Multimodal Superparamagnetic Nanoparticles with Unusually

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Multimodal Superparamagnetic Nanoparticles with Unusually Enhanced Specific Absorption Rate for Synergetic Cancer Therapeutics and Magnetic Resonance Imaging Nanasaheb D Thorat, Raghvendra A Bohara, Victor Malgras, Syed A. M. Tofail, Tansir Ahamad, Saad M. Alshehri, Kevin C.-W. Wu, and Yusuke Yamauchi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 20 May 2016 Downloaded from http://pubs.acs.org on May 20, 2016

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ACS Applied Materials & Interfaces

Multimodal Superparamagnetic Nanoparticles with Unusually Enhanced Specific Absorption Rate for Synergetic Cancer Therapeutics and Magnetic Resonance Imaging

Nanasaheb D. Thorat1,2,3, Raghvendra A. Bohara1, Victor Malgras4, Syed A.M. Tofail2,3, Tansir Ahamad,5 Saad M. Alshehri,*5 Kevin C.-W. Wu*6,7 and Yusuke Yamauchi*4

1

Centre for Interdisciplinary Research, D.Y. Patil University, Kolhapur-416006, India

2

Department of Physics & Energy, University of Limerick, Limerick, Ireland

3

Materials & Surface Science Institute, University of Limerick, Limerick, Ireland

4

World Premier International (WPI) Research Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 3050044, Japan.

5

Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

6

Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan

7

Division of Medical Engineering Research, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan E-mails: [email protected]; [email protected]; [email protected]

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Abstract Superparamagnetic nanoparticles (SPMNPs) used for magnetic resonance imaging (MRI) and magnetic fluid hyperthermia (MFH) cancer therapy frequently face trade off between a high magnetization saturation and their good colloidal stability, high specific absorption rate (SAR) and most importantly biological compatibility. This necessitates the development of new nanomaterials as MFH and MRI are considered to be one of the most promising combined noninvasive treatments. In the present study, we investigated polyethylene glycol (PEG) functionalized La1-xSrxMnO3 (LSMO) SPMNPs for efficient cancer hyperthermia therapy and MRI application. The superparamagnetic nanomaterial revealed excellent colloidal stability and biocompatibility. A high SAR of 390 W/g was observed due to higher colloidal stability leading to an increased Brownian and Neel’s spin relaxation. Cell viability of PEG capped nanoparticles is up to 80% on different cell lines tested rigorously using different methods. PEG coating provided excellent hemocompatibility to human red blood cells as PEG functionalized SPMNPs reduced hemolysis efficiently compared to its uncoated counterpart. Magnetic fluid hyperthermia of SPMNPs resulted in cancer cell death up to 80%. Additionally, improved MRI characteristics were also observed for the PEG capped La1-xSrxMnO3 formulation in aqueous medium compared to the bare LSMO. Taken together, PEG capped SPMNPs can be useful for diagnosis, efficient magnetic fluid hyperthermia and multimodal cancer treatment as the amphiphilicity of PEG can easily be utilized to encapsulate hydrophobic drugs.

Keywords: Superparamagnetic nanoparticles, magnetic fluid hyperthermia, magnetic resonance imaging, polyethylene glycol, drug delivery systems.


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1. Introduction Multifunctional magnetic nanoparticles (MNPs) offer attractive avenues for safe and efficient cancer therapies. Because of their intrinsic magnetic properties and high biocompatibility, MNPs have been successfully used for cancer therapy such as hyperthermia.

1–4

. Chemically functionalized and

colloidally stable MNPs can efficiently convert electromagnetic energy into heat by various mechanisms, including hysteresis, superparamagnetic and eddy current losses. The application of electromagnetic fields on surface functionalized MNPs and the further conversion of such electromagnetic energy into thermal energy can effectively destroy cancer cells, which is so-called hyperthermia.5–7 Extensive efforts are being made to achieve tumor destruction, both in vitro and in vivo, using electromagnetic fields with low frequency.8 This can be possible by using superparamagnetic nanoparticles (SPMNPs) instead of magnetic nanoparticles.9–11 SPMNPs can generate heat under alternating current (AC) applied magnetic field and lose their magnetization when the applied magnetic field is turned off. After removing the external field, they become highly dispersed in the fluid which makes them suitable to treat cancer in clinical therapies.12 Superparamagnetic materials with a Curie temperature (Tc) comprised in the cancer hyperthermia therapeutic range (i.e. 42-50°C, ~ 315-325 K) are most suitable for in vivo applications.12,13 Among various classes of MNPs, La1−xSrxMnO3 (LSMO) is being considered as one of the most adequate candidates for hyperthermia, motivating researchers to study properties such as the superparamagnetic nature, the colloidal stability and the biocompatibility.14–16 The use of LSMO nanoparticles overcomes the drawbacks of metal dopped ferrite nanoparticles (i.e., high Curie temperature and low magnetization). However, low biocompatibility, low colloidal stability and low SAR remained prominent issues for safe and effective in vivo applications. Previous works attempted to surmount these limitations by using surface functionalization with biocompatible polymer (or nonpolymer) including Dextran, Pluronic F127 and silica.17–20 These coating materials were found to be 3 ACS Paragon Plus Environment


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effective in reducing the cytotoxicity and improving the colloidal stability. However, the SAR of LSMO nanocomposites remained far below the ferrite MNPs.21 One of the potential coating for improving biocompatibility, colloidal stability and SAR of MNPs is polyethylene glycol (PEG) because of its highly hydrophilic nature. PEG provides a barrier to the adsorption of protein, reducing the macrophages uptake.22 PEG is also a biocompatible, nonantigenic, and protein-resistant polymer. PEG exhibits neutral hydrophilic segments that leads to homogeneous distribution in aqueous medium. The synthesis of PEG-coated MNPs is, however, a complex and tedious process because PEG tends to form larger aggregates when its molecular weight is too large.23 Thus the selection of an optimum molecular weight of PEG for stabilizing the MNPs is one of the critical factors. The use of LSMO for magnetic fluid hyperthermia (MFH) has several advantages over other ferrite MNPs such as (i) an easily tunable Curie temperature through Sr doping and (ii) the superparamagnetism can be easily attained. The poor biocompatibility, the colloidal instability, and the lower SAR of LSMO remain hindering problems which need to be addressed. In the present investigation, we prepared highly water-dispersible, biocompatible, superparamagnetic LSMO nanoparticles (SPMNPs) by encapsulating them with PEG chains through oleic acid (OA) treatment. To avoid the aggregation of PEG on the SPMNPs, OA was selected to formerly passivate surface prior to PEG coating. The current efforts aimed to improve the SAR of the SPMNPs by improving their colloidal stability and biocompatibility by functionalizing their surface with PEG. The structural, magnetic and colloidal properties of PEG coated SPMNPs sample was studied in details. The performance of the synthesized LSMO@OA-PEG core/shell nanocomposites on the hyperthermia was also studied. We evaluate the biocompatibility by using multiple cell lines and human red blood cells (HRBCs). Furthermore, we studied the interaction between samples and cells by observing confocal


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laser scanning microscopy (CLSM). The toxicity of LSMO@OA-PEG core/shell nanocomposite on HRBCs was evaluated to assess the feasibility of intravenous injection. The effect of hyperthermia was examined in vitro. Finally the efficacy of bare and PEG-capped LSMO nanoparticles as magnetic resonance imaging (MRI) contrast agent is evaluated by magnetic resonance (MR) measurements. The results reveal a significant increase in the transverse relaxation, which reflects the efficacy of T2 contrast agents in MRI. The added benefit of OA and PEG conjugation was found to resist to the nonspecific interactions with biomacromolecules and improved SAR, biocompatibility and MRI properties of the LSMO@OA-PEG core/shell nanoparticles.

2. Result and Discussion 2.1. PEG coating analysis Thermogravimetric analysis was used to measure the existence and the amount of PEG on the OA-LSMO nanocomposites (Fig. 1a). Thermogravimetric analysis of the LSMO@OA-PEG samples reveals 18–20% weight loss as we heated to 600 °C. The weight loss below 100 °C is the evaporation of the water (region a). Then, 9-10% weight loss from 140 to 250 °C can be observed, owing to the disappearance of the PEG molecules on the surface of LSMO and OA (region b). In region c, the loss can be resulted from the decomposition of ther inner OA layer (250 and 375 °C). We observed a kink at 300 °C, that can be explained by the decomposition of the OA layer, and we suggested that there was a further decomposition of OA molecules owing to the remaining weight loss up to 375 °C.24 The obtained weight loss pattern strongly supports the assumption that PEG coating utilize OA as an anchor sites during the synthesis of LSMO@OA-PEG SPMNP, as can also be confirmed in earlier reports.25



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Fig. 1 (a) TGA-DTA analysis of bare, OA capped and PEG-capped LSMO in N2 atmosphere, (b) FTIR spectra of bare LSMO (i), OA-capped LSMO (ii), PEG-capped LSMO MNPs (iii) and only PEG (iv). (c) photographs of the phase transition from bare, to OA- and PEG-capped LSMO MNPs from water to hexane and hexane to water. (d) Schematic diagram showing PEG capping on LSMO MNPs using OA as an anchor.

Surface functionalization of the LSMO MNPs with OA and PEG was further verified by FTIR (shown in Fig. 1b). The FTIR spectrum for the bare LSMO sample was reported in our earlier publication.18 LSMO MNPs simply coated with OA exhibited peaks at 1650 and 3480 cm-1 due to the water absorbed on the LSMO particles. The peaks located around 1210, 1440 and 1529 cm−1 can be regarded as C-O stretching, asymmetric υas (COO-) and symmetric υas (COO-) stretch, respectively. The peaks of 2800–3000 cm−1 are resulted from the symmetric and asymmetric stretching of the CH, CH2 and CH3 groups from oleic acid. After coating LSMO-OA with PEG, we observed peaks at 960 cm−1 for out-of-plance CH bending and 1354 and 1102 cm−1 for C–O–C asymmetric and symmetric 6 ACS Paragon Plus Environment

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stretching, respectively.25 PEG polymer also exhibits a number of prominent peaks between 600-900 cm−1. The magnetic core of the LSMO@OA MNPs did not produce any FTIR signal. Photographs of phase transfer of bare, OA- and PEG-coated LSMO are shown in Fig. 1c. Bare LSMO MNPs easily dispersed in water but not in hexane. The addition of OA in LSMO MNPs solution in similar concentration facilitated their dispersion in hexane. Further coating the LSMO@OA surface with PEG re-established the hydrophilicity, allowing the final LSMO@OA-PEG nanoparticles to disperse in water. A schematic illustrion of the mechanisms involved in the capping of LSMO surface with OA and PEG is shown in Fig. 1d. OA molecules are chemisorbed on the LSMO surface with their hydrophobic segment oriented towards the outside, enabling LSMO@OA to disperse in hexane. After conjugating the OA-capped MNPs with PEG in solution, PEG can provide two functions: (1) the hydrophobic segment can interdigitate with oleic acids that stabilize the structure and (2) the hydrophilic blocks oriented outwards can increase the distribution of LSMO@OA-PEG nanoparticles in water.26 2.2. Size analysis PEG-capped LSMO spherical particles with a diameter around 15-20 nm range was visualized by TEM. The agglomeration of the LSMO MNPs was found due to their magneto-dipole interactions (Fig. 2a). The M vs T measurement (Fig. S1) shows the superparamagnetic nature of PEG capped SPMNPs. The superimposition of FC and ZFC curve implies superparamagnetic nature at room temp as well as the magnetization trend closed towards zero possibly defines Curie temperature (Tc) in between 550-370 K. In our recent publication we found Tc of LSMO is ~ 370 K.21 The M vs H measurements are shown in Fig. 2b. The values of magnetization for bare and PEG-capped SPMNPs were 47.23 and 40.10 emu/g, respectively. This result can be expected as the magnetization is proportional to the mass ratio of magnetic material. The organic layers (OA and PEG) on the samples



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would reduce overall magnetization. We found a negligible magnetization at zero field, which indicates the superparamagentic behavior of the synthesized PEG-capped LSMO.

Fig. 2 Characterization of SPMNPs, particle size and magnetization properties. (a) TEM image of PEG-capped LSMO MNPs. (b) Magnetization data of bare and PEG-capped LSMO SPMNPs at 300 K. The magnetization is slightly reduced after OA and PEG coating. The hysteresis loop is shown in the inset, clearly showing the absence of coercivity in both bare and PEG-capped LSMO. (c) Hydrodynamic diameter distribution of bare and PEG-coated LSMO SPMNPs in water and PBS, (d) Zeta potential vs pH values.

In order to be implemented effectively in biomedical applications, MNPs should remain colloidally stable when encountering physiological ionic strengths as well as a wide range of pH variations in biological systems, and should resist adsorption of proteins present in the bloodstream. We used DLS and Zeta potential to quantify the stability of PEG-capped SPMNPs. Fig. 2c shows the


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hydrodynamic diameter distribution of bare and PEG-capped SPMNPs in water and PBS. Bare SPMNPs possess a large particle size distribution (~ 20-100 nm) which was reduced after PEG coating (~ 20-60 nm in water and ~ 20-40 nm in PBS). The pH dependent colloidal stability of PEG-coated LSMO MNPs showed a value of −23.10 mV at pH 7.4 as well as an isoelectric point at pH 5 (Fig. 2d). The results indicate that the coating of PEG could provide a steric barrier between the LSMO MNPs particles when pH value of the medium is too low (acidic) or too high (basic), thus decreases the agglomeration of the MNPs.23,27 This result indicates that the high coverage of the polymer layer on the OA-coated MNPs surface can stabilize the particles in water media. 2.3. Biocompatibility study 2.3.1. Cell toxicity study It is important to study the safety of new nanomaterials. Therefore, we evaluate the cytotoxicity of the synthesized PEG-capped LSMO MNPs by using different types of cell lines.28 Therefore, to evaluate precise cytotoxicity/biocompatibility of the MNPs, we use various cell-based assays on several cell-lines with variable incubation time and dose-dependent manner of MNPs. Thus, the comparative cytotoxicity assays were performed for MNPs on L929, HeLa and MCF7 cell lines using two different methods: Trypan blue dye exclusion (TBDE) and 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide based colorimetric assay (MTT) with various concentrations of MNPs. Trypan blue can stain cytoplasmic and nuclear components only if the cell membrane is disrupted; therefore, the intracellular components are stained only in dead cells.



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Fig. 3 Biocompatibility of PEG-capped LSMO SPMNPs. (a) Cell viability data obtained by TBDE and MTT assays of PEG-capped LSMO MNPs. (b) Study of the physical interaction between the SPMNPs and the cellular phenotype (Panels A, B and C are cells treated with SPMNPs (0, 0.2 and 1 mg/mL, respectively) (scale 20 µm).

The cytotoxicity data is showed in Fig. 3a. A slight difference can be observed on the cell viability with different cell lines and different assays, which can be attributed to various factors: a) different biophysical nature of various cell lines, b) newly introduced perspective of differential interaction in various cells to same nanoparticle termed as cell “vision” effects 28 and/or c) differential sensitivities of assays. Different cell viability on various cell lines can be explained by different surface propoerty of individual cell line and cellular morphology.29–31 These factors affect the interaction and viability between PEG-capped SPMNPs and cells. The obtained results were coherent, and no extreme changes could be observed. The cell viability of PEG-capped LSMO MNPs (1 mg/mL) by TBDE assay for 24 and 48 h incubation was calculated to be 96% and 85% for L929 cells, 87% and 80% for HeLa cells and 87% and 81% for MCF7 cells, respectively. The consistent cell 10 ACS Paragon Plus Environment

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viability by MTT assay for 24 and 48 h incubation was calculated to be 90 and 85% for L929 cells, 87% and 81% for HeLa cells and 89 and 80% for MCF7 cells, respectively.

2.3.2. Cell-nanoparticle interaction by confocal microscopy In order to support the validity of the TBDE and MTT assays used to determine biocomaptibility and to identify the effect of the PEG-capped LSMO MNPs on the morphology of the cells as well as the induced apoptosis and necrosis, additional confocal microscopy aided with multiple staining was carried out. The MCF7 cells were stained with DAPI, FITC and PI dyes to evaluate the toxicity by MNPs. The multiple staining method used here is to identify the effect of nanoparticles on cellular morphology in an accurate and qualitative way.32–34 The fluorescence imaging of MCF7 incubated with PEG-capped LSMO MNPs (0, 0.2, and 1.0 mg/mL) for 24h observed by CLSM, is shown in Fig. 3b. The fluorescence imaging confirms visually their good biocompatibility. The integrity of the cells appears to not be influenced by high concentrations of MNPs, as the intact green cells are still clearly visible around the blue nuclei. After 24 h of incubation with MNPs, the cells exhibit good proliferative activity as their morphology and nuclear/cytoplasmic ratio were similar as those of the control cells. Even after 24 h of MNPs treatment, the characteristics cell death such as cell shrinkage, cellular extensions or increased floating cells were not observed. However, at very higher concentration (1 mg/mL), cell death could be observed, reflected by an increment in the percentage of cells stained positive for PI. Overall percentage of apoptotic and necrotic cells are statistically insignificant in PEG-capped LSMO MNPs-treated MCF7 cells (1 mg/mL) for 24 h.

2.3.3. Hemolysis activity



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To consider in vivo applications in the future, hemocompatibility is an important issue. Lanthanide elements (La, Gd) have been reported to create pores into the membrane of HRBCs and to produce hemolysis when brought in contact with blood cells.35 In order to study the effect of PEGcapped LSMO MNPs on hemolytic activity and in tegrity of HRBC membrane, hemolysis assay was performed on bare and PEG-capped LSMO MNPs. Fig. 4 shows the hemolytic behavior both samples at various concentrations (0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL). It appears that there was no visible hemolytic effect induced by the PEG-capped LSMO MNPs at 1 mg/mL for 3 h (Fig. 4). The induced hemolysis by these MNPs was 12% and 38% for 3 and 20 h, respectively. However, hemolysis for bare SPMNPs was found to be 40 % at 3 h and reached up to 60% for 20 h. We found that PEG capping reduced significantly the hemolysis of LSMO. The hemolysis observed for PEG-capped LSMO MNPs for 3 h lies near the prescribed permissible limit (5 %) as per ASTMF-756-08.16,36 The negligible hemolytic activity of PEG-capped LSMO MNPs in a wide concentration range (0.1–1 mg/mL) along with their excellent biocompatibility facilitate their use for in vivo applications.


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Fig. 4 Hemolytic activity of bare and PEG-capped LSMO SPMNPs (a) Comparative data of the % hemolytic activity induced by the bare and PEG-capped LSMO MNPs on red blood cells, for 3 and 20 h incubation times using PBS and water as the negative and positive control, respectively. Photograph of HRBC exposed to PEG-capped LSMO MNPs monitored for 20 h is shown in the inset.

2.4. Hyperthermia study The field-dependent temperature kinetics and SAR of PEG-coated LSMO MNPs in water are shown in Fig. 5. Magnetic measurements already exhibited the superparamagnetic nature of PEGcoated LSMO MNPs (Fig. 2). Thus, the primary factor for heat generated by superparamagnetic NPs is attributed to Brownian and Néel’s spin relaxation. 13 ACS Paragon Plus Environment


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Fig. 5 Temperature of the PEG-capped LSMO SPMNPs under AC magnetic field. (a) AMF dependent temperature kinetics of 1 mg/mL PEG-capped LSMO SPMNPs. (b) SAR values of PEG-capped LSMO SPMNPs in water. (c) Schematic illustration describing the mechanisms responsible for improving the SAR of the PEG-capped LSMO MNPs.

This heat generation is associated with power losses related Brownian or Néel’s spin relaxations and can be approximated by: P = µ 0 πχ " fH 2

(1)

where µ0 is the permeability of free space, f the frequency of the applied AC magnetic field, H the amplitude of the applied AMF and χ″ the AC magnetic susceptibility (imaginary part). The AC magnetic susceptibility χ″ contains the action of both relaxation mechanisms and can be expressed as:

χ" =

ωτ eff 1 + (ωτ eff

)

2

χ

(2)


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where χ is the magnetic susceptibility of the material. τeff is the effective relaxation time of the two mechanisms (Brownian and Néel’s spin relaxations) working in parallel and can be estimated by: τ eff =

τ Bτ N τ B +τ N

(3)

where τN and τB are the Néel’s spin relaxation and Brownian relaxation times, respectively. The amount of heat (A) during one magnetic field cycle is the same as the area of the hysteresis loop. We express it as: + H max

A=

∫

µ0 M ( H )dH

(4)

− H max

Then the SAR is: (5)

SAR = A f

Where f is the frequency of the AMF expressed as f = ω/2π, M the magnetization and H the amplitude of the applied magnetic field. Temperature kinetic curves show a gradual increase in temperature with increasing the amplitude of the magnetic field (Fig. 5a). The required hyperthermia temperature (i.e., 42-46 °C) was achieved by the PEG-capped LSMO MNPs within a relatively short time and low applied AC magnetic field. From Fig. 5b, it could be observed that the SAR increased almost linearly with increasing the field. The obtained SAR at 500 Oe for the PEG-capped SPMNPs and bare SPMNPs were ~380 W/g and ~90 W/g (Fig. S2), respectively, which was a drastic increase resulting from the PEG functionalization treatment. The similar observation is observed for chitosan functionalized MNPs and reported in our earlier publication.37 The reasons include: (i) the OA–PEG coating can prevent the particle from aggregation. These functionalized LSMO MNPs have faster relaxation processes, resulting in higher heating efficiency; (ii) the thermo-responsive nature of oleic acid and



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PEG is another advantage for attaining high SAR. The mechanism of enhanced SAR is depicted in Fig. 5c. After evaluating the colloidal stability and hyperthermia properties of the PEG-capped LSMO MNPs, the hyperthermia effect on cancer cells is studied. During in vitro observations, the temperature of the SPMNPs supplemented cell suspension was strictly maintained at 43-44 oC and was made sure to not reach above the upper hyperthermia threshold of 45 oC. MTT results show MCF7 cell killing trends at 1 and 24 h post-hyperthermia (Fig. 6). PEG-capped SPMNPs rapidly suppressed the cell viability after increasing the exposure time under AMF. The killing of MCF7 cells was found to be ~80% under AMF for 1 h while cell viability was measured after 24 h post-hyperthermia.

Fig. 6 Cancer cell killing profile. (a) The percentage of viability of MCF7 cells treated with PEGcapped MNPs. The cells were treated with SPMNPs (1 mg/mL) for 10-60 min under an alternating magnetic field. The proportion of cell viability is calculated by using MTT assay. (b) ROS level after MFH treatment. Values are expressed as mean ± SD, n = 3.

Recently, Moon et al. reported that hyperthermia can generate high level of ROS, killing cancer cells by oxidative burst.38 In order to examine the exact cancer cells killing mechanisms by PEG-capped LSMO MNPs involved in MFH, the ROS level in the cells after the treatment was also 16 ACS Paragon Plus Environment

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estimated. As shown in Fig. 6 b, the hyperthermia induced a significantly higher level of ROS in the cells treated with PEG-capped LSMO MNPs compared to the untreated ones. Also, the treated cancer cells which did not undergo MFH treatment exhibited a much lower ROS level. ROS generation increased with increasing the duration of MFH (10-60 min) and was also significantly enhanced after incubation upto 24 h which indicates a slow increment in the number of dying cells.39 Our studies suggest that MFH combined with PEG-capped LSMO MNPs can significantly enhance ROS generation and improve the cancer cell killing rate. Similar behavior is observed in recent reports.40–42 The rational behind MFH-induced cell death was examined by using DAPI, FITC and PI staining (Fig. 7) coupled with confocal microscopy. As described in an previous section, multiple staining with microscopic visualization helps in assessing the nature of cell death. Changes in the plasma membrane, nucleus fragmentation and shrinkage of the cells were detected through microscopic observations. Before application of MFH, cells are uniformly stained with FITC in order to ascertain their healthy cellular morphology. Post-hyperthermia treatment, time-dependent increment of PI stained cells and simultaneously reduction in DAPI and FITC stained cells indicated cell death by apoptosis, as reported earlier.43 During apoptotic death, cells are broken apart into several vesicles, called apoptotic bodies, with damaged membranes and hence, the staining of FITC is disrupted. The loss of membrane integrity facilitates PI to enter into the dead cells and stains the nuclei (Fig. 7b and c). Thus, observed MFH-induced cell death is due to apoptosis and is confirmed by observing cell shrinkage, cellular extensions and increased numbers of floating cells.



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Fig. 7 Confocal microscopy images of (a) untreated and (b and c) treated MCF7 cells by MFH for 60 min and stained with DAPI (stain nuclei), PI (stain dead cells) and FITC (stain cytoplasm) after (b) 1 h and (c) 24 h incubation (scale 20 µm).

The key factor to enhance cancer cell death is an efficient thermal dose induced by SPMNPs after application of an AMF within a short span of time during MFH. To provide an optimum thermal dose, efficient heat generation by SPMNPs was achieved by synthesizing SPMNPs with high SAR response under AMF. In this study, we focused on increasing the SAR of LSMO MNPs, an unsolved problem to overcome in order to implement them in hyperthermia treatments.44[24] In our earlier publications on LSMO, we provided evidence of small particle providing colloidal stability generating heat efficiently within very short spans of time.19,27,37 This strategy was exploited to developed biocompatible and colloidally stable PEG-capped LSMO MNPs and improved their SAR up to sixfold compared to their bare counterpart reported earlier.21 The improvement of SAR resulted into efficient thermal dose during in vitro MFH and killed the cancer cells. Cancer cell death by apoptosis


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in MFH is a preferred strategy. In our current study, the LSMO@OA-PEG mediated efficient MFH showed enhanced cancer cell killing due to six-fold improved SAR with excellent biocompatibility to different cell lines and red blood cell. 2.5. MRI study To evaluate the usefulness of superparmagnetic bare and PEG-capped LSMO MNPs as MRI contrast agents, we examined the MR relaxivity in aqueous solutions. A linear behavior can be observed in the concentration range from 0.01 to 0.1 mg/mL. T2-weighted images for PEG-capped LSMO MNPs from 0.01 to 0.1 mg/mL are shown in the Fig. 8a. In a representative image, the T2 MR signal became darker when the concentration of PEG-capped LSMO MNPs was increased. PEGcapped LSMO shows a much higher transverse relaxation rate (498. 33 s-1) than bare LSMO (162.33 s1

). As shown in Fig. 8b, we plotted the inverse relaxation time (1/T2) at a constant frequency of 35

MHz as a function of the nanaoparticle concentration.



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Fig. 8 (a) T2-weighted MR imaging of cancer cells incubated with LSMO@PEG at different concentrations. (b) Relaxation rates 1/T2 of bare LSMO and PEG-capped LSMO nanoparticles as a function of their concentration. (c) The intensity of the MR signal of the MCF7 cells before and after hyperthermia treatment with the LSMO@PEG. In the figure (c) traditional water bath hyperthermia is applied at SPMNPs concentration=0.

The value of r2 is increased threefold after PEG coating. A similar behavior can be observed with the PEG capping on Fe3O4 nanoparticles, dynamically improving the T2 contrast and r2 value compared to bare Fe3O4.45 This result indicates that these nanoscale assemblies represent highly


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efficient T2 contrast agents when compared to bare LSMO, Fe3O4 MNPs (196.7 s-1) and ferumoxytol (84.9 s-1), a commonly used contrast agent in MRI.46,47 Furthermore, we investigated the quantiataive analysis of MR imaging of cancer cells incubated with LSMO@PEG with and without hyperthermia. We detected 2×106 cells per mL on a clinical MR imaging system (Fig. 8c). The MR intensity gradually decreases with SPMNPs concentration, suggesting that the SPMNPs can be used as an active targeting contrat agent for other in vivo bio-medicine.

3. Conclusion In conclusion, modifying the surface of LSMO MNPs with OA-PEG improved SAR up to six-fold. PEG-capped MNPs possess a superparamagnetic nature as well as a high colloidal stability in both aqueous and physiological (pH 7.4) media. The surface coating of LSMO MNPs with PEG showed reduced hemolysis below the prescribed permissible limit (5%), unlike its bare counterpart. Under optimized conditions, MFH of PEG-capped LSMO MNPs displayed cancer cell death up to 80% through induced apoptosis. A multimodal cancer therapy which includes both chemotherapy and hyperthermia using this SPMNPs formulation would provide a feasible, relatively simple and cost effective strategy to conjugate anticancer drug in the future.



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Experimental 1. Synthesis of PEG capped LSMO nanocomposites The La0.7Sr0.3MnO3 (LSMO) MNPs were synthesized according to a previous study.21 The LSMO@OA-PEG superparamagnetic nanocomposites were also prepared by a reported method. First, LSMO MNPs were functionalized with oleic acid according to previuos reports.27 Secondly, PEG was coated on the OA-functionalized MNPs surface. Briefly, 20 % PEG solution (molecular weight is 5000) was prepared in water in which the OAcontained LSMO MNPs were then dispersed through ultrasonication with intermittent shaking. The dispersed suspension of OA-coated LSMO and PEG was stirred for 24h in a sealed round bottom flask. The particles were then gathered by a magnet. The sediment was washed using Milli-Q water and magnetic decantation thrice. The LSMO@OA-PEG MNPs (hereafter referred as SPMNPs) were dried in a vacuum overnight.

2. Physical characterization The thermal decomposition behavior was conducted (Dupont 2100). Transmission electron microscopy (TEM, Philips CM200 model) was used to evaluate the particle size and shape. The measurements of magnetization were conducted with a magnetometer (Quantum Design SQUID). Zeta potential was measured in water (ZLS PSS-NICOMP-380, USA). The water-suspended bare and PEGcapped SPMNPs (1 mL) were placed at the coil, and we applied 265 kHz AC current. The particles with different concentrations were ultrasonicated for 20 min to disperse in the carrier fluid. We heated samples for 10 min by applying various currents from 200 to 600 A that can generate magnetic field of 83.8 to 502.8 Oe, respectively.

3. Cell culture The cytotoxicity of PEG-capped SPMNPs was studied by using various kinds of cell lines including L929, HeLa and MCF7 cells.

4. Trypan blue dye exclusion (TBDE) We incubated cells with the concentration of 1×105 cells/mL for 24 h. Cells treated with SPMNPs were incubated at 37 °C before use. The cells were harvested by trypsinization and stained with TrypanBlue dye. We count cells by using a hemocytometer. The cell viability (%) was calculated by the equation: {[Acount]tested / [Acount]control} × 100 5. MTT assay The growth, spent media removal, media replenishment, PEG-capped SPMNPs treatment, incubation time and PBS wash for L929, HeLa and MCF7 cells (2×105 cells/mL) were the same as described previously.27 The relative cell viability (%) was calculated by the equation: {[Aabsorbance] tested / [Aabsorbance] control} × 100. 6. Confocal microscopy


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We transferred MCF7 cells to slide petri dishes. Various concentrations of PEG-capped SPMNPs were added (0, 0.1, 0.2, 0.4, 0.8 and 1 mg/mL).[19]

7. Hemolysis assay We used fresh human blood for studying the hemolytic activity, and the conditions were the same as the previous paper.36

8. In vitro hyperthermia on cancer cells MCF7 cells were mixed with the PEG-capped SPMNPs (1 mg/mL) to evaluate their in vitro hyperthermia performance. During the procedure, AC magnetic fields remained switched on until the temperature reached to 43-44 °C. All in vitro hyperthermia studies were performed (265 kHz) and the AMF was kept at 300 Oe. The media containing PEG-capped SPMNPs suspended with cancer cells (2×105 cells/mL) was put in the coil. The samples were heated for different time periods at 43-44 °C. The cell viability was measured using MTT assay.

9. Reactive oxygen species (ROS) assay 5-(and-6)-chloromethyl-2,7-dichloro-dihydrofluorescein diacetateacetyl ester (H2 DCFDA) assay was used to determine the ROS assay.[42]

10. MRI study The T2 relaxation times of bare and PEG-capped LSMO MNPs samples were measured (General Electric Healthcare, USA). T2-weighted images were obtained according to SE protocol with the following parameters: TR (repetition time) = 5000 ms; (echo time) = 30, 40, 60, 80, 100, 150 and 200 ms.

11. In vitro MRI of cancer cells In vitro MR imaging of cancer cells was performed according to the literature.47

Acknowledgement The author (K. W.) thanks the funding supports from the Ministry of Science and Technology (MOST) of Taiwan (104-2628-E-002-008-MY3), National Taiwan University (102R104100) and National Health Research Institute (NHRI) of Taiwan (03A1-BNMP14-014). T.A., S.M.A., and Y.Y. extend their appreciation to the

Deanship of Scientific Research at King Saud University for funding this work through International Research Group (IRG–14–40).



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Supporting Information The magnetic properties and the SAR values of the synthesized bare and PEG-capped LMSO were supplied as Supporting Information.

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Multimodal Superparamagnetic Nanoparticles with Unusually

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