Effect of core–shell nanoparticle geometry on the enhancement of the proton relaxivity value in a nuclear magnetic resonance experiment

Abstract

This work illustrates the effect of core–shell nanoparticle geometry on the enhancement of the proton relaxivity value in a nuclear magnetic resonance experiment. Chemically synthesized CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles were chosen as a candidate material. A two step methodology was used to synthesize the core–shell nanoparticles. In the first step, CoFe₂O₄ seed nanoparticles were synthesized and in the second step a MnFe₂O₄ phase was grown over seed CoFe₂O₄ nanoparticles to form the core–shell geometry. Characterization of the as-synthesized nanoparticles by diffraction methods, electron microscopy and X-ray photoelectron spectroscopy confirmed the formation of uniform core–shell nanoparticles. Magnetic measurement revealed the superparamagnetic nature of the as-synthesized core–shell nanoparticles. The transverse proton relaxivity values obtained by the nuclear magnetic resonance experiment conducted at room temperature using a field of 9.4 T in the presence of single phase CoFe₂O₄, MnFe₂O₄ and CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles were 60.9 mM⁻¹ s⁻¹, 83.2 mM⁻¹ s⁻¹ and 194.8 mM⁻¹ s⁻¹ respectively. This result clearly illustrated that a greater magnetic inhomogeneity induced in the medium surrounding the core–shell nanoparticles containing two different magnetic phases yields the highest value for the transverse proton relaxivity.

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1 Introduction

Multi-phase nano-solids with core–shell geometry have attracted considerable attention due to their potential application in various fields such as catalysis, bio-technology, energy storage, optical devices etc.^1–3 Three microstructural components of the core–shell geometry are (a) phase forming the core, (b) phase forming the shell and (c) the core–shell interface. Alteration of the volume fraction, size and shape of the core and the shell phases and the width, structure, and composition of the core–shell interface can be used to engineer the properties and, therefore, the functionalities of the core–shell nanoparticles.^4–6 Among the core–shell nanoparticles, magnetic core–shell nanoparticles containing hard and soft magnetic phases have received attention due to their potential application in various biomedical fields like molecular imaging, drug delivery, and cancer therapy.^7,8 In the particular case of cancer therapy, the exchange coupling between the hard and soft magnetic phases in the core–shell nanoparticles provides two important functionalities. They are: (a) intercellular hyperthermia which involves destruction of the cancer cells due to the production of heat when the core–shell nanoparticles attached to the malignant tissue are subjected to an AC magnetic field⁹ and (b) effective imaging of the malignant tissue by magnetic resonance imaging (MRI) in which the core–shell geometry of the nanoparticle induces high magnetic inhomogeneity in the surrounding medium.^7,10,11 Apart from magnetic core–shell nanoparticles there are reports in which the synergistic effect of core–shell geometry to enhance photo catalytic activity, sensing and antimicrobial activity is reported.^12–16

In this paper, we demonstrate the potential of magnetically hard–soft, core–shell nanoparticle system for application as contrast agent in MRI. CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles were used as candidate material. It is illustrated that the difference in the magnetocrystalline anisotropy between the two magnetic phases in the CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles leads to a greater magnetic inhomogeneities in the liquid medium in the vicinity of these core–shell nanoparticles when compared to the extent of magnetic inhomogeneity induced by single phase nanoparticles of the core and shell phases.^6,10 High magnetic inhomogeneity increased the transverse relaxivity value in the NMR experiment.

2 Experiment: synthesis of core–shell nanoparticles

2.1 First step: synthesis of seed CoFe₂O₄ nanoparticle

To synthesize CoFe₂O₄ seed nanoparticles, Co(acac)₂ (1 mmol), Fe(acac)₃ (2 mmol), 1,2-hexadecanediol (5 mmol), oleic acid (6 mmol), and oleylamine (6 mmol) and diphenyl ether (20 mL) were mixed. This solution was poured in a three neck round bottom flask. The synthesis reaction was conducted in argon atmosphere. The reaction mixture was initially heated to 150 °C for 30 min. The temperature of the reaction mixture was then raised to its reflux temperature (∼260 °C) and kept there for 30 min. After 30 min, the reaction mixture containing precipitated nanoparticles was allowed to cool down to the room temperature. At room temperature, 40 mL of ethanol was added into the reaction mixture to sediment the nanoparticles which were subsequently isolated by centrifugation.

2.2 Second step: synthesis of CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles

To synthesize CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles, 40 mg of as-synthesized seed CoFe₂O₄ nanoparticles were dispersed in 20 mL of hexane by sonication. Into this solution, 20 mL benzyl ether, Mn(acac)₂ (1 mmol), Fe(acac)₃ (3 mmol), oleic acid (6 mmol), oleylamine (6 mmol) and 1,2-hexadecanediol (5 mmol) were added. This reaction mixture was poured into a three neck round bottom flask. The synthesis reaction was carried out under argon atmosphere. The reaction mixture was initially heated at 100 °C for 60 min, to evaporate away the hexane. The temperature of the reaction mixture was then increased to 200 °C and kept at this temperature for 60 min. After 60 min, the temperature of the reaction mixture was finally raised to 290 °C and maintained at this temperature for another 60 min. After this the reaction mixture containing precipitated nanoparticles was cooled to the room temperature. At room temperature, 40 mL of ethanol was added into the reaction mixture to sediment the nanoparticles which were subsequently isolated by centrifugation.

As-synthesized nanoparticles were coated with oleic acid and oleyl amine. To make these nanoparticles water dispersible, a surfactant exchange reaction was conducted in which oleyl amine and oleic acids on the nanoparticle surface were replaced by chitosan. The details of this process have been reported earlier.^11,17

3 Characterization techniques

X-ray diffraction (XRD) profiles were obtained using the X-Pert PAN Analytical machine using Cu K-alpha radiation source. Energy dispersive spectroscopy (EDS) was used to determine the elemental composition of the nanoparticles using the FEI ESEM Quanta scanning electron microscope (SEM) operating at 20 kV. A 300 keV field emission FEI Tecnai F-30 transmission electron microscope (TEM) was used for obtaining TEM bright field images and selected area electron diffraction (SAD) patterns from as-synthesised nanoparticles. Samples for the TEM-based analysis were prepared by drop drying a dilute dispersion of the as-synthesised nanoparticles onto an electron transparent carbon-coated Cu grid. Magnetic measurement data was obtained by using the Lakeshore vibrating sample magnetometer (VSM). The concentration of iron in dispersions used in the NMR experiment was determined by atomic absorption spectroscopy (AAS) technique conducted using the Thermo Electron Corporation M-series machine. X-ray photoelectron spectroscopy (XPS) profiles were obtained from the as-synthesized samples using an AXIS Ultra DLD (KRATOS ANALYTICAL) instrument. Zeta potential and hydrodynamic radius was measured using Zetasizer Nano-ZS 90 MALVERN instrument. Transverse relaxivity of water protons (prepared by mixing 99% D₂O and 1% H₂O) in the presence of as-synthesized composites was measured using Bruker Avance-III spectrometer operating at 400 MHz ¹H resonance frequency. Transverse relaxation of water was measured using CPMG/T₂-filter (Carr Purucell Meiboom Gill) NMR experiment. The relaxation delay time ‘τ’ was varied between 10 ms and 1 s collecting 12 data points to get decay curve to extract T₂ relaxation time constant. The 16k complex points were collected with 1.1 s acquisition time and 7000 Hz spectral width. The relaxation delay of 15 s was given between the scans. Only one scan was used to acquire the data to avoid radiation damping.

4 Results and discussion

Elemental analysis of nanoparticles synthesized in the first and second step of the synthesis process was conducted using the SEM-EDS technique. Peaks corresponding to the Fe, Co and O were observed in the EDS profile obtained from the seed nanoparticles synthesized in the first step. Peaks corresponding to Mn, Fe, Co and O were observed in the EDS profile obtained from the core–shell nanoparticles synthesized in the second step of the synthesis process. Representative EDS profiles are provided in Fig. 1.

Fig. 1 Representative SEM-EDS profile obtained from (a) CoFe₂O₄ seeds and (b) CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles.

XRD profiles obtained from the seed and core–shell nanoparticles are shown in Fig. 2. XRD profiles for both cases show ferrite phase peaks only.^17,18 Average crystallite size for the nanoparticles were determined from the FWHM (full width at half maximum) of the (311) peak and the Scherrer formula.¹⁹ Calculated average crystallite size values were 8 nm for the seed nanoparticles and 12.5 nm for the core–shell nanoparticles.

Fig. 2 XRD profiles obtained from CoFe₂O₄ seeds and CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles.

TEM bright field image and SAD pattern obtained from seed and core–shell nanoparticles are shown in Fig. 3(a) and (b) respectively. Both SAD patterns contained diffraction rings corresponding only to the ferrite phase.²⁰ TEM micrographs clearly revealed the presence of a nearly monodisperse nanoparticle population for both cases and an increase in size of the nanoparticles after the second step of the synthesis process. Both these points are illustrated in the nanoparticle size distribution histogram provided in Fig. 3(c) and (d). The size distribution histograms were obtained from the sizes of at least 500 individual nanoparticles. The sizes of the majority of nanoparticles for both seed and core–shell nanoparticles as observed in the histograms is in agreement with the crystallite size that was obtained using the Scherrer analysis. Size distribution histogram reveal the absence of nanoparticles with sizes less than ∼7 nm in the particle dispersion obtained from the second step of the synthesis process. This confirmed the coating of the seed nanoparticles to form the core–shell nanoparticles. The molar ratio between core and shell phases in the core–shell nanoparticles as calculated using the average sizes of seed and core–shell nanoparticles was 1 : 3.5.

Fig. 3 TEM bright field image and SAD pattern obtained from (a) CoFe₂O₄ seeds and (b) CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles. Size distribution histogram for (c) CoFe₂O₄ seeds (d) CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles.

Confirmation of the core–shell geometry was done using the XPS based analysis of the as-synthesized nanoparticles. XPS spectra of Co 2p, Fe 2p and Mn 2p obtained from seed and core–shell nanoparticles are provided in Fig. 4(a)–(c). Whole XPS spectrum obtained from the seed and the core–shell nanoparticles in also provided in Fig. 4(d). In Fig. 4(a), the XPS spectrum obtained from the CoFe₂O₄ seed nanoparticles contains two peaks at 775.7 and 794 eV corresponding²¹ to the Co²⁺ whereas the core–shell nanoparticles do not show any peaks corresponding to Co²⁺. This confirmed the absence of cobalt on the surface of the core–shell nanoparticles. The Fe 2p spectrums in Fig. 4(b) reveal peaks at ∼710 and ∼723.3 eV corresponding²² to Fe³⁺ for both seed and core of the nanoparticles. Mn 2p spectrum (Fig. 4(c)) obtained from the core–shell nanoparticles reveal peaks at 641 and 653 eV corresponding to Mn 2p_3/2 and Mn 2p_1/2 indicating²³ the presence of Mn²⁺ on the particle surface. The XPS results therefore also revealed the formation of CoFe₂O₄–MnFe₂O₄ core–shell geometry nanoparticles.

Fig. 4 XPS spectrums showing (a) Co, (b) Fe, (c) Mn peaks and (d) full spectrum of CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles.

Magnetic hysteresis curves obtained from CoFe₂O₄ seed and CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles using an applied field of 2 tesla are shown in Fig. 5(a). Both the magnetic hysteresis curves showed negligible coercivity and no magnetic saturation. Both these attributes indicated that the as-synthesized nanoparticles were superparamagnetic in nature.¹⁸ The saturation magnetization value obtained at 2 tesla field for seed and core–shell nanoparticles was 55.4 emu g⁻¹ and 72.7 emu g⁻¹ respectively. ZFC (zero field cooled) measurements were conducted on seed and core–shell nanoparticles. ZFC curves are shown in Fig. 5(b). CoFe₂O₄ seed nanoparticles exhibited a broad peak in the ZFC curve. Whereas the ZFC curve of the CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles exhibited a broad hump at higher value of temperature. Shift of the ZFC curve peak to higher temperature values was essentially due to an increase in the nanoparticle size. The narrow distribution of nanoparticles sizes and high broadness of the ZFC curve in case of MnFe₂O₄–CoFe₂O₄ core–shell nanoparticles indicated towards an interaction between magnetic spins at the MnFe₂O₄ and CoFe₂O₄ interface in the core–shell nanoparticles.²⁴

Fig. 5 (a) M–H curves and (b) zero-field cooled curves obtained from CoFe₂O₄ seeds and CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles.

Transverse relaxivity of water protons was determined in the presence of different concentrations of chitosan-coated CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles. The hydrodynamic diameter of the core–shell nanoparticles measured by dynamic light scattering method was 400 nm. This revealed a chitosan coating thickness of ∼200 nm over the core–shell nanoparticles. The chitosan coated core–shell nanoparticles formed stable dispersion in water. The zeta potential of the chitosan coated core nanoparticles was 40 mV. Fitting of the exponentially decaying spin echo data was done to obtain the T₂ of water protons at different concentrations of iron. 1/T₂ vs. concentration of iron plots shown in Fig. 6(a) and (b) were used to derive the transverse relaxivity value form the slope of the line fitted to the data points. The transverse relaxivity values obtained were 194.8 mM⁻¹ s⁻¹ and 60.9 mM⁻¹ s⁻¹ respectively for CoFe₂O₄ seed and CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles. In an earlier work¹¹ the authors have illustrated that the transverse relaxivity value (measure using similar NMR parameters) obtained from 9 nm MnFe₂O₄ nanoparticle showing a saturation magnetization value of 61.5 emu g⁻¹ was 83.2 mM⁻¹ s⁻¹ saturation magnetization values and the transverse proton relaxivity values for CoFe₂O₄, MnFe₂O₄ and CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles are listed in Table 1. Table 1 clearly illustrates that the relaxivity value for the CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles is significantly higher that the transverse relaxivity value exhibited by the single phase CoFe₂O₄ and MnFe₂O₄ nanoparticles with similar values for saturation magnetization and size. This clearly illustrates that the difference in the magnetocrystalline anisotropy of the phases in the core–shell nanoparticles creates greater magnetic inhomogeneity which considerably enhances the proton relaxivity value.

Fig. 6 (a) 1/T₂ vs. Fe concentration curve for 8 nm CoFe₂O₄ and (b) 1/T₂ vs. Fe concentration curve for CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles.

Table 1 List of average particle size, saturation magnetization and transverse relaxivity values

Sample	Average size (nm)	Saturation magnetization (emu g^−1)	Transverse relaxivity (mM^−1 s^−1)
MnFe2O4	9	61.5	83.2
CoFe2O4	8	55.54	60.9
CoFe2O4–MnFe2O4 core–shell	12.5	72.73	194.8

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4.1 Cytotoxicity of CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles

MTT assay was performed using MCF-7 (human breast cancer cells) to determine the cytotoxicity of CoFe₂O₄–MnFe₂O₄ core–shell nanoparticles. Iron concentration of the chitosan coated nanoparticles was determined by AAS (Atomic Absorption Spectroscopy). For MTT analysis, the chitosan-coated core–shell nanoparticles were dispersed in water. Results from the cytotoxicity experiment are presented in Fig. 7. The cores–shell nanoparticles with different concentrations (100, 50, 25, 12.5 and 6.25 μg mL⁻¹) showed biocompatibility towards the MCF-7 cell line.

Fig. 7 MTT assay of chitosan coated MnFe₂O₄–CoFe₂O₄ core–shell nanoparticles.

5 Conclusions

This work illustrates that for similar values of saturation magnetization, the proton relaxivity value obtained in the dispersion of core–shell nanoparticles is considerably greater than the proton relaxivity value obtained in the presence of single phase nanoparticles of the core and shell phases. Core–shell nanoparticles created highest magnetic inhomogeneity in the water medium surrounding it resulting in highest value of proton relaxivity. The origin of the greater magnetic inhomogeneity is the difference in the magnetocrystalline anisotropy of the core and shell material in the core–shell nanoparticles.

Acknowledgements

Authors acknowledge the electron microscopy facilities available at the Advanced Centre for Microscopy and Microanalysis (AFMM) IISc Bangalore. The MCF-7 cell line were provided by Professor Paturu Kondaiah. Cell toxicity analysis was done using the facilities available in Professor Paturu Kondaiah's laboratory in IISc, Bangalore. C. Srivastava acknowledges the research grant received from SERB, Govt. of India. The NMR facility at NMR Research Centre, supported by DST, is gratefully acknowledged.

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