a). Thus, we obtained a pool of nanoparticles with an average size range of 10 to 40 nm (Figure 1b-i and S2). The shape of the bigger particles, above the limit of 25 nm, became more irregular, losing the quasi-spherical structure that the smaller iron oxide nanoparticles had. From 30 to 40 nm, various defects were detected in the crystals, with the inception of the formation of dimers or flower-like structures (Figure 1g-i and S2f-h). The obtained nanoparticles were characterized by vibrating-sample magnetometer (VSM) in order to correlate the different core sizes to a different response to a magnetic stimulus. As expected, increasing the diameter of the nanoparticles induced a progressive opening of the hysteresis loop (Figure S3a). In particular, this phenomenon was evident when the particles exceeded the hypothetical threshold of 19-20 nm. This result is in accordance with some previous scientific reports on the subject, although this value ranges from 18 to 25 nm depending on the shape or purity of the particles.11-13 Interestingly, analysis of the magnetization curves revealed that not all the analyzed nanoparticles can reach saturation under the magnetic fields


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applied in the analysis (Figure S3b). This effect could be due to the presence of crystalline defects as vacancies and/or symmetry breaking at the surface originating non colinear magnetic structure resulting from frustrated topology, and/or of different phases in the same nanoparticle. Thus, the size of these nanoparticles do not influence the average saturation, which is close to 40 Am2/KgFe and is quite far from the reference value of bulk material.14 In this regard, the nanoparticles have been exposed to an oxidation process in air at high temperatures (> 250 °C) in order to uniform the multiple magnetic phases to a single pure maghemite. This treatment enables the annealing of the crystal defects within the nanoparticle structure, while the size of the synthesized magnetic core remains unaltered. This postprocessing step is usually conducted in air, fluxed oxygen or by adding an organic oxidizing agent.15-16 The following data were obtained by oxidizing iron oxide nanoparticles with an average diameter of 12 nm. From the hysteresis curves, one can note that both samples maintained a superparamagnetic profile (Figure 2a), but the oxidized nanoparticles had substantially higher magnetic susceptibility (Figure 2c). The magnetic saturation curves are very different for the magnetization of the saturation level (58 Am2/KgFe at 3 T for nanoparticles not oxidized, whereas 98 Am2/KgFe had already been obtained at 0.4 T for the oxidized nanoparticles) (Figure 2b). As described above, the presence of defects in non-oxidated nanoparticles affected dramatically the magnetic profile, since the saturation value is quite far from the corresponding Fe3O4 in bulk.9, 17 Additional data acquired with 17 nm nanoparticles are reported in Figure S4. X-ray photoelectron spectroscopy (XPS) analyses were performed to investigate the chemical modification of two different sized magnetic nanoparticles (12 nm and 17 nm) upon an oxidation


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process. XPS has been used since long time to distinguish and to quantify Fe2+ and Fe3+ species.18 In fact, these oxidation states can be easily evaluated by Fe 2p region, employing chemical shift and multiplet splitting occurring for these species and evaluating characteristic satellites which increase identification capability of the technique. Several examples of XPS application to iron oxide species have been reported including nanoparticles and magnetic systems.19-26 Fitting of Fe 2p region requires the knowledge of standard spectra of potential present iron oxide species such as FeO, Fe3O4 and Fe2O3 (including α-Fe2O3 and γ-Fe2O3). In this respect a very accurate work is represented by ref.19 which was used for interpreting our results. In the survey spectra (data not shown), the major peaks are represented by the C 1s and O 1s that originated mostly from the oleic capping agent on the nanoparticle surface and the Fe 2p that originated from iron oxides. For a more detailed analysis of the chemical modifications that occurred during the treatments, high resolution XPS Fe2p spectra were recorded. These spectra were fitted using multiplet peaks, as showed in Figure 3 and S5. The only differences are in the Fe3+/Fe2+ ratio for magnetite which is very close to the theoretical value (2) and the so called surface peak and pre-peak which are more abundant than those recorded by Grosvenor et al.19, probably due to the nanosize of the present systems. Figure 3a and S5a report the XPS results respectively for the 17 nm and 12 nm nanoparticles before treatment. The data show that, in both cases, the nanoparticles mainly consisted of Fe3O4 (magnetite). The Fe 2p XPS spectra for nanoparticles after the oxidation process are shown in Figure 3b and S5b. Fe 2p signals are modified and can be fitted to the peak model of ϒ-Fe2O3 (maghemite). Therefore, the treatment apparently transforms both 12 nm and 17 nm nanoparticles from a magnetite phase to maghemite one.


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Although the XPS could be considered in this case as a bulk technique because of the size of the small nanoparticles,27 a Mössbauer spectrometry analysis was carried out on prepared nanoparticles for confirming and completing the magnetic phase characterization. The main interest of 57Fe Mössbauer spectrometry is to discriminate the different Fe species in a Fe-containing sample and to determine their respective oxidation states from the values of isomer shift. In the case of nanoparticles, it allows also the static and dynamic magnetic states to be distinguished, particularly those related to superparamagnetic relaxation phenomena. The Mössbauer spectra obtained on as-prepared and oxidized samples are compared in Figure 4 (bottom and top, respectively). The spectrum corresponding to the as-prepared sample consists of a magnetic sextet with non-homogeneous broadening, particularly at the outer lines. In addition, one distinguishes a splitting in the 1st line which can be a priori assigned to a mixture of magnetite and maghemite while the broadening is attributed to the presence of relaxation phenomena originated from the non-interacting nanoparticles. The description of such a component requires a combined distribution of hyperfine field and isomer shift, the range of which is characteristic of valency states varying from Fe3+ and Fe2+. In addition, the quadrupolar component occurring at the central part is probably due to the presence of ultra-fine non interacting Fe3+ containing nanoparticles. The spectrum of the oxidized sample differs as the magnetic sextet lines are narrower than those of the as-prepared sample. In addition to the quadrupolar component assigned to the presence of non-interacting Fe3+ containing small nanoparticles, the fitting model involves two magnetic components which are attributed to Fe3+ species located in tetrahedral and octahedral environment. The values of the hyperfine parameters and the proportions of the two main phases


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allow to conclude to the exclusive presence of maghemite. It is important to emphasize that the total mean values are respectively 0.49 and 0.40 mm/s for the as-prepared and oxidized samples, that is consistent with an oxidation process. The magnetic performance of the resulting nanoparticles was also assessed for their eventual application in magnetic resonance imaging, evaluating the nanoparticle relaxation times T1 and T2.28-29 First, the nanoparticles were transferred from chloroform to water by enwrapping them within a polymer coating, using a procedure previously optimized by the research unit based on the amphiphilic polymer poly(maleic anhydride-alt-1-octadecene).10 For both tested sizes (12 and 17 nm) an increase in T2 relaxivity and a subsequent slight increase in the r2 / r1 ratio were observed (Figure 5). This behavior is in line with the increase of MS in the particles that influences the spin-spin relaxation more than the spin lattice one. In order to exploit these nanoparticles in nanomedicine, it is necessary to cover the surface with polymers or replace the surfactant molecules deriving from the synthesis with biocompatible molecules. Herein, a new protocol for the coating of nanoparticles with dextran molecules was developed. The choice of this molecule lies in its documented biocompatibility and in the possibility to compare the most common magnetic contrast agents used in clinics with nanoparticles, which are the subject of this study. The fundamental difference is that in the case of Resovist, Endorem, etc., the nanoparticles were synthesized by the alkaline co-precipitation of iron salts with the presence of dextran. Starting with the nanoparticles obtained using the thermal decomposition approach, only modified dextrans were used for the coating of the hydrophobic nanocrystals, to the best of our knowledge.30-31 In this study, the nanoparticles were transferred to water using a dextran with an average molecular weight of 70 kDa, with no further chemical modifications. Moreover, the presence of several hydroxyl groups could provide a versatile


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synthetic handle, allowing the polymer molecules to anchor to the surface of the nanoparticles.32 Finally, the dextran composites possess an intrinsic antifouling behavior that could contribute to the water-transferred nanoparticles achieving an excellent colloidal stability.33 All the procedure details are reported in Experimental section. The water-transferred nanoparticles showed exceptional colloidal stability in water and in cell culture medium supplemented with serum (TEM images and DLS analysis in Figure 6). The hydrodynamic diameter did not particularly increase with protein adsorption, probably as a result of the stealth effect produced by the dextran molecules on the surface.34 These nanoparticles were also monitored in terms of long term storage, pH and lyophilization: no modifications of stability were observed in comparison to starting nanoparticles (Figure S7). The procedure was first developed on non-oxidized nanoparticles and did not present any critical aspects. When it was applied to the oxidized nanoparticles, some aggregation phenomena or a reduction in the transferred portion in the aqueous phase was observed. This was due to a greater cohesion between the surfactant molecules and the surface of the nanoparticles during the synthesis / oxidation. This excess of organic molecules interfered with the interaction of the dextran functional groups with the surface of the particle, decreasing or preventing complete covering. Therefore, it was necessary to set a more aggressive washing procedure before the polymeric coating step, through an emulsion of hexane, acetic acid and ethanol, to induce the detachment of the surfactant molecules from the surface. After the additional purification step, the nanoparticles could still be dispersed in chloroform, but only after the application of a large dose of high frequency ultrasound. The clean nanoparticles were transferred in aqueous phase, following the aforementioned protocol with no further modifications.


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Initially, a surface coating with dextran was designed, with the purpose of obtaining an outer layer by absorbing multiple molecules. However, the data obtained by thermogravimetric analysis (TGA) suggested that the molecules of dextran tend to unseat the oleic acid molecules on the surface of the nanocrystals as a result of the hydroxyl groups which derived from the glucose units that are present in the polymer, resulting in a stable affinity exchange. The nanoparticles that were transferred to water with the two described methods were analyzed by TGA. The particle coated with poly(maleic anhydride-alt-1-octadecene) had some peaks in common with the bare hydrophobic particles and with the oleic acid. However, the particles transferred with dextran showed a single peak at 290 °C, which is close to the profile of pure dextran. No contribution from oleic acid was detected in the curves of these nanoparticles, confirming that the dextran molecules are directly conjugated to the surface of the iron oxide (Figure 7). In order to assess the effective biocompatibility of dextran-iron oxide nanoparticles, we performed preliminary in vitro tests to evaluate the acute toxicity and the internalization of the nanocrystals in cells. Two neuronal cell lineages of rat, C6 and RSC96, were tested for these experiments. The nanoparticles showed good dispersibility in culture medium, without any formation of visible aggregates. Furthermore, after 24 hours of incubation, no toxicity was detected for doses of up to 10 mM of iron (Figure S9a). The cells incubated with nanoparticles were analyzed by ICP-OES and TEM to evaluate the quantity of nanocrystals that were uptaken and their intracellular localization. Under the above described incubation conditions, no internalization was noticed in either of the cell lineages (Figure 8a and S10). To increase the interaction of the nanocrystals with cell membranes and to allow the inclusion or the adsorption of molecules/moieties on their surface, a chitosan cloak was assembled, in


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addition to the first layer of dextran, by electrostatic interactions. Among many varieties of NPs, chitosan-based ones are promising for use in biological applications on account of the advantages of their matrix, such as biodegradability, biocompatibility, mucus adhesion, and low cost.35 For instance, chitosan NPs have been used as carriers in the targeted delivery of anticancer drugs as well as in plasmid DNA delivery.36-37 Remarkably, positively charged chitosan-based NPs have been exposed to several cell lines (epithelial, fibroblastic, endothelial and blood cells) that show a higher internalization rate and a further increase in the internalization amount than those that are negatively charged.38 Positive NPs may, in principle, display a favorable value in most therapy cases, overcoming the cell membrane barrier quickly, thereby increasing cargo concentration at the lesion site. In contrast, negatively charged NPs might be more appropriate for delivering drugs that require a longer half-life or, as it is also reported, may accomplish a better delivery of drugs deep into tissue.39 The functional groups present in this different polysaccharide allow the surface charge of the nanoparticles to vary; the nanoparticles coated with dextran were slightly negative but those coated with chitosan were positive (Figure 8b). The nanoparticles coated with chitosan were tested on the aforementioned cell types in order to investigate any change in cell uptake. The positive surface should force interaction with the plasma membrane and boost cellular endocytosis.40 As expected, the particles with the additional chitosan layer showed a higher level of cell internalization, as was revealed by the ICP-OES and TEM analyses. The images show that the nanoparticles are confined in dense endosomes for both lineages (Figure 8c-d and S10). In particular, these vesicles seem to be formed by different subunits, which probably results from a strong interaction between the nanoparticles and the protrusions of the cell membranes. It is known that, in general, cationic NPs are prone to induce


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more pronounced disruption of plasma-membrane integrity, stronger mitochondrial and lysosomal damage, acidification of endosomes and increased number of autophagosomes.40 These occurrences resulted in significant cytotoxicity for chitosan-coated NPs (Figure S9a). Particularly, at an iron concentration of 2 mM, a drop of 35-40% in C6 cells viability was observed after 24h of incubation with 17 nm dextran@chitosan coated iron oxide NPs. This behavior could be as well motivated by an enhanced internalization of positive-charged NPs in comparison to dextran coated NPs (Figure 8a and S9b). As previously stated, positive charge promotes the internalization rate and further increases the cellular uptake of chitosan NPs.38

CONCLUSION Our initial goal was to prepare different sizes of superparamagnetic nanoparticles by applying a single synthesis method by modifying a few parameters. One of the requirements of the selected method was the use of a one-pot approach to avoid the formation of crystalline mismatches that would affect the magnetic properties of nanoparticles.9 Compared to the original protocol,8 we have shown here that the introduction of a vacuum step helps to increase the reproducibility of the synthesis and to effectively achieve control of size. Apart from the nanoparticle that was bigger that 20 nm, we initially assumed that the smaller crystals were not affected by structural defects. Instead, they were composed of a mixed phase of magnetite and maghemite. Such a composition leads to a poor magnetic capacity of the synthesized magnetic nanocrystals. Moreover, the hysteresis curves showed a low susceptibility for all the particles, which limits their use in biomedical imaging. Hence, we decided to introduce a high temperature oxidation step to obtain an annealing of the crystalline phases and to also make the particles


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stable over time. In fact, the slow oxidation of the outer layer of magnetite on the surface layer could represent a problem in maintaining the characteristics of nanoparticles when exposed to air or dispersed in aqueous media.41 The application of an aggressive oxidation step, performed at the end of the synthesis, helped to transform the crystal structure to a pure maghemite phase, as revelead by XPS and Mössbauer spectrometry. These particles showed a higher magnetic saturation which was close to the bulk value for the material. The nanoparticles possess a layer of surfactants on the surface that avoid their dispersion in water. Mimicking the characteristics of the commercial formulations of magnetic nanoparticles (Endorem, Resovist, etc.), we decided to coat the nanoparticles in a layer of polysaccharides. Specifically, dextran is a biocompatible polymer widely used in clinical environments. Our main idea was to use a non-derivatized dextran. By exploiting the affinity of the hydroxyl groups to the iron oxide surface, we obtained an exchange of the oleic acid surfactants with the dextran molecules. The coating with polysaccharides guaranteed a stable and efficient water-transfer of hydrophobic nanocrystals. Interestingly, the dextran-coated nanoparticles were not passively internalized by cells. The functionalization with specific ligands, with an affinity for cellular membrane domains (e.g. antibody), could boost the internalization of the nanoparticles, thus promoting an active recognition of the target and a more precise delivery. As proof of concept, a supplementary polysaccharide layer, based on chitosan, was grafted onto the dextran layer. Chitosan provides a positive charge on the surface of the nanoparticles, thus it enhances their interaction with membranes and increases cellular internalization. On the other hand, such prepared particles could be improved with regard to dextran/chitosan complex stability in blood as well to endosome escape efficiency when the so called native chitosan proton sponge effect fails, and tested for nucleic acid delivery efficiency.42


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FIGURES

Figure 1. a) In the graph, the measured diameters of nanoparticles obtained by varying the ratio between the surfactant (oleic acid) and the iron precursor are reported. b-i) TEM analyses of different nanoparticles, with a corresponding diameter of 9.7±1.3, 11.8±1.2, 14.7±1.4, 19.6±1.0, 22.5±1.3, 24.2±1.8, 32.0±2.6 and 40.5±5.5 nm.


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Figure 2. VSM magnetic characterization of 12 nm-iron oxide nanoparticles after oxidation processing. a) Hysteresis curves acquired at 300K. The insets show the TEM images of analyzed nanoparticles b-c) Magnetic saturation curves acquired at 300K, normalized to iron weight for b) and to the maximum observed MS for c).


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Figure 3. High resolution spectra of Fe 2p region for 17 nm nanoparticles (a) pre-oxidation and (b) after oxidation step.

Figure 4. 77K transmission Mössbauer spectra of the non-oxidized (bottom) and oxidized (top) samples.


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Figure 5. a) Influence of oxidation processing on magnetic relaxivities of water-transferred iron oxide nanoparticles of 12 and 17 nm. b) MR images of 12 and 17 nm iron oxide nanoparticles in gelatin phantoms, analyzed at 1 T with a T2 MSME sequence (TR: 3000ms, TE:51ms), before and after the oxidation processing. Relaxivities of dextran coated nanoparticles are reported in Figure S6.


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Figure 6. a) Sketch depicting the main steps of synthesis and functionalization of iron oxide nanoparticles. In the transferring of magnetic nanoparticles in aqueous media, the addition of dextran unseats the surfactant molecules from the particle, because of the higher affinity of the hydroxyl moieties towards the iron oxide surface. The resulting particles were easily dispersed in water or biological media, as shown by TEM analysis (panel b, 17 nm iron oxide nanoparticles transferred to water by dextran coating) and by DLS analysis (panel c, comparison of hydrodynamic diameters of iron oxide nanoparticles coated with dextran, dispersed in water,


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blue line, and 10% FBS supplemented DMEM, red line). In panel b, the scale bar corresponds to 100 nm length.

Figure 7. TGA curves of hydrophobic nanoparticles (NP CHCl3), dextran-transferred nanoparticles (NP Dextran), poly(maleic anhydride-alt-1-octadecene) coated nanoparticles (NP Polymer). As a reference, the curves of oleic acid, dextran and polymer were reported as dashed lines. TGA curves (weight vs. temperature) are reported in Figure S8.


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Figure 8. a) ICP analysis of C6 and RSC96 cells incubated with iron oxide nanoparticles coated with dextran or dextran@chitosan. The diameters of the evaluated particles were 12 and 17 nm. b) Surface charge analysis of bare dextran coated nanoparticle (blue line) before and after coating with chitosan (red line). c-d) TEM analysis of C6 cells incubated with 12 and 17 nm nanoparticles, coated with dextran@chitosan (iron concentration 2 mM, 24h incubation). ASSOCIATED CONTENT Supporting Information is available from the ACS Publications website. Additional data for TEM images, magnetization curves, XPS, MRI, TGA and DLS analyses, cellular internalization and cytotoxicity, characterization data summary. AUTHOR INFORMATION Corresponding Author E-mail: [email protected]


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was funded by Italian PON project AMIDERHA (PON02_00576_3329762). T.P. thanks the European Union (ERC-StG project ICARO, Contract No. 678109) for partial funding. We are grateful to Prof. G. Maruccio and Dr. A. Monteduro for help in VSM measurements and for their fruitful discussion.

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TOC FIGURE


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Maghemite Nanoparticles with Enhanced Magnetic Properties: One

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