Journal of Colloid and Interface Science 425 (2014) 118–127
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Characterization and cytotoxicity studies on liposome–hydrophobic magnetite hybrid colloids Alice Floris a, Chiara Sinico a,⇑, Anna Maria Fadda a, Francesco Lai a, Francesca Marongiu a, Alessandra Scano b, Martina Pilloni b, Fabrizio Angius c, Carlos Vázquez-Vázquez d, Guido Ennas b,⇑ a
Dipartimento di Scienze della Vita e dell’Ambiente, Sezione Scienze del Farmaco, Università di Cagliari, Via Ospedale 72, 09124 Cagliari (CA), Italy Dipartimento di Scienze Chimiche e Geologiche, Cagliari Unità di Ricerca del Consorzio Nazionale di Scienze e Tecnologie dei Materiali (INSTM), Cittadella Universitaria di Monserrato, SS 554 bivio Sestu , 09042 Monserrato (CA), Italy c Dipartimento di Scienze Biomediche, Sezione Patologia, Università di Cagliari, Via Porcell 4, 09124 Cagliari (CA), Italy d Departamento de Química Física, Facultad de Química, Universidad de Santiago de Compostela, Santiago de Compostela, 15782 Galicia, Spain b
a r t i c l e
i n f o
Article history: Received 18 December 2013 Accepted 15 March 2014 Available online 25 March 2014 Keywords: Magnetoliposomes Nanoparticles synthesis SPION Magnetite Stealth liposomes
a b s t r a c t The aim of this study was to highlight the main features of magnetoliposomes prepared by TLE, using hydrophobic magnetite, and stabilized with oleic acid, instead of using the usual hydrophilic magnetite surrounded by sodium citrate. These biocompatible magnetoliposomes (MLs) were prepared with the purpose of producing a magnetic carrier capable of loading either hydrophilic or lipophilic drugs. The effect of different liposome/magnetite weight ratios on the stability of magnetoliposomes was evaluated by monitoring the mean diameter of the particles, their polydispersity index, and zeta potential over time. The prepared magnetoliposomes showed a high liposome–magnetite association, with magnetoliposomes containing PEG (polyethylene glycol) showing the best magnetite loading values. To verify the position of magnetite nanoparticles in the vesicular structures, the morphological characteristics of the structures were studied using transmission electron microscopy (TEM). TEM studies showed a strong affinity between hydrophobic magnetite nanoparticles, the surrounding oleic acid molecules, and phospholipids. Furthermore, the concentration above which one would expect to find a cytotoxic effect on cells as well as morphological cell–nanoparticle interactions was studied in situ by using the trypan blue dye exclusion assay, and the Prussian Blue modified staining method. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The high biocompatibility and versatile nature of liposomes have made these particles unique nanometric systems, with a wide spectrum of biomedical applications. In particular, due to their size and surface tailoring, they have proved to be efficient drugdelivery systems. Their amphiphilic properties permit different types of drugs, both hydrophilic (contained within their internal aqueous compartments) or hydrophobic (contained inside their lipid bilayer shell) molecules to be incorporating within their structures. Liposomes that have been sterically stabilized by grafting hydrophilic polyethylene glycol (PEG) chains onto their lipid ⇑ Corresponding authors. Fax: +39 070 6754388. E-mail addresses: alicefl@tiscali.it (A. Floris), [email protected] (C. Sinico), mfadda@ unica.it (A.M. Fadda), [email protected] (F. Lai), [email protected] (F. Marongiu), [email protected] (A. Scano), [email protected] (M. Pilloni), fangius@ unica.it (F. Angius), [email protected] (C. Vázquez-Vázquez), ennas@ unica.it (G. Ennas). http://dx.doi.org/10.1016/j.jcis.2014.03.046 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
bilayer, also called StealthÒ liposomes, allow for a prolonged circulation time and a reduced uptake by the mononuclear phagocyte system (MPS) [1–4]. To limit systemic toxic effects related to the non-specific distribution of a drug within the body, and to deliver drugs to a precise lesion or region, different types of targeting mechanisms have been developed. Such mechanisms include active targeting, such as coupling to the PEG chain coated surface special ligands in order to increase liposome–cell association [5], and physical targeting, such as inclusion of magnetic nanoparticles in order to permit guidance by an external magnetic field [6–8]. Physical targeting can be exploited by including magnetite or other magnetizable materials (e.g. maghemite or gadolinium ions) in the vesicular structure of the liposome. Different magnetic behaviors can be observed depending on the size of the magnetic grains. At sizes 90
– – – – – 0.45 ± 0.08 – 2.44 ± 0.07 – 2.10 ± 0.32 – 4.62 ± 0.83 – 6.97 ± 1.10 – 10.42 ± 0.43 – >19.02
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Each formulation was prepared and purified in three replicates and the magnetite amount obtained values were used to calculate LMA and LC mean values (±standard deviation). 2.2. Zeta potential and particle size analysis Mean diameter and polydispersity of MLP were determined by light scattering method. The scattered light intensity of the solution at 173° was measured by light scattering photometer (ZEN3600 Zetasizer ZS, Malvern Instruments, England). The scattered light intensity was converted to the diffusion coefficient (D, m2/s). The size of the nanoparticle was calculated from the diffusion coefficient of the particles using Stoke–Einstein equation by PCS software version. The polydispersity index (PI) was used as a measure of the width of the size distribution. PI less than 0.2 indicates a homogenous and monodisperse population. Zeta potential was measured as the particle electrophoretic mobility by means of laser microelectrophoresis in a thermostated cell. Every sample was diluted with phosphate buffer. Measurement was repeated three times and results are expressed as mean ± standard deviation. 2.3. TEM analysis Microscopical analysis was carried out by transmission electron microscope (Philips CM-12 and Jeol JEM-1011) operating at 100 kV. Liposomes and magnetoliposomes were observed depositing a drop of their colloidal dispersions over carbon-coated copper grids and using as stain a 1% (w/w) aqueous solution of phosphotungstic acid. Magnetite nanoparticles were observed by placing a drop of chloroform dispersion onto a Formwar coated cooper grid and dried at room temperature. 2.4. Cytotoxicity assays
Fig. 3. (a) Hydrodynamic mean diameter, (b) zeta potential and (c) polydispersity index of MLP* up to 5 months.
h LMA ð%Þ ¼ ðFe3 O4 weightÞsupernatant þ ðFe3 O4 weightÞlight brown phase = i ðFe3 O4 weightÞtotal 100 ð1Þ The total amount of magnetite is referred to amount of magnetite present in the non-purified sample. The magnetite loading capacity (%) of magnetoliposomes was calculated as presented below:
h LC ð%Þ ¼ ðFe3 O4 molÞsupernatant þ ðFe3 O4 molÞlight brown phase = i ðlipid molÞtotal 100
ð2Þ
Cytotoxicity of nanoparticles was screened in CCRF-CEM (human T cell leukemia) cells (ATCC, Rockville, MD). Stock cultures of CEM cells in stationary suspension were maintained in exponential growth (between 2.0 105 cells/mL and 1.5 106 cells/mL) at 37 °C, 5% CO2, in T25 culture flasks (Falcon, Milano, Italy) containing RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), streptomycin (100 mg/mL), penicillin (100 U/mL) and L-glutamine (2 mM). At the start of the experiments, cells from the stock cultures were diluted and seeded in 12-well plates at the density of 2.0 105 cells/mL. After dilution cells were allowed to grow for 24 h before the treatments. Thereafter, OA–Fe3O4 or MLP*_6A dispersion amount were added to the culture medium to obtain the final magnetite concentrations of 10.30, 3.72, 1.86 or 0.93 lg/mL. The experimental session has taken from 0 (baseline) to 48 h of treatment. At each time point and for each treatment, the number of viable cells was determined with a Bürker chamber by the direct count of stained dead and unstained live cells by trypan blue (TB) dye exclusion method. All data were expressed as the mean ± standard deviation (SD) of experiments in triplicate and analyzed by linear regression test and one-way ANOVA with Newman-Keuls post hoc test when required using GraphPad Prism 5 package (GraphPad software Inc., USA). 2.5. Association with cells and microscopy In situ cell–nanoparticles interaction was visualized by an adapted Prussian Blue (PB, sometimes called Perl’s reagent) staining method, that is particularly useful in studying conditions accompanied by varying degree of excess iron or hemosiderosis of the marrow. The stainable iron is all virtually the same color (blue) and it is visible in normoblastic erythrocytes as well as in
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Fig. 4. (a) Hydrodynamic mean diameter, (b) polydispersity index and (c) zeta potential of LP*A sample, up to 5 months; (a1) hydrodynamic mean diameter, (b1) polydispersity index and (c1) zeta potential of MLP*A sample, up to 5 months.
Fig. 5. Hydrodynamic mean diameter of LP* MLP*_3 and MLP*_4 samples before and after lyophilization with increasing trehalose:lipid ratios.
macrophages and in sections subjected to the PB reaction. In the presence of ferrocyanide the ferric ions precipitate as a highly colored and water insoluble complex, potassium ferric ferrocyanide (Prussian Blue). Ferrous ions do not produce a colored reaction product, and thus are excluded from the visualization. In our experiments, cells from stock cultured suspension were seeded at the density of 1.0 106 cells/mL and allowed to incubate for 2 h with an appropriate amount of OA–Fe3O4 or MLP*_6A suspension to obtain the final magnetite concentration of 155.00 lg/mL. They were successively washed with PBS to eliminate non-internalized
nanoparticles and resuspended in FCS (50 ll). A drop of the FCS cellular suspension was placed on one end of a glass slide and using a spreader slide at an angle of approximately 25–45°, a wedge type smear was made and allowed to air dry. Then it was fixed adding methanol and drying (few minutes). The slides were stained in PB reagent up to 30 min, rinsed with distilled water and counterstained with Nuclear Fast Red (NFR) for 5 min. Rinsed, dried and mounted with Vecta Mount for microscope observation. This modified method has allowed to highlight iron like blue-spots, nuclei in red and cytoplasmic membrane in pink. Observations were made using a Zeiss-Jena light microscope equipped with a 20 and a 40 planapochromatic water immersion objective (UPlanSApo series), with an efficient chromatic and spherical correction. Images were taken with a high-resolution live imaging microscopy camera (Moticam2000, Motic, Wetzlar, Germany). The nominal resolution of images was 0.8 and 0.2 lm/pixel for 20 and 40 objectives, respectively. For each experimental group at least six microscopic fields were captured. Image analysis and measurements were performed with the ImagePro Plus package (MediaCybernetics, Silver Springs, MD, USA). 3. Results and discussion Characterization of magnetic nanoparticles. The TEM image (Fig. 1), shows dispersed spherical shaped particles with a narrow distribution of particle sizes.
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Fig. 6. TEM images of (a) a non-centrifuged MLP*_3 sample; (b) supernatant (S), (c–e) brown precipitate phase (BP) and (f) dark precipitate phase of a centrifuged MLP*_3 sample (DP).
The average calculated particle size (Fig. SI 1) was 10.8 nm, ±2.7. This result was in good agreement with DLS measurements (Fig. SI 2), considering that the hydrodynamic size is always greater than the real size. X-ray diffraction pattern for OA–Fe3O4 nanoparticles (Fig. SI 3) shows nanoparticle phase corresponded to crystalline magnetite, and was determined by matching the diffraction peaks with the magnetite pattern (vertical lines). The sample, dissolved in HCl, was first checked by using a rapid ferricyanide test to confirm the presence of Fe(II) and exclude the presence of a maghemite phase [27]. XRD Rietveld refinement (cubic, space group Fd3m), leads to a lattice parameter of a0 = 0.8354 nm, with an average crystal size of 12 nm, and no significant strain. The average crystal size was in agreement with the TEM results. The FTIR spectrum (Fig. SI 4) shows peaks in the high wavenumber region, characteristic of the alkyl chains of oleic acid. Peaks at 2924, 2853, and 2956 cm1 were assigned to CAH stretching
(ma(CH2)), (ms(CH2)), and (ma(CH3)), respectively. The peak at 3006 cm1 was consistent with the presence of the double bond in the oleic acid, and assigned to (CAH) stretching. The carboxylate bound to the nanoparticle surface showed as symmetric and asymmetric (COO) stretching bands at 1407 and 1525 cm1. The presence of symmetric and asymmetric vibration modes suggested that carboxylate was bound to a single surface Lewis acid Fe3+in a chelating bidentate configuration [28]. The IR spectrum showed an absorption band at 590 cm1, which was assigned to FeAO stretching of the magnetite lattice [29]. The presence of peaks at 1750 cm1 for C@O stretching suggested the presence of free oleic acid in the samples. TGA analysis was conducted on a previously dried sample of OA–Fe3O4 at room temperature for several hours in order to eliminate chloroform. TGA thermogram and its derivate dTGA (Fig. SI5) show an initial loss of water or residue solvents, followed at higher temperatures, by degradation of the organic shell. The presence of water in the sample was also confirmed by the FTIR bands
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Fig. 7. (a and b) TEM images of LP*A sample; (c–g) TEM images of MLP*_6A sample.
(3500 cm1). TGA analysis indicated that the dry weight of the sample was comprised of 2% moisture, 25% oleic acid, and 73% magnetite. Magnetization (M) versus applied field (H) measurements of OA–Fe3O4 nanoparticles (Fig. SI6) showed superparamagnetic behavior at room temperature, with a saturation magnetization of 50 emu/g (of sample); no hysteretic behavior was observed. 3.1. Magnetoliposome characterization The stability of formulations was initially evaluated by simple visual observation. Three hours after preparation, the non-sonicated MLP samples (MLPs_1-6) showed a phase separation phenomenon, that later produced two phases: a more or less brown precipitate (depending on the magnetite concentration), and a supernatant. Sonicated MLP formulations (MLPs*_1-6) showed the separation of a small sediment after a longer period of time, (6 h) compared to the non-sonicated samples (Fig. 2a and b). This different behavior could be due to the small size of the dispersed phase of the sonicated samples, that remains stable for a longer period of time (Table 2). In
all samples, the separated phases were easily dispersed by manual or mechanical agitation. The sonicated formulation prepared from a mixture PC:DSPE-PEG (MLP*_6A) showed a phase separation after a rest period of 7–14 days (compared to 6 h for MLP*_1-6 samples), thus showing a much higher stability. A dynamic laser light scattering (DLS) analysis of MLP*_1-6 samples showed that mean particle size, zeta potential, and polydispersity index values were stable for up to 2 months; however, they became unstable and variable after that time period (Fig. 3a–c). A correlation between zeta potential and the trends for mean diameter was also detected beginning at the second month, and the mean size increased, whereas the zeta potential decreased (Fig. 3a and b). LP*_6A and MLP*_6A samples showed stable mean particle size, zeta potential, and polydispersity index values for up to 5 months (Fig. 4a, a1, b, b1, c, and c1). The effects of lyophilization on magnetoliposomes were also evaluated. The study showed good stability of formulations following the freeze-drying treatment, and the best results were obtained for dimensional stability when trehalose (2 mg trehalose/mg
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Fig. 8. Trypan blue dye exclusion assay during a period of 48 h of treatments of OA–Fe3O4 and MPL* at the same concentration (10.30 lg/mL) (a) and different concentrations of MPL* (b); representative microscopic fields of control (c) and OA–Fe3O4 (d) and MPL* (e) treated cells stained with Prussian Blue/Nuclear Fast Red. In the bottom of each image (c–e) the first line reports selected represents single cell samples. In the bottom of each image (c–e) the upper line reports single cells selected from representative samples. Moreover, in order to make clear the presence of magnetite internalized or not into the cells, in the bottom line we reported the same cells with the highlighted blue spots by using a imaging method based on the blue grade (RGB color space). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
lipids) was added before lyophilization (Fig. 5). Given the absence of studies on magnetoliposomes prepared using hydrophobic magnetite, and the accompanying purification procedures, we chose centrifugation as the purification method, because it would not depend on the hydrophilic or hydrophobic nature of the involved materials. This technique was already reported for separation of hydrophilic magnetite from magnetoliposomes [7,20,22,30]. The centrifugation of MLP*_1-6 samples generally led to the separation of three distinct phases – DP, BP, and S, which by means of a specific TEM investigation (Fig. 6a–f), were correlated with the presence of magnetite, magnetoliposomes, and small liposomes/ magnetoliposomes, respectively. Moreover, this separation allowed us to calculate a correct liposome–magnetite association percentage by using (Eq. (1)). Formulations used for MLP*_1-6 samples showed liposome–magnetite association values ranging from 55% to 93%, and these values also showed a decreasing trend beginning from sample MLP*_2 and continuing to sample MLP*_6. However, the loading capacities of MLP*_1 to MLP*_6 increased (Table 2), resulting in an overall positive result. The MLP*_6A sample showed rather different behaviors following centrifugation, by separating into only 2 phases – a supernatant and a brown sediment. The underneath very dark phase was not present, and it was therefore not possible to precisely calculate the liposome–magnetite association of MLP*_6A samples. However, we could only presume an association percentage greater than those calculated for MLP*_1-6 samples, which were >90%.
A TEM micrograph of a non-centrifuged MLP*_3 sample revealed the presence of magnetite, both in the vesicular structures and in the surrounding environment, (Fig. 6a). Furthermore, two main types of entities could be recognized, vesicular structures with magnetite nanoparticles only present in the phospholipid bilayer (black circles in fig 6a), and smaller vesicular structures with nanoparticles present in the phospholipid bilayer and also in the inner space (black arrows in fig 6a), thus demonstrating a high degree of liposome–magnetite association. All three phases of a centrifuged MLP*_3 sample were analyzed. Fig. 6b shows the supernatant phase that is characterized by clusters of small nanoparticles partially inserted into the phospholipid bilayer and partly exposed outside of the vesicular structure (magnetoliposomes), and also the presence of small empty liposomes. Fig. 6c–e show the brown precipitate phase that is characterized by the presence of magnetoliposomes. In this phase, magnetite nanoparticles were mainly found in the phospholipid bilayers, but in some cases, they were also visible in the central area, corresponding to the aqueous core area. This could be due to the presence of nanoparticles at different depths within the phospholipid bilayers; moreover, in this phase are barely detectable free magnetite nanoparticles, which mainly characterized the dark phase (Fig. 6f). To verify the influence of both the PC:DSPE-PEG phospholipid mixture and a high magnetite concentration (1 mg/mL) on the hybrid magnetic colloidal structures, the LP*A and MLP*_6A samples were observed by TEM. As illustrated in Fig. 7a and b, empty
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liposomes (LP*A) showed a regular and multilamellar structure, typical of the classical vesicular bilayer structure. Fig. 7c–g shows the main magnetoliposomal structures. In Fig. 7a and b, it is possible to observe magnetoliposomal structures with magnetic nanoparticles clearly incorporated into phospholipid bilayers, confirming observations obtained with MLP*_1-6 samples. Fig. 7e–g shows the second configuration obtained, which was mainly characterized by aggregates of nanoparticles, which were directly stabilized by phospholipids. The latter configuration was very similar to the configuration reported in our previous study [24], where we described magnetoliposomes prepared by hydrophobic magnetite differently synthesized and surrounded by a higher percentage of oleic acid. In the case of OA–Fe3O4 nanoparticles, this particular magnetoliposomal configuration occurred mainly in the formulations prepared using a high concentration of magnetite (1 mg/mL), and with the PC:DSPE-PEG mixture (MLP*_6A). However, in our previous study, this configuration was obtained even at a low concentration of magnetite (0.2 mg/mL) and using P90G phospholipids [24]. This emphasizes the great impact of the different oleic acid:magnetite ratios and the involved phospholipids on the final magnetoliposomal configuration. The nanoparticle aggregates directly stabilized by one or more phospholipid bilayers seem to be a characteristic configuration for magnetoliposomes prepared using hydrophobic magnetite and stabilized with oleic acid. Since SAXS investigations of multicompartment and hierarchically organized lipid carriers have introduced new directions in the research on stealth lipid nanoparticles, further research will be focused on the characterization of MLP*_6A configuration by means of this technique [31–33.]. Considering their high stability, high liposome–magnetite association percentage, and greater suitability for vivo applications, magnetoliposomes prepared using the PC:DSPE-PEG mixture and the LP*A and MLP*_6A formulations were considered the most suitable for conducting the cytotoxicity assay. 3.2. Cytotoxicity and cell–nanoparticle interaction To assess the capability of MLPs* to interact with cells, we conducted a study using cells growing in suspension (CEM cells), rather than cells adhering to a substrate, because this condition better reflects the blood environment of a hypothetical in vivo treatment using intravenous administration. Our first study was conducted to determine the effect of nanoparticles on cell viability. Preliminary data suggested that OA–Fe3O4 nanoparticles at a concentration of 10.3 lg/mL, were unable to reduce the number of viable cells as compared to untreated cells (control). However, the same magnetite concentration contained in MLPs* strongly reduced the number of viable cells (p < 0.01), suggesting that the carrier delivery system had a significant impact on cell viability (Fig. 8a). Interestingly, OA–Fe3O4 alone and OA–Fe3O4 associated with liposomes (MLPs*) appeared to interact with cells in different ways. Using a modified Prussian Blue/Nuclear Fast Red staining method, modified for our experimental conditions (Fig. 8c–e) (see Section 2 for more details), it was possible to observe different associations of OA–Fe3O4 nanoparticles and MLP* with cells. The OA–Fe3O4 nanoparticles were not internalized into the cells (majority of blue spots were on the white background), and this was probably due to aggregation and precipitation of OA–Fe3O4 in the environment. In contrast, when the cells were treated with MLPs*, the nanoparticles appeared to be incorporated into the cells because the pinkish cytoplasmic membranes showed blue spots compatible with the presence of iron in that compartment. In some cases, we observed a diffuse blue–green color of the cytoplasm, that could be attributed to the more soluble and physiologically ubiquitous form of iron (ferritin) in cells; which however, did not
affect our analysis. Furthermore, to evaluate a dose-dependent effect of magnetite on cell viability, in the second step of our experiments, the cells were treated with different amounts of MLP*, as specified in Section 2. Our data suggested that there were not significant differences between treated cells and control cells (Fig. 8b), but a statistical analysis of the linear regression slopes (with intercept 0) obtained from the growth curves of each group showed that a large concentration of MLP* (corresponding to a magnetite concentration of 3.72 lg/mL) produced a significant decrease in the growth curve of treated cells, compared to the growth curve for control cells (p < 0.05). This result indicated that 3.72 lg/mL may be the threshold concentration for producing a cytotoxic effect on cells, in terms of inhibiting proliferation or viability. 4. Conclusion In this work, we presented new magnetic stealth liposomes prepared by means of the simple, versatile, and reproducible TLE procedure and loaded with hydrophobic magnetite stabilized with oleic acid. Overall results have shown the great impact of both the different oleic acid:magnetite ratios and the involved phospholipids on the final magnetoliposomal configuration and features. Moreover, we found that the incorporation of magnetite into liposomal structures had a significant impact on cell toxicity. Indeed, the vesicular delivery system allowed magnetite nanoparticles to interact with cells and, as a consequence, to be easily internalize thus exerting a dose-dependent effect on cell viability. The ability of these structures to incorporate high amount of magnetite nanoparticles as well as to penetrate cell membrane, led us to conclude that they can be the object of further study as potential and useful carrier in biomedical field. Acknowledgments This work was financed by University of Cagliari and by Fondazione Banco di Sardegna. Authors gratefully thanks NANOGAP (Santiago de Compostela, Spain) for kind supplying magnetite (brand name: NGAP FeO-03#01). Appendix A. Supplementary material Six additional figures are provided. OA–Fe3O4 nanoparticles size distribution obtained by DLS; size distribution by volume of OA–Fe3O4 nanoparticles; X-ray diffraction patterns of OA–Fe3O4 nanoparticles with rietveld simulation; FTIR spectrum of OA–Fe3O4 nanoparticles; TGA and dTGA curves for OA–Fe3O4 nanoparticles; Hysteresis curve OA–Fe3O4 nanoparticles. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.03.046. References [1] T.M. Allen, P.R. Cullis, Adv. Drug Del. Rev. 65 (2013) 36–48. [2] D.D. Lasic, F.J. Martin, A. Gabizon, S.K. Huang, D.P. Papahdjopoulos, Biochim. Biophys. Acta 1070 (1991) 187–192. [3] V.E. Trubetskoy, V.P. Torchilin, Adv. Drug Del. Rev. 16 (1995) 311–320. [4] L. Cattel, M. Ceruti, F. Dosio, J. Chemother. 16–4 (2004) 94–97. [5] L.D. Leserman, J. Barbet, F. Kourilsky, J.N. Weinstein, Nature 288 (1980) 602– 604. [6] M. Shinkai, A. Ito, Adv. Biochem. Eng. Biotechnol. 91 (2004) 191–220. [7] H. Nobuto, T. Sugita, S. Shimose, Y. Yasunaga, T. Murakami, M. Ochi, Int. J. Cancer 109 (2004) 627–635. [8] A.A. Kuznetsov, V.I. Filippov, R.N. Alyautdin, N.L. Torshina, O.A. Kuznetsov, J. Magn. Magn. Mater. 225 (2001) 95–100. [9] P. Tartaj, M.P. Morales, S. Veintemillas-Verdaguer, T. Gonzalez-Carrenõ, C.J. Serna, J. Phys. D: Appl. Phys. 36 (2003) 182–197. [10] M. Hoehn, E. Kustermann, J. Blunk, Proc. Natl. Acad. Sci. USA 99 (2002) 16267– 16272. [11] C.C. Berry, A.S.G. Curtis, J. Phys. 36 (2003) R198–R206.
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Contents lists available at ScienceDirect
Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Characterization and cytotoxicity studies on liposome–hydrophobic magnetite hybrid colloids Alice Floris a, Chiara Sinico a,⇑, Anna Maria Fadda a, Francesco Lai a, Francesca Marongiu a, Alessandra Scano b, Martina Pilloni b, Fabrizio Angius c, Carlos Vázquez-Vázquez d, Guido Ennas b,⇑ a
Dipartimento di Scienze della Vita e dell’Ambiente, Sezione Scienze del Farmaco, Università di Cagliari, Via Ospedale 72, 09124 Cagliari (CA), Italy Dipartimento di Scienze Chimiche e Geologiche, Cagliari Unità di Ricerca del Consorzio Nazionale di Scienze e Tecnologie dei Materiali (INSTM), Cittadella Universitaria di Monserrato, SS 554 bivio Sestu , 09042 Monserrato (CA), Italy c Dipartimento di Scienze Biomediche, Sezione Patologia, Università di Cagliari, Via Porcell 4, 09124 Cagliari (CA), Italy d Departamento de Química Física, Facultad de Química, Universidad de Santiago de Compostela, Santiago de Compostela, 15782 Galicia, Spain b
a r t i c l e
i n f o
Article history: Received 18 December 2013 Accepted 15 March 2014 Available online 25 March 2014 Keywords: Magnetoliposomes Nanoparticles synthesis SPION Magnetite Stealth liposomes
a b s t r a c t The aim of this study was to highlight the main features of magnetoliposomes prepared by TLE, using hydrophobic magnetite, and stabilized with oleic acid, instead of using the usual hydrophilic magnetite surrounded by sodium citrate. These biocompatible magnetoliposomes (MLs) were prepared with the purpose of producing a magnetic carrier capable of loading either hydrophilic or lipophilic drugs. The effect of different liposome/magnetite weight ratios on the stability of magnetoliposomes was evaluated by monitoring the mean diameter of the particles, their polydispersity index, and zeta potential over time. The prepared magnetoliposomes showed a high liposome–magnetite association, with magnetoliposomes containing PEG (polyethylene glycol) showing the best magnetite loading values. To verify the position of magnetite nanoparticles in the vesicular structures, the morphological characteristics of the structures were studied using transmission electron microscopy (TEM). TEM studies showed a strong affinity between hydrophobic magnetite nanoparticles, the surrounding oleic acid molecules, and phospholipids. Furthermore, the concentration above which one would expect to find a cytotoxic effect on cells as well as morphological cell–nanoparticle interactions was studied in situ by using the trypan blue dye exclusion assay, and the Prussian Blue modified staining method. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction The high biocompatibility and versatile nature of liposomes have made these particles unique nanometric systems, with a wide spectrum of biomedical applications. In particular, due to their size and surface tailoring, they have proved to be efficient drugdelivery systems. Their amphiphilic properties permit different types of drugs, both hydrophilic (contained within their internal aqueous compartments) or hydrophobic (contained inside their lipid bilayer shell) molecules to be incorporating within their structures. Liposomes that have been sterically stabilized by grafting hydrophilic polyethylene glycol (PEG) chains onto their lipid ⇑ Corresponding authors. Fax: +39 070 6754388. E-mail addresses: alicefl@tiscali.it (A. Floris), [email protected] (C. Sinico), mfadda@ unica.it (A.M. Fadda), [email protected] (F. Lai), [email protected] (F. Marongiu), [email protected] (A. Scano), [email protected] (M. Pilloni), fangius@ unica.it (F. Angius), [email protected] (C. Vázquez-Vázquez), ennas@ unica.it (G. Ennas). http://dx.doi.org/10.1016/j.jcis.2014.03.046 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
bilayer, also called StealthÒ liposomes, allow for a prolonged circulation time and a reduced uptake by the mononuclear phagocyte system (MPS) [1–4]. To limit systemic toxic effects related to the non-specific distribution of a drug within the body, and to deliver drugs to a precise lesion or region, different types of targeting mechanisms have been developed. Such mechanisms include active targeting, such as coupling to the PEG chain coated surface special ligands in order to increase liposome–cell association [5], and physical targeting, such as inclusion of magnetic nanoparticles in order to permit guidance by an external magnetic field [6–8]. Physical targeting can be exploited by including magnetite or other magnetizable materials (e.g. maghemite or gadolinium ions) in the vesicular structure of the liposome. Different magnetic behaviors can be observed depending on the size of the magnetic grains. At sizes 90
– – – – – 0.45 ± 0.08 – 2.44 ± 0.07 – 2.10 ± 0.32 – 4.62 ± 0.83 – 6.97 ± 1.10 – 10.42 ± 0.43 – >19.02
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Each formulation was prepared and purified in three replicates and the magnetite amount obtained values were used to calculate LMA and LC mean values (±standard deviation). 2.2. Zeta potential and particle size analysis Mean diameter and polydispersity of MLP were determined by light scattering method. The scattered light intensity of the solution at 173° was measured by light scattering photometer (ZEN3600 Zetasizer ZS, Malvern Instruments, England). The scattered light intensity was converted to the diffusion coefficient (D, m2/s). The size of the nanoparticle was calculated from the diffusion coefficient of the particles using Stoke–Einstein equation by PCS software version. The polydispersity index (PI) was used as a measure of the width of the size distribution. PI less than 0.2 indicates a homogenous and monodisperse population. Zeta potential was measured as the particle electrophoretic mobility by means of laser microelectrophoresis in a thermostated cell. Every sample was diluted with phosphate buffer. Measurement was repeated three times and results are expressed as mean ± standard deviation. 2.3. TEM analysis Microscopical analysis was carried out by transmission electron microscope (Philips CM-12 and Jeol JEM-1011) operating at 100 kV. Liposomes and magnetoliposomes were observed depositing a drop of their colloidal dispersions over carbon-coated copper grids and using as stain a 1% (w/w) aqueous solution of phosphotungstic acid. Magnetite nanoparticles were observed by placing a drop of chloroform dispersion onto a Formwar coated cooper grid and dried at room temperature. 2.4. Cytotoxicity assays
Fig. 3. (a) Hydrodynamic mean diameter, (b) zeta potential and (c) polydispersity index of MLP* up to 5 months.
h LMA ð%Þ ¼ ðFe3 O4 weightÞsupernatant þ ðFe3 O4 weightÞlight brown phase = i ðFe3 O4 weightÞtotal 100 ð1Þ The total amount of magnetite is referred to amount of magnetite present in the non-purified sample. The magnetite loading capacity (%) of magnetoliposomes was calculated as presented below:
h LC ð%Þ ¼ ðFe3 O4 molÞsupernatant þ ðFe3 O4 molÞlight brown phase = i ðlipid molÞtotal 100
ð2Þ
Cytotoxicity of nanoparticles was screened in CCRF-CEM (human T cell leukemia) cells (ATCC, Rockville, MD). Stock cultures of CEM cells in stationary suspension were maintained in exponential growth (between 2.0 105 cells/mL and 1.5 106 cells/mL) at 37 °C, 5% CO2, in T25 culture flasks (Falcon, Milano, Italy) containing RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), streptomycin (100 mg/mL), penicillin (100 U/mL) and L-glutamine (2 mM). At the start of the experiments, cells from the stock cultures were diluted and seeded in 12-well plates at the density of 2.0 105 cells/mL. After dilution cells were allowed to grow for 24 h before the treatments. Thereafter, OA–Fe3O4 or MLP*_6A dispersion amount were added to the culture medium to obtain the final magnetite concentrations of 10.30, 3.72, 1.86 or 0.93 lg/mL. The experimental session has taken from 0 (baseline) to 48 h of treatment. At each time point and for each treatment, the number of viable cells was determined with a Bürker chamber by the direct count of stained dead and unstained live cells by trypan blue (TB) dye exclusion method. All data were expressed as the mean ± standard deviation (SD) of experiments in triplicate and analyzed by linear regression test and one-way ANOVA with Newman-Keuls post hoc test when required using GraphPad Prism 5 package (GraphPad software Inc., USA). 2.5. Association with cells and microscopy In situ cell–nanoparticles interaction was visualized by an adapted Prussian Blue (PB, sometimes called Perl’s reagent) staining method, that is particularly useful in studying conditions accompanied by varying degree of excess iron or hemosiderosis of the marrow. The stainable iron is all virtually the same color (blue) and it is visible in normoblastic erythrocytes as well as in
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Fig. 4. (a) Hydrodynamic mean diameter, (b) polydispersity index and (c) zeta potential of LP*A sample, up to 5 months; (a1) hydrodynamic mean diameter, (b1) polydispersity index and (c1) zeta potential of MLP*A sample, up to 5 months.
Fig. 5. Hydrodynamic mean diameter of LP* MLP*_3 and MLP*_4 samples before and after lyophilization with increasing trehalose:lipid ratios.
macrophages and in sections subjected to the PB reaction. In the presence of ferrocyanide the ferric ions precipitate as a highly colored and water insoluble complex, potassium ferric ferrocyanide (Prussian Blue). Ferrous ions do not produce a colored reaction product, and thus are excluded from the visualization. In our experiments, cells from stock cultured suspension were seeded at the density of 1.0 106 cells/mL and allowed to incubate for 2 h with an appropriate amount of OA–Fe3O4 or MLP*_6A suspension to obtain the final magnetite concentration of 155.00 lg/mL. They were successively washed with PBS to eliminate non-internalized
nanoparticles and resuspended in FCS (50 ll). A drop of the FCS cellular suspension was placed on one end of a glass slide and using a spreader slide at an angle of approximately 25–45°, a wedge type smear was made and allowed to air dry. Then it was fixed adding methanol and drying (few minutes). The slides were stained in PB reagent up to 30 min, rinsed with distilled water and counterstained with Nuclear Fast Red (NFR) for 5 min. Rinsed, dried and mounted with Vecta Mount for microscope observation. This modified method has allowed to highlight iron like blue-spots, nuclei in red and cytoplasmic membrane in pink. Observations were made using a Zeiss-Jena light microscope equipped with a 20 and a 40 planapochromatic water immersion objective (UPlanSApo series), with an efficient chromatic and spherical correction. Images were taken with a high-resolution live imaging microscopy camera (Moticam2000, Motic, Wetzlar, Germany). The nominal resolution of images was 0.8 and 0.2 lm/pixel for 20 and 40 objectives, respectively. For each experimental group at least six microscopic fields were captured. Image analysis and measurements were performed with the ImagePro Plus package (MediaCybernetics, Silver Springs, MD, USA). 3. Results and discussion Characterization of magnetic nanoparticles. The TEM image (Fig. 1), shows dispersed spherical shaped particles with a narrow distribution of particle sizes.
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Fig. 6. TEM images of (a) a non-centrifuged MLP*_3 sample; (b) supernatant (S), (c–e) brown precipitate phase (BP) and (f) dark precipitate phase of a centrifuged MLP*_3 sample (DP).
The average calculated particle size (Fig. SI 1) was 10.8 nm, ±2.7. This result was in good agreement with DLS measurements (Fig. SI 2), considering that the hydrodynamic size is always greater than the real size. X-ray diffraction pattern for OA–Fe3O4 nanoparticles (Fig. SI 3) shows nanoparticle phase corresponded to crystalline magnetite, and was determined by matching the diffraction peaks with the magnetite pattern (vertical lines). The sample, dissolved in HCl, was first checked by using a rapid ferricyanide test to confirm the presence of Fe(II) and exclude the presence of a maghemite phase [27]. XRD Rietveld refinement (cubic, space group Fd3m), leads to a lattice parameter of a0 = 0.8354 nm, with an average crystal size of 12 nm, and no significant strain. The average crystal size was in agreement with the TEM results. The FTIR spectrum (Fig. SI 4) shows peaks in the high wavenumber region, characteristic of the alkyl chains of oleic acid. Peaks at 2924, 2853, and 2956 cm1 were assigned to CAH stretching
(ma(CH2)), (ms(CH2)), and (ma(CH3)), respectively. The peak at 3006 cm1 was consistent with the presence of the double bond in the oleic acid, and assigned to (CAH) stretching. The carboxylate bound to the nanoparticle surface showed as symmetric and asymmetric (COO) stretching bands at 1407 and 1525 cm1. The presence of symmetric and asymmetric vibration modes suggested that carboxylate was bound to a single surface Lewis acid Fe3+in a chelating bidentate configuration [28]. The IR spectrum showed an absorption band at 590 cm1, which was assigned to FeAO stretching of the magnetite lattice [29]. The presence of peaks at 1750 cm1 for C@O stretching suggested the presence of free oleic acid in the samples. TGA analysis was conducted on a previously dried sample of OA–Fe3O4 at room temperature for several hours in order to eliminate chloroform. TGA thermogram and its derivate dTGA (Fig. SI5) show an initial loss of water or residue solvents, followed at higher temperatures, by degradation of the organic shell. The presence of water in the sample was also confirmed by the FTIR bands
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Fig. 7. (a and b) TEM images of LP*A sample; (c–g) TEM images of MLP*_6A sample.
(3500 cm1). TGA analysis indicated that the dry weight of the sample was comprised of 2% moisture, 25% oleic acid, and 73% magnetite. Magnetization (M) versus applied field (H) measurements of OA–Fe3O4 nanoparticles (Fig. SI6) showed superparamagnetic behavior at room temperature, with a saturation magnetization of 50 emu/g (of sample); no hysteretic behavior was observed. 3.1. Magnetoliposome characterization The stability of formulations was initially evaluated by simple visual observation. Three hours after preparation, the non-sonicated MLP samples (MLPs_1-6) showed a phase separation phenomenon, that later produced two phases: a more or less brown precipitate (depending on the magnetite concentration), and a supernatant. Sonicated MLP formulations (MLPs*_1-6) showed the separation of a small sediment after a longer period of time, (6 h) compared to the non-sonicated samples (Fig. 2a and b). This different behavior could be due to the small size of the dispersed phase of the sonicated samples, that remains stable for a longer period of time (Table 2). In
all samples, the separated phases were easily dispersed by manual or mechanical agitation. The sonicated formulation prepared from a mixture PC:DSPE-PEG (MLP*_6A) showed a phase separation after a rest period of 7–14 days (compared to 6 h for MLP*_1-6 samples), thus showing a much higher stability. A dynamic laser light scattering (DLS) analysis of MLP*_1-6 samples showed that mean particle size, zeta potential, and polydispersity index values were stable for up to 2 months; however, they became unstable and variable after that time period (Fig. 3a–c). A correlation between zeta potential and the trends for mean diameter was also detected beginning at the second month, and the mean size increased, whereas the zeta potential decreased (Fig. 3a and b). LP*_6A and MLP*_6A samples showed stable mean particle size, zeta potential, and polydispersity index values for up to 5 months (Fig. 4a, a1, b, b1, c, and c1). The effects of lyophilization on magnetoliposomes were also evaluated. The study showed good stability of formulations following the freeze-drying treatment, and the best results were obtained for dimensional stability when trehalose (2 mg trehalose/mg
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Fig. 8. Trypan blue dye exclusion assay during a period of 48 h of treatments of OA–Fe3O4 and MPL* at the same concentration (10.30 lg/mL) (a) and different concentrations of MPL* (b); representative microscopic fields of control (c) and OA–Fe3O4 (d) and MPL* (e) treated cells stained with Prussian Blue/Nuclear Fast Red. In the bottom of each image (c–e) the first line reports selected represents single cell samples. In the bottom of each image (c–e) the upper line reports single cells selected from representative samples. Moreover, in order to make clear the presence of magnetite internalized or not into the cells, in the bottom line we reported the same cells with the highlighted blue spots by using a imaging method based on the blue grade (RGB color space). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
lipids) was added before lyophilization (Fig. 5). Given the absence of studies on magnetoliposomes prepared using hydrophobic magnetite, and the accompanying purification procedures, we chose centrifugation as the purification method, because it would not depend on the hydrophilic or hydrophobic nature of the involved materials. This technique was already reported for separation of hydrophilic magnetite from magnetoliposomes [7,20,22,30]. The centrifugation of MLP*_1-6 samples generally led to the separation of three distinct phases – DP, BP, and S, which by means of a specific TEM investigation (Fig. 6a–f), were correlated with the presence of magnetite, magnetoliposomes, and small liposomes/ magnetoliposomes, respectively. Moreover, this separation allowed us to calculate a correct liposome–magnetite association percentage by using (Eq. (1)). Formulations used for MLP*_1-6 samples showed liposome–magnetite association values ranging from 55% to 93%, and these values also showed a decreasing trend beginning from sample MLP*_2 and continuing to sample MLP*_6. However, the loading capacities of MLP*_1 to MLP*_6 increased (Table 2), resulting in an overall positive result. The MLP*_6A sample showed rather different behaviors following centrifugation, by separating into only 2 phases – a supernatant and a brown sediment. The underneath very dark phase was not present, and it was therefore not possible to precisely calculate the liposome–magnetite association of MLP*_6A samples. However, we could only presume an association percentage greater than those calculated for MLP*_1-6 samples, which were >90%.
A TEM micrograph of a non-centrifuged MLP*_3 sample revealed the presence of magnetite, both in the vesicular structures and in the surrounding environment, (Fig. 6a). Furthermore, two main types of entities could be recognized, vesicular structures with magnetite nanoparticles only present in the phospholipid bilayer (black circles in fig 6a), and smaller vesicular structures with nanoparticles present in the phospholipid bilayer and also in the inner space (black arrows in fig 6a), thus demonstrating a high degree of liposome–magnetite association. All three phases of a centrifuged MLP*_3 sample were analyzed. Fig. 6b shows the supernatant phase that is characterized by clusters of small nanoparticles partially inserted into the phospholipid bilayer and partly exposed outside of the vesicular structure (magnetoliposomes), and also the presence of small empty liposomes. Fig. 6c–e show the brown precipitate phase that is characterized by the presence of magnetoliposomes. In this phase, magnetite nanoparticles were mainly found in the phospholipid bilayers, but in some cases, they were also visible in the central area, corresponding to the aqueous core area. This could be due to the presence of nanoparticles at different depths within the phospholipid bilayers; moreover, in this phase are barely detectable free magnetite nanoparticles, which mainly characterized the dark phase (Fig. 6f). To verify the influence of both the PC:DSPE-PEG phospholipid mixture and a high magnetite concentration (1 mg/mL) on the hybrid magnetic colloidal structures, the LP*A and MLP*_6A samples were observed by TEM. As illustrated in Fig. 7a and b, empty
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liposomes (LP*A) showed a regular and multilamellar structure, typical of the classical vesicular bilayer structure. Fig. 7c–g shows the main magnetoliposomal structures. In Fig. 7a and b, it is possible to observe magnetoliposomal structures with magnetic nanoparticles clearly incorporated into phospholipid bilayers, confirming observations obtained with MLP*_1-6 samples. Fig. 7e–g shows the second configuration obtained, which was mainly characterized by aggregates of nanoparticles, which were directly stabilized by phospholipids. The latter configuration was very similar to the configuration reported in our previous study [24], where we described magnetoliposomes prepared by hydrophobic magnetite differently synthesized and surrounded by a higher percentage of oleic acid. In the case of OA–Fe3O4 nanoparticles, this particular magnetoliposomal configuration occurred mainly in the formulations prepared using a high concentration of magnetite (1 mg/mL), and with the PC:DSPE-PEG mixture (MLP*_6A). However, in our previous study, this configuration was obtained even at a low concentration of magnetite (0.2 mg/mL) and using P90G phospholipids [24]. This emphasizes the great impact of the different oleic acid:magnetite ratios and the involved phospholipids on the final magnetoliposomal configuration. The nanoparticle aggregates directly stabilized by one or more phospholipid bilayers seem to be a characteristic configuration for magnetoliposomes prepared using hydrophobic magnetite and stabilized with oleic acid. Since SAXS investigations of multicompartment and hierarchically organized lipid carriers have introduced new directions in the research on stealth lipid nanoparticles, further research will be focused on the characterization of MLP*_6A configuration by means of this technique [31–33.]. Considering their high stability, high liposome–magnetite association percentage, and greater suitability for vivo applications, magnetoliposomes prepared using the PC:DSPE-PEG mixture and the LP*A and MLP*_6A formulations were considered the most suitable for conducting the cytotoxicity assay. 3.2. Cytotoxicity and cell–nanoparticle interaction To assess the capability of MLPs* to interact with cells, we conducted a study using cells growing in suspension (CEM cells), rather than cells adhering to a substrate, because this condition better reflects the blood environment of a hypothetical in vivo treatment using intravenous administration. Our first study was conducted to determine the effect of nanoparticles on cell viability. Preliminary data suggested that OA–Fe3O4 nanoparticles at a concentration of 10.3 lg/mL, were unable to reduce the number of viable cells as compared to untreated cells (control). However, the same magnetite concentration contained in MLPs* strongly reduced the number of viable cells (p < 0.01), suggesting that the carrier delivery system had a significant impact on cell viability (Fig. 8a). Interestingly, OA–Fe3O4 alone and OA–Fe3O4 associated with liposomes (MLPs*) appeared to interact with cells in different ways. Using a modified Prussian Blue/Nuclear Fast Red staining method, modified for our experimental conditions (Fig. 8c–e) (see Section 2 for more details), it was possible to observe different associations of OA–Fe3O4 nanoparticles and MLP* with cells. The OA–Fe3O4 nanoparticles were not internalized into the cells (majority of blue spots were on the white background), and this was probably due to aggregation and precipitation of OA–Fe3O4 in the environment. In contrast, when the cells were treated with MLPs*, the nanoparticles appeared to be incorporated into the cells because the pinkish cytoplasmic membranes showed blue spots compatible with the presence of iron in that compartment. In some cases, we observed a diffuse blue–green color of the cytoplasm, that could be attributed to the more soluble and physiologically ubiquitous form of iron (ferritin) in cells; which however, did not
affect our analysis. Furthermore, to evaluate a dose-dependent effect of magnetite on cell viability, in the second step of our experiments, the cells were treated with different amounts of MLP*, as specified in Section 2. Our data suggested that there were not significant differences between treated cells and control cells (Fig. 8b), but a statistical analysis of the linear regression slopes (with intercept 0) obtained from the growth curves of each group showed that a large concentration of MLP* (corresponding to a magnetite concentration of 3.72 lg/mL) produced a significant decrease in the growth curve of treated cells, compared to the growth curve for control cells (p < 0.05). This result indicated that 3.72 lg/mL may be the threshold concentration for producing a cytotoxic effect on cells, in terms of inhibiting proliferation or viability. 4. Conclusion In this work, we presented new magnetic stealth liposomes prepared by means of the simple, versatile, and reproducible TLE procedure and loaded with hydrophobic magnetite stabilized with oleic acid. Overall results have shown the great impact of both the different oleic acid:magnetite ratios and the involved phospholipids on the final magnetoliposomal configuration and features. Moreover, we found that the incorporation of magnetite into liposomal structures had a significant impact on cell toxicity. Indeed, the vesicular delivery system allowed magnetite nanoparticles to interact with cells and, as a consequence, to be easily internalize thus exerting a dose-dependent effect on cell viability. The ability of these structures to incorporate high amount of magnetite nanoparticles as well as to penetrate cell membrane, led us to conclude that they can be the object of further study as potential and useful carrier in biomedical field. Acknowledgments This work was financed by University of Cagliari and by Fondazione Banco di Sardegna. Authors gratefully thanks NANOGAP (Santiago de Compostela, Spain) for kind supplying magnetite (brand name: NGAP FeO-03#01). Appendix A. Supplementary material Six additional figures are provided. OA–Fe3O4 nanoparticles size distribution obtained by DLS; size distribution by volume of OA–Fe3O4 nanoparticles; X-ray diffraction patterns of OA–Fe3O4 nanoparticles with rietveld simulation; FTIR spectrum of OA–Fe3O4 nanoparticles; TGA and dTGA curves for OA–Fe3O4 nanoparticles; Hysteresis curve OA–Fe3O4 nanoparticles. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.03.046. References [1] T.M. Allen, P.R. Cullis, Adv. Drug Del. Rev. 65 (2013) 36–48. [2] D.D. Lasic, F.J. Martin, A. Gabizon, S.K. Huang, D.P. Papahdjopoulos, Biochim. Biophys. Acta 1070 (1991) 187–192. [3] V.E. Trubetskoy, V.P. Torchilin, Adv. Drug Del. Rev. 16 (1995) 311–320. [4] L. Cattel, M. Ceruti, F. Dosio, J. Chemother. 16–4 (2004) 94–97. [5] L.D. Leserman, J. Barbet, F. Kourilsky, J.N. Weinstein, Nature 288 (1980) 602– 604. [6] M. Shinkai, A. Ito, Adv. Biochem. Eng. Biotechnol. 91 (2004) 191–220. [7] H. Nobuto, T. Sugita, S. Shimose, Y. Yasunaga, T. Murakami, M. Ochi, Int. J. Cancer 109 (2004) 627–635. [8] A.A. Kuznetsov, V.I. Filippov, R.N. Alyautdin, N.L. Torshina, O.A. Kuznetsov, J. Magn. Magn. Mater. 225 (2001) 95–100. [9] P. Tartaj, M.P. Morales, S. Veintemillas-Verdaguer, T. Gonzalez-Carrenõ, C.J. Serna, J. Phys. D: Appl. Phys. 36 (2003) 182–197. [10] M. Hoehn, E. Kustermann, J. Blunk, Proc. Natl. Acad. Sci. USA 99 (2002) 16267– 16272. [11] C.C. Berry, A.S.G. Curtis, J. Phys. 36 (2003) R198–R206.
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