Shell Superparamagnetic Nanoparticle

Article pubs.acs.org/Langmuir

Nitric Oxide Delivery by Core/Shell Superparamagnetic Nanoparticle Vehicles with Enhanced Biocompatibility X. F. Zhang,†,‡,∥ S. Mansouri,†,‡,∥ D. A. Mbeh,§ L’H. Yahia,§ E. Sacher,§ and T. Veres*,†,‡ †

National Research Council of Canada, 75 Boulevard de Mortagne, Boucherville, Québec, Canada J4B 6Y4 INRS Énergie, Matériaux et Télécommunications, 1650 Boulevard Lionel Boulet, Varennes, Québec, Canada J3X 1S2 § ́ Ecole Polytechnique de Montréal, Case Postale 6079, succursale Centre-Ville, Montréal, Québec, Canada H3C 3A7 ‡

ABSTRACT: We report the synthesis of Fe3O4/silica core/shell nanoparticles and their functionalization with S-nitrosothiols. These nanoparticles are of immense interest because of their nitric oxide (NO) release capabilities in human alveolar epithelial cells. Moreover, they act as large storage reservoirs of NO that can be targeted magnetically to the specific site with a sustainable release of NO for up to 50 h. Such nanoparticles provide an enhancement of the biocompatibility with released NO while allowing intracellular accumulation ascribed to their small size.

OA), were synthesized on the basis of a thermal decomposition process.18 Octyl ether (20 mL) was mixed with 1.92 mL of oleylamine at room temperature for ∼10 min and subsequently heated to 100 °C within 20 min under a flow of Ar. At 100 °C, 0.4 mL of iron pentacarbonyl was injected, and the temperature was increased to reflux for 2 h and then cooled to room temperature by removing the heating mantle. The product was precipitated by adding excess anhydrous ethanol, separated by centrifugation (9000 rpm), and further washed with cyclohexane and ethanol at least three times. The core/shell Fe3O4/silica nanoparticles were synthesized in a water-in-oil microemulsion. Fe3O4/OA nanoparticles in cyclohexane (0.5 mL, 1 mg/mL) were injected into a mixture of 1.77 g of Triton X-100, 1.6 mL of anhydrous 1-hexanol, and 7 mL of cyclohexane under strong vortex mixing for about 1 h, and then 0.5 mL of an ammonia solution (6%) was added with stirring for another 1 h. To synthesize the fluorescent Fe3O4/silica nanoparticles, 20 mg of Ru(bpy)32+ complex dyes was added to the above mixture with additional mixing for 30 min. TEOS (25 μL) was added and left to react for 24 h. Subsequently, 2 μL of 3-mercaptopropyltrimethoxysilane was added to the reaction mixture and left for another 24 h. The result was thiolfunctionalized Fe3O4/silica nanoparticles (denoted as Fe3O4/silica(−SH) nanoparticles, which were separated by centrifugation at 9000 rpm, washed with ethanol and distilled water, and dried under vacuum for use. 2.3. Synthesis of Core/Shell Fe3O4/Silica(−SNO) Nanoparticles. The addition of NO groups, transforming −SH groups to −SNO groups of Fe3O4/silica(−SH) NPs, occurs by reacting the −SH functional groups with t-butyl nitrite. The detailed procedure consisted of 20 mg of Fe3O4/silica(−SH) nanoparticles and excess t-butyl nitrite (about 20 mg) being dispersed in 10 mL of 10% methanol/90% toluene (v/v) and stirred for 24 h at room temperature under N2 flow. The resultant nanoparticles with −SNO groups were centrifuged at 9000 rpm, washed three times with anhydrous ethanol and deionized

1. INTRODUCTION Nitric oxide (NO) is an extremely important biological agent that has the ability to protect cardiovascular tissue by inhibiting thrombosis and enzymes, to mediate neurotransmission, and to repair wounds.1−3 The development of nitric oxide (NO) delivery vehicles has attracted a considerable amount of attention because of such physiological features.4−13 However, the delivery of NO molecules to specific tissue sites that provide therapeutically effective release while reducing the toxic side effects of delivery vehicles remains challenging. The ideal delivery vehicle should be an integration of biocompatibility, targeted release, and the ability to pass through the cell membrane wall for efficient cell uptake. In this article, Fe3O4/ silica core/shell nanoparticles have been synthesized to store and release NO chemically by functionalizing the silica shells with S-nitrosothiols that can spontaneously release NO through the homolytic bond cleavage of the S−N bond under physiological conditions.14−17 The integrated superparamagnetic cores of such nanoparticles exhibit a superior potential for magnetic targeting, and the silica shells increase their biocompatibility and allow better permeability for cellular uptake. 2. EXPERIMENTAL DETAILS 2.1. Materials. Oleic acid (OA, 90%), anhydrous 1-hexaneol (99%), octyl ether (98%), ammonia solution (NH4OH, 28−30 wt % in water), Triton X-100, hexane (95%), cyclohexane (99.5%), tetraethoxysilane (TEOS, 99.999%), and Ru(bpy)32+ complex dyes were purchased from Sigma-Aldrich. Iron pentacarbonyl (99.5%) was purchased from Strem Chemicals (Newburyport, MA). N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3, ≥ 90%) and 3mercaptopropyltrimethoxysilane (≥97%) were purchased from Gelest (Tullytown, PA). 2.2. Synthesis of Core/Shell Fe3O4/Silica (SH) Nanoparticles. Fe3O4 nanoparticles, with a thin protective layer of oleic acid (Fe3O4/ © 2012 American Chemical Society

Received: June 11, 2012 Revised: August 10, 2012 Published: August 15, 2012 12879

dx.doi.org/10.1021/la302357h | Langmuir 2012, 28, 12879−12885

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Figure 1. (a, b) TEM image of Fe3O4/silica(−SH) nanoparticles and Fe3O4/silica(−SNO) nanoparticles. (c) Scheme of nitric oxide grafting showing the protocol to transform −SH functional groups into −SNO groups of Fe3O4/silica(−SH) nanoparticles by reacting the −SH functional groups with t-butyl nitrite. water, rapidly dried in vacuum for 5 min (a longer drying time will cause −SNO decomposition), and finally stored at low temperature (−20 °C) under light-shielded conditions. 2.4. Characterization of the Nanoparticles. The microstructures of the nanoparticles were characterized by X-ray diffraction (Siemens D-500 X-ray diffractometer with Cu Kα radiation (λ = 0.154 nm) and transmission electron microscopes (Hitachi S-4700 at 30 kV and JEOL 2010F at 200 kV)). The surface chemistry was characterized by Fourier transform infrared (FTIR) spectra collected with a Nicolet Fourier spectrophotometer at 600 and 4000 cm−1. The magnetic properties were determined with a Quantum Design PPMS model 6000 magnetometer. 2.5. Quantitive Evaluation of the NO Release of Fe3O4/ Silica(−SNO) Nanoparticles. The detection of the NO molecules released from the nanoparticles was carried out by amperometric analysis using a nitric oxide detector (World Precision Instruments). The ISO-NOP sensor (World Precision Instruments Ltd.) was calibrated by the addition of 50 μM (100 μL for each step) NaNO2 in 20 mL of 0.1 M H2SO4 + 0.1 M KI solution. The detection sensitivity was determined to be 3.478 pA/nM at 37 °C. The temperature-triggering release of the NO molecules from the decomposition of the S-nitrosothiol groups in Fe3O4/silica(−SNO) nanoparticles was carried out at normal body temperature (37 °C) under light exposure. The details are the following: 0.7 mg of nanoparticles was dispersed into 1 mL of a PBS pH 7.2 buffer, forming a stable suspension, and then rapidly injected into 19 mL of a PBS pH 7.2 buffer, at which time the ISO-NOP sensor had reached a low, stable current level. The NO probe was immersed about 2 cm into the

suspension with magnetic stirring at 600 rpm, and the measurement temperature was fixed at 37 °C. 2.6. Cell Culture. The in situ NO release and cytotoxicity of the Fe3O4/silica(−SNO) nanoparticles were evaluated using adenocarcinomic human alveolar basal epithelial cells (A549, American Type Culture Collection, ATCC, USA). The medium used was Ham’s F-12 (ATCC, USA) supplemented with penicillin (100 IU/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS). The cells were cultured at a density of 1 × 105 cells per/mL of medium in 24-well culture plates at 37 °C in a 5% CO2 atmosphere. After 20 h of culturing, the medium in the wells was replaced with fresh medium containing Fe3O4/silica(−SNO) nanoparticles (1, 5, and 10 μg/mL) and was further cultured for 48 h. In control cultures, the cells were placed in the medium, without nanoparticles, at the same cell density. 2.7. Intracellular Release of NO. Cell line A549 (1 × 105 cells/ mL) was incubated with Fe3O4/silica(−SNO) nanoparticles (10 μg/ mL) for 3, 24, and 48 h. After three washes with culture medium, 10 μm of 4-amino-5-methylamino-2′,7′-difluorofluorescein, DAF-FM (Sigma), was added and incubated for 2 h at 37 °C. After incubation, the cells were then washed three times with culture medium. Fluorescent images were captured with a computer-controlled charged-coupled device (CCD) camera using an image-capturing system consisting of a Nikon Eclipse microscope equipped with a filter set and a 20× objective. 2.8. In Vitro Cell Viability. The cell viability test was carried out via the reduction of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Invitrogen). After 48 h of culturing with the Fe3O4/silica(−SNO) nanoparticles (1, 5, and 10 12880


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μg/mL), 100 μL of an MTT dye solution (5 mg/mL in phosphate buffer pH 7.4) was added to each well and incubated for 4 h at 37 °C in 5% CO2. The medium was removed, and formazan crystals were solubilized with 150 μL of dimethyl sulfoxide (DMSO). The absorbance of each well was read using a spectrophotometer (Biotek, USA) at 570 nm, and the relative cell viability (%) related to control wells containing cell culturing medium without nanoparticles was calculated as [A]test/[A]control × 100. Three replicates were measured, and the results are presented as the mean ± the standard deviation.

3. RESULTS AND DISCUSSION Figure 1a shows the TEM image of Fe3O4/silica(−SH) nanoparticles. The nanoparticles are all spherical in shape with a narrow size distribution consisting of a core diameter of 10 nm and a shell thickness of 20 nm. The conjugation of NO molecules is carried out by reacting the −SH functional groups of Fe3O4/silica(−SH) nanoparticles with t-butyl nitrite, as shown in Figure 1c, and the product, Fe3O4/silica(−SNO) nanoparticles, are as shown in the TEM image of Figure 1b. The surface functional groups of the silica shells have been confirmed by Fourier transform infrared (FTIR) spectra, as shown in Figure 2. The formation of the −S−NO groups on

Figure 2. Fourier transform infrared (FTIR) spectra of (a) Fe3O4/ silica(−OH), (b) Fe3O4/silica(−SH), and (c) Fe3O4/silica(−SNO) nanoparticles.

the surface of the Fe3O4/silica nanoparticles is demonstrated by the presence of the characteristic peaks of −S−N, −CH2− S−, and −NO at 764, 1237, and 1505 cm−1.19−22 However, the transformation rate (loading rate) from −SH and −S−NO groups cannot be quantitatively estimated. Although a small number of −SH groups unavoidably remain, the reaction time (24 h) and excess use of t-butyl nitrite ensure that the loading of −S−NO groups approaches its saturation. The surface functional groups and the shell topology of silica coatings weakened the magnetic interactions between the nanoparticles, thus resulting in nanoparticles having good monodispersibility and stability in aqueous solution. The assynthesized Fe3O4/silica(−SNO) nanoparticles exhibit typical superparamagnetic behavior as shown in Figure 3a−c. The hysteretic loops present a saturation magnetization (Ms) of ∼3.2 emu/g and a coercive field (HC) of ∼19 Oe at 5 K and Ms ≈ 2.7 emu/g and HC = 0 Oe at 300 K. Considering the saturation magnetization (Ms) of 73 emu/g for Fe3O4/OA nanoparticles,23 it is estimated that the nonmagnetic silica composition is about 96.8 wt %. The ferromagnetic−super-

Figure 3. Magnetic properties of Fe3O4/silica(−SNO) nanoparticles. (a, b) Hysteresis loops and the enlargements at the origin at 5 and 300 K. (c) ZFC-FC magnetization curves cooled under an applied field of 100 Oe at 10 K.

paramagnetic transition onset appears at around 191 K in the zero-field-cooling (ZFC) and field-cooling (FC) magnetization curves at an applied magnetic field of 100 Oe. Figure 4 shows a series of images of the magnetic manipulation process of the Fe3O4/silica nanoparticles doped by Ru(bpy)32+ complex dyes flowing in a plastic tube with 0.75 mm diameter. The concentration of Fe3O4/silica nanoparticles and its flow velocity were fixed to be 50 μg/mL and 20 cm/s, which are roughly based on the normal blood circulation in body.24 When a magnetic field of ∼1 T was applied to one side of the plastic tube, we found that the Fe3O4/silica nanoparticles were rapidly captured. After the magnetic field was removed, the Fe3O4/silica nanoparticles were immediately released 12881


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Figure 4. Magnetic manipulation of 100 μL/mL Fe3O4/silica nanoparticles doped with Ru(bpy)32+ complex dyes flowing in a plastic tube with 0.75 mm diameter. (a) Optical image, (b) fluorescent image under green optical irradiation, (c) image after adding a small magnet beside the tube, showing the immediate capture of nanoparticles (green). (d, e) As the capture time increased, more and more nanoparticles were captured. (f) Image after the magnet was removed, showing the rapid release of nanoparticles.

without any physical adsorption onto the tube wall. These advantages endow the Fe3O4/silica nanoparticles with the potential for the magnetically targeted delivery of NO molecules at specific sites. Quantitative detection of the NO release of nanoparticles was carried out by amperometric analysis using the nitric oxide detector (ISO-NOP sensor, World Precision Instruments Ltd.). The results revealed an immediate NO release following the addition of nanoparticles. From Figure 5a, it is evident that the release rate of NO is relatively stable, exhibiting an initial peak of 46.7 nM/mg at 6.55 h, followed by a gradual decrease with time for up to 50 h. The cumulative amount of NO released is approximately 1.34 μmol of NO/mg of nanoparticles with a standard deviation of 5.7% for three sample batches. It should be noted that, at 50 h, the cumulative release is already close to a plateau, accompanied by a very low release rate, which probably implies a total NO storage amount in the nanoparticles. The release capacity of Fe3O4/silica(−SNO) nanoparticles is comparable to that of silica(−SNO) nanoparticles (0.09−4.39 μmol/mg).10 The duration of the NO release is about 50 h with a half-life (t1/2) of 16.9 h. It is noteworthy that the samples stored for up to 4 months exhibited release kinetics (time and cumulative total amount) comparable to that of the fresh samples. Additionally, we addressed the stability of the product at room temperature as shown in Figure 5b, indicating that only 18.3% of the cumulative amount of NO was released at 20 °C after 50 h compared to that at 37 °C. One critical aspect of NO delivery vehicles is their permeability through cell membranes, which can significantly enhance the cellular uptake at targeted sites. Human alveolar epithelial cells were used to assess this issue. Intracellular fluorescence imaging of NO production was achieved using a commercial NO indicator, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), with a limit of detection of ∼3 nM.25 Figure 6 presents the bright-field and fluorescence microscopy images of cells incubated with 10 μg/mL Fe3O4/ silica(−SNO) nanoparticles at various times. After 3 h of incubation, the fluorescence inside the cells was weak as an indication of low NO uptake. The nanoparticles internalized in the cells were quite limited, and the released NO was therefore distributed mainly in the extracellular medium

Figure 5. Quantitative evaluation of NO release for Fe3O4/ silica(−SNO) nanoparticles in PBS 7.4 buffer at (top) 37 and (bottom) 20 °C.

that had been washed away. In comparison, after 24 h of incubation, the Fe3O4/silica(−SNO) nanoparticles were localized in the whole cell (cytoplasm, subcellular vesicles, and even nucleus), as indicated by the visible fluorescence micrograph (Figure 6d). The uptake process of nanoparticles by the cells occurs through endocytosis that requires a nanoparticle size smaller than 100 nm. The intracellular release of NO was attributed to the distinct characteristics of the 12882


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Figure 6. Bright-field and fluorescence microscopy images of the NO release detection of 10 μg/mL Fe3O4/silica(−SNO) nanoparticles in cells with various incubation times of (a, b) 3, (c, d) 24, and (e, f) 48 h. Bright-field and fluorescence microscopy images of the NO detection of cells without nanoparticles after incubation for (g, h) 3, (i, j) 24, and (k, l) 48 h.

nanoparticles, such as the monodispersibility and the small size distribution of around 40 nm, which can enhance the cellular uptake and accumulation. However, the cells did not present obvious fluorescence intensity after 48 h of incubation in Figure 6f, indicating that the Fe3O4/silica(−SNO) nanoparticles cannot sustain a detectable release of NO in cells for more than 48 h, which is consistent with that measured by amperometric analysis in Figure 5. The viability of human alveolar epithelial cells, affected by the NO released from the Fe3O4/silica(−SNO) nanoparticles, was quantitatively determined by the MTT assay, as shown in Figure 7. Although many studies have reported that NO has no influence on cell viability or manifold cytotoxicity,26−28 our experiments indicate that at low concentrations of nanoparticles (1 and 5 μg/mL) the viability was slightly increased compared to that of the control cell lines and gradually decreased as the concentrations increased, presenting a viability of 70% at 100 μg/mL. It has been demonstrated that the cytoprotective role

for low NO concentration is to act directly as an antioxidant to scavenge peroxyl radicals. Moreover, NO can significantly inhibit lipid peroxidation by ferrous compounds/H2O2 and reactive oxygen intermediates such as Fe2+.29−33 From Figure 7, the cell viability as a percentage of the number of Fe3O4/ silica(−SNO) nanoparticles was greater than the number of Fe3O4/silica(−SH) nanoparticles, suggesting that released NO from S-nitrosothiols can suppress the negative effects of silicacoated nanoparticles. Additionally, Fe3O4/silica(−SNO) has shown a better cell viability compared to that of Fe3O4/silica nanoparticles with primary amine groups in our recent study.23 These results suggest that S-nitrosothiols, as surface functional groups of biomedical vehicles, can improve biocompatibility. In summary, we reported the synthesis of Fe3O4/silica(−SNO) core/shell nanoparticles and the concept of their use as NO delivery vehicles. Their relatively small size provides the possibility to permeate the cell membrane subsequently to release the molecule of interest. The surface modification of the 12883


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(7) Polizzi, M. A.; Stasko, N. A.; Schoenfisch, M. H. Water-soluble nitric oxide-releasing gold nanoparticles. Langmuir 2007, 23, 4938− 4943. (8) Robbins, M. E.; Hopper, E. D.; Schoenfisch, M. H. Synthesis and characterization of nitric oxide-releasing sol-gel microarrays. Langmuir 2004, 20, 10296−10302. (9) Shin, J. H.; Metzger, S. K.; Schoenfisch, M. H. Synthesis of nitric oxide-releasing silica nanoparticles. J. Am. Chem. Soc. 2007, 129, 4612− 4619. (10) Riccio, D. A.; Nugent, J. L.; Schoenfisch, M. H. Stöber synthesis of nitric oxide-releasing S-nitrosothiol-modified silica particles. Chem. Mater. 2011, 23, 1727−1735. (11) Jo, Y. S.; van der Vlies, A. J.; Gantz, J.; Thacher, T. N.; Antonijevic, S.; Cavadini, S.; Demurtas, D.; Stergiopulos, N.; Hubbell, J. A. Micelles for delivery of nitric oxide. J. Am. Chem. Soc. 2009, 131, 14413−14418. (12) Pinto, M. L.; Rocha, J.; Gomes, J. R. B.; Pires, J. Slow release of NO by microporous titanosilicate ETS-4. J. Am. Chem. Soc. 2011, 133, 6396−6402. (13) Riccio, D. A.; Dobmeier, K. P.; Hetrick, E. M.; Privett, B. J.; Paul, H. S.; Schoenfisch, M. H. Nitric oxide-releasing S-nitrosothiolmodified xerogels. Biomaterials 2009, 30, 4494−4502. (14) Al-Sa’doni, H.; Ferro, A. S-nitrosothiols: a class of nitric oxidedonor drugs. Clin. Sci. 2000, 98, 507−520. (15) Frost, M. C.; Meyerhoff, M. E. Synthesis, characterization, and controlled nitric oxide release from S-nitrosothiol-derivatized fumed silica polymer filler particles. J. Biomed. Mater. Res. A 2005, 15, 409− 419. (16) Josephy, P. D.; Rehorek, D.; Janzen, E. D. Electron spin resonance spin trapping of thiyl, radicals from the decomposition of thionitrites. Tetrahedron Lett. 1984, 25, 1685−1688. (17) Oliveira, M. G.; Shishido, S. M.; Seabra, A. B.; Morgon, N. H. Thermal stability of primary S-nitrosothiols: roles of autocatalysis and structural effects on the rate of nitric oxide release. J. Phys. Chem. A 2002, 106, 8963−8970. (18) Woo, K.; Hong, J.; Choi, S.; Lee, H. W.; Ahn, J. P.; Kim, C. S.; Lee, S. W. Easy synthesis and magnetic properties of iron oxide nanoparticles. Chem. Mater. 2004, 16, 2814−2818. (19) Goto, K.; Hino, Y.; Kawashima, T.; Kaminaga, M.; Yano, E.; Yamamoto, G.; Takagi, N.; Nagase, S. Synthesis and crystal structure of a stable S-nitrosothiol bearing a novel steric protection group and of the corresponding S-nitrothiol. Tetrahedron Lett. 2000, 41, 8479− 8483. (20) Williams, D. L. H. The Chemistry of S-Nitrosothiols. Acc. Chem. Res. 1999, 32, 869−876. (21) The Sadtler Handbook of Infrared Spectra; Sadtler Research Laboratories: Philadelphia, PA, 1978; p 82. (22) Perissinotti, L. L.; Leitus, G.; Shimon, L.; Estrin, D.; Doctorovich, F. A unique family of stable and water-soluble Snitrosothiol complexes. Inorg. Chem. 2008, 47, 4723−4733. (23) Zhang, X. F.; Mansouri, S.; Clime, L.; Ly, H. Q.; Yahia, L. ’H.; Veres, T. Fe3O4-silica core−shell nanoporous particles for highcapacity pH-triggered drug delivery. J. Mater. Chem. 2012, 22, 14450− 14457. (24) Wexler, L.; Bergel, D. H.; Gabe, I. T.; Makin, G. S.; Mills, C. J. Velocity of blood flow in normal human venae cavae. Circ. Res. 1968, 23, 349−359. (25) Kojima, H.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Hirata, Y.; Nagano, T. Fluorescent indicators for imaging nitric oxide production. Angew. Chem., Int. Ed. 1999, 38, 3209−3212. (26) Sun, M. H.; Han, X. C.; Jia, M. K.; Jiang, W. D.; Wang, M.; Zhang, H.; Han, G.; Jiang, Y. Expressions of inducible nitric oxide synthase and matrix metalloproteinase-9 and their effects on angiogenesis and progression of hepatocellular carcinoma. World J. Gastroenterol. 2005, 11, 5931−5937. (27) Lipton, S. A.; Choi, Y. B.; Pan, Z. H.; Lei, S. Z.; Chen, H. S. V.; Sucher, N. J.; Loscalzo, J.; Singel, D. J.; Stamler, J. S. A redox-based mechanism for the neuroprotective and neurodestructive effects of

Figure 7. Cell viability percentage incubated after 48 h by Fe3O4/ silica(−SNO) nanoparticles with various concentrations of 0, 1, 5, 10, 50, and 100 μg/mL. The control cell viability is normalized to be 100%.

nanoparticles with S-nitrosothiols increased their biocompatibility. In addition, such hybrid nanostructures, with superparamagnetic cores, have great application potential in magnetically targeted manipulation, magnetic resonance imaging, and hyperthermia.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: + 450 641-5105. Author Contributions ∥

These authors made equal contributions to this article.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was jointly supported by the Natural Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research, and the National Research Council of Canada, Industrial Materials Institute (IMI-NRC). We are grateful to Nitric Medical Devices (NMD) Inc. for financial support, and Dr. Blaise Gilbert and Dr. Omar Quraishi for their insightful advice on the use of magnetic carriers for biomedical applications. We are grateful to Dr. Lidija Malic for the technical revision of the article and significant discussions.

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REFERENCES

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