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Dextrin-coated zinc substituted cobalt-ferrite nanoparticles as an MRI contrast agent: In vitro and in vivo imaging studies
Colloids and Surfaces B: Biointerfaces 129 (2015) 15–20
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Dextrin-coated zinc substituted cobalt-ferrite nanoparticles as an MRI contrast agent: In vitro and in vivo imaging studies N. Sattarahmady a,b , T. Zare a,b , A.R. Mehdizadeh a,b , N. Azarpira c , M. Heidari a,b , M. Lotfi d , H. Heli b,e,∗ a
Department of Medical Physics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran c Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran d Department of Radiology, Shiraz University of Medical Sciences, Shiraz, Iran e Department of Nanomedicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran b
a r t i c l e
i n f o
Article history: Received 10 November 2014 Received in revised form 3 March 2015 Accepted 6 March 2015 Available online 14 March 2015 Keywords: Superparamagnetic nanoparticles Relaxivity Contrast agent Zinc substituted cobalt ferrite MRI
a b s t r a c t Application of superparamagnetic iron oxide nanoparticles (NPs) as a negative contrast agent in magnetic resonance imaging (MRI) has been of widespread interest. These particles can enhance contrast of images by altering the relaxation times of the water protons. In this study, dextrin-coated zinc substituted cobalt-ferrite (Zn0.5 Co0.5 Fe2 O4 ) NPs were synthesized by a co-precipitation method, and the morphology, size, structure and magnetic properties of the NPs were investigated. These NPs had superparamagnetic behavior with an average size of 3.9 (±0.9, n = 200) nm measured by transmission electron microscopy. Measurements on the relaxivities (r2 and r2∗ ) of the NPs were performed in vitro by agarose phantom. In addition, after subcutaneous injection of the NPs into C540 cell line in C-57 inbred mice, the relaxivities were measured in vivo by a 1.5 T MRI system. These NPs could effectively increase the image contrast in both T2 -and T2∗ -weighted samples.
1. Introduction Today, magnetic resonance imaging (MRI) is widely used as a powerful and noninvasive imaging modality in clinical centers with several advantages, such as excellent spatial resolution [1,2], no radiation exposure [2], and no limitation in tissue depth. However, in comparison with nuclear medicine and fluorescence imaging, the sensitivity of MRI must be improved by enhancement in its signal contrast, when small tissue lesions are scanned or cellular or molecular activities are monitored [3,4]. The contrast of MRI signals depends on the longitudinal/spin-lattice relaxation time (T1 ) and transverse/spin relaxation times (T2 , T2∗ ) of proton. Relaxation times
Abbreviations: CA, contrast agent; ZCFNPs, zinc substituted cobalt ferrite nanoparticles; FA, flip angle; FOV, field of view; FTIR, Fourier transform infrared; MRI, magnetic resonance imaging; NPs, nanoparticles; SEM, scanning electron microscope; SI, signal intensity; SPIONs, superparamagnetic iron oxide nanoparticles; TEM, transmission electron microscope; TR, repetition time; VSM, vibrating sample magnetometer; XRD, X-ray diffraction; FBS, fetal bovine serum. ∗ Corresponding author at: Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Tel.: +98 71 32 34 93 32; fax: +98 71 32 34 93 32. E-mail addresses: [email protected], [email protected] (H. Heli). http://dx.doi.org/10.1016/j.colsurfb.2015.03.021 0927-7765/© 2015 Elsevier B.V. All rights reserved.
© 2015 Elsevier B.V. All rights reserved.
of water proton in pathological tissues are different and depend on the environment [5]. Finding ultrasensitive contrast agents (CAs) for earlier detection of pathology has attracted a lot of attention in recent years [6,7]. CAs reduce the relaxation times and improve the MRI sensitivity [8,9]. Relaxivities (r1 , r2 and r2∗ ) are defined as the T1 , T2 and T2∗ shortening by an efficient contrast agent and expressed as: 1 1 = + ri C Tim Ti
(1)
where the subscript i = 1 or 2, and 1/Tim and 1/Ti are the observed relaxation rates in the presence and absence of CA, respectively. Also, r is the relaxivity and C is the CA concentration [10,11]. MRI CAs are divided into two groups of positive and negative. Positive CAs, such as gadolinium chelating ligands, are paramagnetic molecules that decrease T1 of water proton in the adjacent tissues, resulting in a low r2 /r1 ratio and causing positive contrast in MRI [12]. On the other hand, negative CAs produce dark image by shortening in T2 resulted from a higher r2 /r1 ratio [12]. Superparamagnetic iron oxide nanoparticles (SPIONs) act as negative CAs in a concentration-dependent manner. SPIONs have high magnetic moments, interact with the surrounding proton, and enhance both T1 and T2 relaxation rates [12,13]. Therefore, lower concentrations
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of SPIONs provide higher relaxation rates in specific organs, compared to paramagnetic materials [14,15]. SPIONs can be used as CAs in sensitive imaging of cellular and molecular components, reticuloendothelial systems, lymph nodes, and tumors [3,13]. Ferrite NPs (AFe2 O4 , A: Co, Ni, Zn, etc.), similar to SPIONs, are widely used in medicine [16,17], including MRI CAs [18]. Ferrite NPs have intermediate magnetization [19] and produce negative contrast in MRI [17]. The properties of ferrite NPs significantly depend on the synthesis conditions [20,21]. Two different types of zinc substituted/doped cobalt ferrite with formulas of Znx Co1−x Fe2 O4 [22–34] and CoZnx Fe2−x O4 [35] have been synthesized. For the synthesis of the former, the methods of microwave heating [22], solution combustion [23,24], thermal decomposition [25–27], solid-state reaction [28,29], sol–gel [30,31], forced hydrolysis [32], and co-precipitation [33,34] have been reported. On the other hand, CoZnx Fe2−x O4 was synthesized by a combustion technique using Co-, Fe-, and Zn-nitrate salts as precursors [35]. In the present study, zinc substituted cobalt ferrite NPs (Zn0.5 Co0.5 Fe2 O4 , ZCFNPs) with a biocompatible coating were synthesized by the aqueous co-precipitation method. ZCFNPs were characterized by different physical and chemical methods, and relaxivities r2 and r2∗ were measured with a clinical 1.5 T MRI scanner in vitro. Mice were scanned before and after the injection of the nanoparticles into their melanoma tumors, and the efficiency of dextrin-coated ZCFNPs was also evaluated in vivo.
adhesive tape, followed by gold vapor sputtering. The crystal structure of dextrin-coated ZCFNPs was determined using a Philips X’Pert diffractometer (the Netherlands) equipped with Cu/K␣ radiation source ( = 0.1540 nm) at a scanning rate of 1 ◦ min−1 in a 2 range of 10–90◦ . FTIR spectra were acquired by a Burker Tensor 27 (Germany) spectrometer. The samples were pelletized with KBr powder before measurement. VSM was performed by Meghnatis Daghigh Kavir Co. instrument (Iran) at room temperature. The external magnetic field varied from −20 to 20 kOe. 2.4. MRI study using a phantom
2. Materials and methods
Agarose phantom was used to measure the MRI relaxivities. For this purpose, different concentrations of dextrin-coated ZCFNPs were suspended in agarose gel (1%, w/v) in a 96-well plate ELISA. The MRI relaxometry was performed using a 1.5 T Magnetom Avanto, Siemens (Germany) MRI instrument equipped with a knee coil with Turbo Spin-Echo and FLASH pulse sequence, to obtain r2 and r2∗ . For T2 determination, eight different times of echo (TE) from 22 to 98 ms were used, with time of repetition (TR) = 1800 ms. For T2∗ determination, eight different TEs were used ranging from 11 to 39 ms, with TR = 350 ms. Other scan parameters were set as follows: field of view (FOV) = 160 × 160 mm2 , slice thickness = 3 mm, matrix = 320 × 256 and flip angle (FA) = 30◦ . The values of r2 and r2∗ were calculated by the curve fitting of plots of 1/T2 and 1/T2∗ (in s−1 ) versus the sum of the concentration (in mmol L−1 ) of cobalt and iron species in the dextrin-coated ZCFNPs.
2.1. Chemicals
2.5. MRI study using tumor-bearing mice
All chemicals were purchased form Merck (Germany) or Scharlau (Spain) and used without further purification. Dextrin was obtained by roasting pure corn starch at 200 ◦ C for 2 h. The employed drugs were purchased from a local drugstore. Redistilled water was used throughout the study.
Six to eight week old female inbred/C57 mice (n = 1, body weight of ∼20 g) were obtained from center of comparative and experimental medicine (Shiraz, Iran). The animals were housed in special cages at a controlled temperature (24 ± 2 ◦ C) and humidity (40–70%) with weekly floor exchange. They had free access to water and standard pelleted laboratory animal diets. A 12:12 light:dark cycle was followed in the mentioned Animal House Center. The mice received care in compliance with the standard ethical guidelines approved by the Ethics Committee of Shiraz University of Medical Sciences (approval no. 93-01-57-8884). The tumors were induced by subcutaneous injection of C540 cell line (1 × 106 cells in 100 L 10% fetal bovine serum, FBS). During implantation, the mice were anesthetized with the mixture of ketamin (10%) and xylazine (2%) by intramuscular injection. Two weeks after the tumor was established, the mice were anaesthetized with the mixture of ketamin (10%) and xylazine (2%) by intramuscular injection. The animals were imaged using a 1.5 T Magnetom Avanto, Siemens (Germany) MRI instrument before and after intratumoral injection of dextrin-coated ZCFNPs. T2 - and T2∗ -weighted images were acquired using a Turbo Spin-Echo pulse sequence and FLASH pulse sequence, respectively. Scan parameters were FOV = 103 × 210 mm2 , slice thickness = 5 mm, TE = 80 ms, TR = 2600 ms, matrix = 320 × 179, and FA = 150◦ for T2 -weighted. Scan parameters of T2∗ -weighted images were FOV = 98 × 210 mm2 , slice thickness = 5 mm, TE = 10 ms, TR = 135 ms, matrix = 256 × 132, and FA = 90◦ .
2.2. Synthesis of dextrin-coated ZCFNPs Dextrin-coated ZCFNPs were synthesized by an aqueous co-precipitation route. The individual metal chlorides in an appropriate stoichiometric proportions were dissolved in a dilute hydrochloric acid (0.1 mol L−1 HCl) solution, and heated to 80 ◦ C. A mixed solution of 4 mol L−1 solution of sodium hydroxide and 12.5 g dextrin was prepared separately and heated to 80 ◦ C. These hot solutions were then rapidly mixed with magnetic stirring (final pH 12.0). The temperature was then increased to 100 ◦ C and stirring was continued for 1 h. This time is needed for crystallization of the ferrite samples. After that time, the solution was cooled to room temperature, the precipitate was collected by a permanent magnet and washed several times with water to neutralize the supernatant. The ferrite samples were dried at room temperature. 2.3. Characterizations of dextrin-coated ZCFNPs The size, morphology and structure of the dextrin-coated ZCFNPs were measured using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The magnetic properties of ZCFNPs were also measured by vibrating sample magnetometer (VSM). TEM was performed by a Zeiss, EM10C (Germany) transmission electron microscope at an accelerating voltage of 80 kV. For the sample preparation, a drop of the diluted and horn-ultrasonicated sample in water was dropped on a copper grid followed by air drying. SEM was done using a Philips X-30 instrument (Germany). For sample preparation, a small portion of dried sample was placed on a piece of silver
3. Results 3.1. Characterization of dextrin-coated ZCFNPs Fig. 1A shows SEM images of dextrin-coated ZCFNPs. The NPs are agglomerated due to their magnetic property because the images were recorded from a dried sample. From the SEM images, the mean diameter of individual agglomerated particles was 37.8 (±5.6, n = 200) nm. Fig. 1B represents TEM images of the dextrin-coated
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Fig. 1. (A) SEM images of dextrin-coated ZCFNPs with different magnifications. (B) TEM images of the dextrin-coated ZCFNPs with different magnifications. (C) An XRD pattern of ZCFNPs. (D) A FTIR spectrum of dextrin-coated ZCFNPs.
ZCFNPs with different magnifications. TEM images clearly show that the synthesized ZCFNPs are uniform spheres with a narrow distribution size and a mean diameter of 3.9 (±0.9, n = 200) nm. This ultra-small sized particles are interested in application of ZCFNPs in biological studies and nanomedicine. An XRD pattern of ZCFNPs is presented in Fig. 1C. In the diffractogram, the peaks appeared at 2 values of 17.9, 29.8, 35.3, 42.8, 57.3 and 62.5◦ , which are compatible with the cobalt ferrite (JCPDs card number 22-1012). These are assigned to (1 1 1), (2 2 0), (3 1 1), (4 0 0), (5 1 1) and (4 4 0) diffractions. In addition, the mean crystalline size of ZCFNPs was obtained as 1.1 (±0.15, n = 6) nm, based on the full width at half maximum of diffraction peaks using the Scherrer equation. This indicates a very fine crystal size and that the ZCFNPs comprised several single crystals. A FTIR spectrum of dextrin-coated ZCFNPs is shown in Fig. 1D. In the spectrum, a strong band at around 3420 cm−1 was related to O H stretching vibration. The peak at 2924 cm−1 was characterized as the symmetric vibration of CH2 group. The peak at around 1045 cm−1 was related to the CO vibration of the alcohol functional group. These results indicated that the surface of ZCFNPs
was covered by a dextrin layer. In addition, a sharp absorption band at 576 cm−1 was associated to the stretching vibration mode of Fe O. The stability of dextrin-coated ZCFNPs was also investigated by recording FTIR spectra. It was found that coating of the nanoparticles was stable for at least four months. Fig. 2 represents the magnetization curves of ZCFNPs and dextrin-coated ZCFNPs. The patterns of the hysteresis loops confirm the superparamagnetic behavior of the samples. The magnetic moments per formula unit in Bohr magneton for ZCFNPs and dextrin-coated ZCFNPs were calculated from saturation magnetization of the hysteresis loops using the following equation [36]: nB =
Mw Ms 5585
(2)
where nB is the magnetic moment, Mw is the molecular weight, Ms is saturation magnetization and the coefficient of 5585 is the magnetic factor. The magnetic properties of ZCFNPs and dextrincoated ZCFNPs were obtained from hysteresis data and reported in the Supplementary material S1. The relatively small value of
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Based on the slopes of the plots (8.5 (±0.5, n = 6) mmol L−1 s−1 and 79 (±3.8, n = 6) mmol L−1 s−1 as r2 and r2∗ , respectively), the relaxivities of dextrin-coated ZCFNPs are comparable or higher than those reported previously. A comparison of the relaxivities of some different NPs was made in the Supplementary material S2. 3.3. MRI using tumor-bearing mice In vivo contrast enhancement of dextrin-coated ZCFNPs was evaluated via T2 - and T2∗ -weighted MRI. Fig. 4 represents the T2 - and T2∗ -weighted images before and after ZCFNPs injection into induced tumors in the mice. ZCFNPs induced negative contrast enhancement in the images of tumor area due to T2 and T2∗ relaxation times shortening. Before and after intratumoral injection of ZCFNPs, the average values of SIs of the images of the tumor volume were calculated from T2 - and T2∗ -weighted images, and are shown in the Supplementary material S3. Using the plots, decrements in SIs (%) after NPs injection in the tumor area were calculated based on the following equation [40]: Fig. 2. Magnetization curves of ZCFNPs and dextrin-coated ZCFNPs.
magnetization of dextrin-coated ZCFNPs was related to the dextrin surface coating, and also to the ultra-small size of the NPs [37–39]. 3.2. MRI using a phantom A 1.5 T clinical MRI was used to understand the relaxivity of dextrin-coated ZCFNPs as the contrast agent using agar phantom. Fig. 3A shows T2 -(spin-echo pulse sequence, TR = 1800 ms, TE = 88 ms) and T2∗ -(FLASH pulse sequence, TR = 350 ms, TE = 15 ms) weighted images of dextrin-coated ZCFNPs. Based on the images, the signal intensity (SI) of T2 - and T2∗ -weighted images decreased, upon increase in the ZCFNPs concentration. This is due to the T2 and T2∗ shortening. In Fig. 3B, the dependencies of the relaxation rates (R2 and R2∗ ) on the Fe + Co concentration in ZCFNPs are shown. The relaxation rates are increased linearly with the concentration.
Decrements in SIs (%) = −100
SIpre − SIpost SIpre
(3)
where SIpre and SIpost are the SIs before and after ZCFNPs injection. The results indicated that the MRI signal intensities for the tumor area after the ZCFNPs injection decreased by 12.5% (±0.6, n = 3) and 66.5% (±0.5, n = 3) for T2 - and T2∗ -weighted images, respectively. Although both the decrements in SIs are interested, the decrement in SI for T2∗ -weighted image is more favorable. The results suggest that dextrin-coated ZCFNPs can be considered as a proper contrast enhancement agent in MRI. 4. Discussion Today, magnetic NPs have great applications in biology and medicine, such as biomolecular separation [41], drug delivery [42] and MRI [43]. For these purposes, ultra-small sized NPs are highly desirable. However, NPs should be coated with a biocompatible
Fig. 3. (A) T2 - and T2∗ -weighted images of dextrin-coated ZCFNPs at different Fe + Co concentrations. (B) The dependencies of the relaxation rates (R2 and R2∗ ) on the Fe + Co concentration in ZCFNPs. The error amounts for data points as a percentage of the values of that data points (95% confidence interval error bars) are shown.
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Fig. 4. T2 - and T2∗ -weighted images before (left) and after (right) ZCFNPs injection into induced tumors in mice.
and non-toxic material to be used in vivo. In addition, particle size plays the major role for their times of blood circulation and cell internalization [44,45]. Recently, different magnetic NPs were synthesized and coated with different polymers [46], surfactants [47] and proteins [48]. In the present study, ZCFNPs were synthesized and coated by a dextrin layer. Dextrin is a polymer of ␣-(1 → 4) d-glucose that is produced by partial hydrolysis of starch. Dextrin was used in food, medicine and industry, because of its biocompatible and biodegradable characteristics [49,50]. FTIR studies showed that the surface of ZCFNPs is coated with a hydrophilic dextrin layer that is significant for the stability of ZCFNPs in human plasma. It also has an impact on interactions of ZCFNPs with biomolecules and cells. Dextrin bears hydroxyl functional groups and its interaction with proteins via hydrogen bonds can be predicted. Also, dextrin can shield the electrostatic interactions between ZCFNPs [51]. Although the presence of dextrin as a non-magnetic material on the ZCFNPs surface reduces the magnetization property of the dextrin-coated ZCFNPs, they retain their utility as a MRI contrast agent. In vitro, the value of r2∗ was higher than r2 , confirming the effectiveness of dextrin-coated ZCFNPs to create a huge contrast in the T2∗ -weighted images. In vivo, these NPs induced a negative contrast in T2 - and T2∗ -weighted images. In addition, calculation of the decrement in SIs in the tumor area indicates that the NPs have grater impact on T2∗ -weighted images.
5. Conclusion In summary, monodispersed dextrin-coated ZCFNPs were synthesized by a co-precipitation method. The size of NPs was 3.9 (±0.9, n = 200) nm with a narrow size distribution and a spherical morphology. FTIR studies confirmed the presence of dextrin on the surface of NPs. The magnetic property, and efficiency of dextrincoated ZCFNPs in MRI were evaluated both in vitro and in vivo. The NPs showed superparamagnetic behavior with contrast enhancement in T2∗ -weighted images. The dextrin-coated ZCFNPs have a great potential in MRI, and the present synthesis method and evaluation approaches of the NPs are extendable as a new route to other magnetic nanoparticles. Acknowledgments We would like to thank the Research Council of Shiraz University of Medical Sciences (8884), and the Iran National Science Foundation (INSF) for supporting this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2015.03.021.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Dextrin-coated zinc substituted cobalt-ferrite nanoparticles as an MRI contrast agent: In vitro and in vivo imaging studies N. Sattarahmady a,b , T. Zare a,b , A.R. Mehdizadeh a,b , N. Azarpira c , M. Heidari a,b , M. Lotfi d , H. Heli b,e,∗ a
Department of Medical Physics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran c Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran d Department of Radiology, Shiraz University of Medical Sciences, Shiraz, Iran e Department of Nanomedicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran b
a r t i c l e
i n f o
Article history: Received 10 November 2014 Received in revised form 3 March 2015 Accepted 6 March 2015 Available online 14 March 2015 Keywords: Superparamagnetic nanoparticles Relaxivity Contrast agent Zinc substituted cobalt ferrite MRI
a b s t r a c t Application of superparamagnetic iron oxide nanoparticles (NPs) as a negative contrast agent in magnetic resonance imaging (MRI) has been of widespread interest. These particles can enhance contrast of images by altering the relaxation times of the water protons. In this study, dextrin-coated zinc substituted cobalt-ferrite (Zn0.5 Co0.5 Fe2 O4 ) NPs were synthesized by a co-precipitation method, and the morphology, size, structure and magnetic properties of the NPs were investigated. These NPs had superparamagnetic behavior with an average size of 3.9 (±0.9, n = 200) nm measured by transmission electron microscopy. Measurements on the relaxivities (r2 and r2∗ ) of the NPs were performed in vitro by agarose phantom. In addition, after subcutaneous injection of the NPs into C540 cell line in C-57 inbred mice, the relaxivities were measured in vivo by a 1.5 T MRI system. These NPs could effectively increase the image contrast in both T2 -and T2∗ -weighted samples.
1. Introduction Today, magnetic resonance imaging (MRI) is widely used as a powerful and noninvasive imaging modality in clinical centers with several advantages, such as excellent spatial resolution [1,2], no radiation exposure [2], and no limitation in tissue depth. However, in comparison with nuclear medicine and fluorescence imaging, the sensitivity of MRI must be improved by enhancement in its signal contrast, when small tissue lesions are scanned or cellular or molecular activities are monitored [3,4]. The contrast of MRI signals depends on the longitudinal/spin-lattice relaxation time (T1 ) and transverse/spin relaxation times (T2 , T2∗ ) of proton. Relaxation times
Abbreviations: CA, contrast agent; ZCFNPs, zinc substituted cobalt ferrite nanoparticles; FA, flip angle; FOV, field of view; FTIR, Fourier transform infrared; MRI, magnetic resonance imaging; NPs, nanoparticles; SEM, scanning electron microscope; SI, signal intensity; SPIONs, superparamagnetic iron oxide nanoparticles; TEM, transmission electron microscope; TR, repetition time; VSM, vibrating sample magnetometer; XRD, X-ray diffraction; FBS, fetal bovine serum. ∗ Corresponding author at: Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran. Tel.: +98 71 32 34 93 32; fax: +98 71 32 34 93 32. E-mail addresses: [email protected], [email protected] (H. Heli). http://dx.doi.org/10.1016/j.colsurfb.2015.03.021 0927-7765/© 2015 Elsevier B.V. All rights reserved.
© 2015 Elsevier B.V. All rights reserved.
of water proton in pathological tissues are different and depend on the environment [5]. Finding ultrasensitive contrast agents (CAs) for earlier detection of pathology has attracted a lot of attention in recent years [6,7]. CAs reduce the relaxation times and improve the MRI sensitivity [8,9]. Relaxivities (r1 , r2 and r2∗ ) are defined as the T1 , T2 and T2∗ shortening by an efficient contrast agent and expressed as: 1 1 = + ri C Tim Ti
(1)
where the subscript i = 1 or 2, and 1/Tim and 1/Ti are the observed relaxation rates in the presence and absence of CA, respectively. Also, r is the relaxivity and C is the CA concentration [10,11]. MRI CAs are divided into two groups of positive and negative. Positive CAs, such as gadolinium chelating ligands, are paramagnetic molecules that decrease T1 of water proton in the adjacent tissues, resulting in a low r2 /r1 ratio and causing positive contrast in MRI [12]. On the other hand, negative CAs produce dark image by shortening in T2 resulted from a higher r2 /r1 ratio [12]. Superparamagnetic iron oxide nanoparticles (SPIONs) act as negative CAs in a concentration-dependent manner. SPIONs have high magnetic moments, interact with the surrounding proton, and enhance both T1 and T2 relaxation rates [12,13]. Therefore, lower concentrations
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of SPIONs provide higher relaxation rates in specific organs, compared to paramagnetic materials [14,15]. SPIONs can be used as CAs in sensitive imaging of cellular and molecular components, reticuloendothelial systems, lymph nodes, and tumors [3,13]. Ferrite NPs (AFe2 O4 , A: Co, Ni, Zn, etc.), similar to SPIONs, are widely used in medicine [16,17], including MRI CAs [18]. Ferrite NPs have intermediate magnetization [19] and produce negative contrast in MRI [17]. The properties of ferrite NPs significantly depend on the synthesis conditions [20,21]. Two different types of zinc substituted/doped cobalt ferrite with formulas of Znx Co1−x Fe2 O4 [22–34] and CoZnx Fe2−x O4 [35] have been synthesized. For the synthesis of the former, the methods of microwave heating [22], solution combustion [23,24], thermal decomposition [25–27], solid-state reaction [28,29], sol–gel [30,31], forced hydrolysis [32], and co-precipitation [33,34] have been reported. On the other hand, CoZnx Fe2−x O4 was synthesized by a combustion technique using Co-, Fe-, and Zn-nitrate salts as precursors [35]. In the present study, zinc substituted cobalt ferrite NPs (Zn0.5 Co0.5 Fe2 O4 , ZCFNPs) with a biocompatible coating were synthesized by the aqueous co-precipitation method. ZCFNPs were characterized by different physical and chemical methods, and relaxivities r2 and r2∗ were measured with a clinical 1.5 T MRI scanner in vitro. Mice were scanned before and after the injection of the nanoparticles into their melanoma tumors, and the efficiency of dextrin-coated ZCFNPs was also evaluated in vivo.
adhesive tape, followed by gold vapor sputtering. The crystal structure of dextrin-coated ZCFNPs was determined using a Philips X’Pert diffractometer (the Netherlands) equipped with Cu/K␣ radiation source ( = 0.1540 nm) at a scanning rate of 1 ◦ min−1 in a 2 range of 10–90◦ . FTIR spectra were acquired by a Burker Tensor 27 (Germany) spectrometer. The samples were pelletized with KBr powder before measurement. VSM was performed by Meghnatis Daghigh Kavir Co. instrument (Iran) at room temperature. The external magnetic field varied from −20 to 20 kOe. 2.4. MRI study using a phantom
2. Materials and methods
Agarose phantom was used to measure the MRI relaxivities. For this purpose, different concentrations of dextrin-coated ZCFNPs were suspended in agarose gel (1%, w/v) in a 96-well plate ELISA. The MRI relaxometry was performed using a 1.5 T Magnetom Avanto, Siemens (Germany) MRI instrument equipped with a knee coil with Turbo Spin-Echo and FLASH pulse sequence, to obtain r2 and r2∗ . For T2 determination, eight different times of echo (TE) from 22 to 98 ms were used, with time of repetition (TR) = 1800 ms. For T2∗ determination, eight different TEs were used ranging from 11 to 39 ms, with TR = 350 ms. Other scan parameters were set as follows: field of view (FOV) = 160 × 160 mm2 , slice thickness = 3 mm, matrix = 320 × 256 and flip angle (FA) = 30◦ . The values of r2 and r2∗ were calculated by the curve fitting of plots of 1/T2 and 1/T2∗ (in s−1 ) versus the sum of the concentration (in mmol L−1 ) of cobalt and iron species in the dextrin-coated ZCFNPs.
2.1. Chemicals
2.5. MRI study using tumor-bearing mice
All chemicals were purchased form Merck (Germany) or Scharlau (Spain) and used without further purification. Dextrin was obtained by roasting pure corn starch at 200 ◦ C for 2 h. The employed drugs were purchased from a local drugstore. Redistilled water was used throughout the study.
Six to eight week old female inbred/C57 mice (n = 1, body weight of ∼20 g) were obtained from center of comparative and experimental medicine (Shiraz, Iran). The animals were housed in special cages at a controlled temperature (24 ± 2 ◦ C) and humidity (40–70%) with weekly floor exchange. They had free access to water and standard pelleted laboratory animal diets. A 12:12 light:dark cycle was followed in the mentioned Animal House Center. The mice received care in compliance with the standard ethical guidelines approved by the Ethics Committee of Shiraz University of Medical Sciences (approval no. 93-01-57-8884). The tumors were induced by subcutaneous injection of C540 cell line (1 × 106 cells in 100 L 10% fetal bovine serum, FBS). During implantation, the mice were anesthetized with the mixture of ketamin (10%) and xylazine (2%) by intramuscular injection. Two weeks after the tumor was established, the mice were anaesthetized with the mixture of ketamin (10%) and xylazine (2%) by intramuscular injection. The animals were imaged using a 1.5 T Magnetom Avanto, Siemens (Germany) MRI instrument before and after intratumoral injection of dextrin-coated ZCFNPs. T2 - and T2∗ -weighted images were acquired using a Turbo Spin-Echo pulse sequence and FLASH pulse sequence, respectively. Scan parameters were FOV = 103 × 210 mm2 , slice thickness = 5 mm, TE = 80 ms, TR = 2600 ms, matrix = 320 × 179, and FA = 150◦ for T2 -weighted. Scan parameters of T2∗ -weighted images were FOV = 98 × 210 mm2 , slice thickness = 5 mm, TE = 10 ms, TR = 135 ms, matrix = 256 × 132, and FA = 90◦ .
2.2. Synthesis of dextrin-coated ZCFNPs Dextrin-coated ZCFNPs were synthesized by an aqueous co-precipitation route. The individual metal chlorides in an appropriate stoichiometric proportions were dissolved in a dilute hydrochloric acid (0.1 mol L−1 HCl) solution, and heated to 80 ◦ C. A mixed solution of 4 mol L−1 solution of sodium hydroxide and 12.5 g dextrin was prepared separately and heated to 80 ◦ C. These hot solutions were then rapidly mixed with magnetic stirring (final pH 12.0). The temperature was then increased to 100 ◦ C and stirring was continued for 1 h. This time is needed for crystallization of the ferrite samples. After that time, the solution was cooled to room temperature, the precipitate was collected by a permanent magnet and washed several times with water to neutralize the supernatant. The ferrite samples were dried at room temperature. 2.3. Characterizations of dextrin-coated ZCFNPs The size, morphology and structure of the dextrin-coated ZCFNPs were measured using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The magnetic properties of ZCFNPs were also measured by vibrating sample magnetometer (VSM). TEM was performed by a Zeiss, EM10C (Germany) transmission electron microscope at an accelerating voltage of 80 kV. For the sample preparation, a drop of the diluted and horn-ultrasonicated sample in water was dropped on a copper grid followed by air drying. SEM was done using a Philips X-30 instrument (Germany). For sample preparation, a small portion of dried sample was placed on a piece of silver
3. Results 3.1. Characterization of dextrin-coated ZCFNPs Fig. 1A shows SEM images of dextrin-coated ZCFNPs. The NPs are agglomerated due to their magnetic property because the images were recorded from a dried sample. From the SEM images, the mean diameter of individual agglomerated particles was 37.8 (±5.6, n = 200) nm. Fig. 1B represents TEM images of the dextrin-coated
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Fig. 1. (A) SEM images of dextrin-coated ZCFNPs with different magnifications. (B) TEM images of the dextrin-coated ZCFNPs with different magnifications. (C) An XRD pattern of ZCFNPs. (D) A FTIR spectrum of dextrin-coated ZCFNPs.
ZCFNPs with different magnifications. TEM images clearly show that the synthesized ZCFNPs are uniform spheres with a narrow distribution size and a mean diameter of 3.9 (±0.9, n = 200) nm. This ultra-small sized particles are interested in application of ZCFNPs in biological studies and nanomedicine. An XRD pattern of ZCFNPs is presented in Fig. 1C. In the diffractogram, the peaks appeared at 2 values of 17.9, 29.8, 35.3, 42.8, 57.3 and 62.5◦ , which are compatible with the cobalt ferrite (JCPDs card number 22-1012). These are assigned to (1 1 1), (2 2 0), (3 1 1), (4 0 0), (5 1 1) and (4 4 0) diffractions. In addition, the mean crystalline size of ZCFNPs was obtained as 1.1 (±0.15, n = 6) nm, based on the full width at half maximum of diffraction peaks using the Scherrer equation. This indicates a very fine crystal size and that the ZCFNPs comprised several single crystals. A FTIR spectrum of dextrin-coated ZCFNPs is shown in Fig. 1D. In the spectrum, a strong band at around 3420 cm−1 was related to O H stretching vibration. The peak at 2924 cm−1 was characterized as the symmetric vibration of CH2 group. The peak at around 1045 cm−1 was related to the CO vibration of the alcohol functional group. These results indicated that the surface of ZCFNPs
was covered by a dextrin layer. In addition, a sharp absorption band at 576 cm−1 was associated to the stretching vibration mode of Fe O. The stability of dextrin-coated ZCFNPs was also investigated by recording FTIR spectra. It was found that coating of the nanoparticles was stable for at least four months. Fig. 2 represents the magnetization curves of ZCFNPs and dextrin-coated ZCFNPs. The patterns of the hysteresis loops confirm the superparamagnetic behavior of the samples. The magnetic moments per formula unit in Bohr magneton for ZCFNPs and dextrin-coated ZCFNPs were calculated from saturation magnetization of the hysteresis loops using the following equation [36]: nB =
Mw Ms 5585
(2)
where nB is the magnetic moment, Mw is the molecular weight, Ms is saturation magnetization and the coefficient of 5585 is the magnetic factor. The magnetic properties of ZCFNPs and dextrincoated ZCFNPs were obtained from hysteresis data and reported in the Supplementary material S1. The relatively small value of
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Based on the slopes of the plots (8.5 (±0.5, n = 6) mmol L−1 s−1 and 79 (±3.8, n = 6) mmol L−1 s−1 as r2 and r2∗ , respectively), the relaxivities of dextrin-coated ZCFNPs are comparable or higher than those reported previously. A comparison of the relaxivities of some different NPs was made in the Supplementary material S2. 3.3. MRI using tumor-bearing mice In vivo contrast enhancement of dextrin-coated ZCFNPs was evaluated via T2 - and T2∗ -weighted MRI. Fig. 4 represents the T2 - and T2∗ -weighted images before and after ZCFNPs injection into induced tumors in the mice. ZCFNPs induced negative contrast enhancement in the images of tumor area due to T2 and T2∗ relaxation times shortening. Before and after intratumoral injection of ZCFNPs, the average values of SIs of the images of the tumor volume were calculated from T2 - and T2∗ -weighted images, and are shown in the Supplementary material S3. Using the plots, decrements in SIs (%) after NPs injection in the tumor area were calculated based on the following equation [40]: Fig. 2. Magnetization curves of ZCFNPs and dextrin-coated ZCFNPs.
magnetization of dextrin-coated ZCFNPs was related to the dextrin surface coating, and also to the ultra-small size of the NPs [37–39]. 3.2. MRI using a phantom A 1.5 T clinical MRI was used to understand the relaxivity of dextrin-coated ZCFNPs as the contrast agent using agar phantom. Fig. 3A shows T2 -(spin-echo pulse sequence, TR = 1800 ms, TE = 88 ms) and T2∗ -(FLASH pulse sequence, TR = 350 ms, TE = 15 ms) weighted images of dextrin-coated ZCFNPs. Based on the images, the signal intensity (SI) of T2 - and T2∗ -weighted images decreased, upon increase in the ZCFNPs concentration. This is due to the T2 and T2∗ shortening. In Fig. 3B, the dependencies of the relaxation rates (R2 and R2∗ ) on the Fe + Co concentration in ZCFNPs are shown. The relaxation rates are increased linearly with the concentration.
Decrements in SIs (%) = −100
SIpre − SIpost SIpre
(3)
where SIpre and SIpost are the SIs before and after ZCFNPs injection. The results indicated that the MRI signal intensities for the tumor area after the ZCFNPs injection decreased by 12.5% (±0.6, n = 3) and 66.5% (±0.5, n = 3) for T2 - and T2∗ -weighted images, respectively. Although both the decrements in SIs are interested, the decrement in SI for T2∗ -weighted image is more favorable. The results suggest that dextrin-coated ZCFNPs can be considered as a proper contrast enhancement agent in MRI. 4. Discussion Today, magnetic NPs have great applications in biology and medicine, such as biomolecular separation [41], drug delivery [42] and MRI [43]. For these purposes, ultra-small sized NPs are highly desirable. However, NPs should be coated with a biocompatible
Fig. 3. (A) T2 - and T2∗ -weighted images of dextrin-coated ZCFNPs at different Fe + Co concentrations. (B) The dependencies of the relaxation rates (R2 and R2∗ ) on the Fe + Co concentration in ZCFNPs. The error amounts for data points as a percentage of the values of that data points (95% confidence interval error bars) are shown.
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Fig. 4. T2 - and T2∗ -weighted images before (left) and after (right) ZCFNPs injection into induced tumors in mice.
and non-toxic material to be used in vivo. In addition, particle size plays the major role for their times of blood circulation and cell internalization [44,45]. Recently, different magnetic NPs were synthesized and coated with different polymers [46], surfactants [47] and proteins [48]. In the present study, ZCFNPs were synthesized and coated by a dextrin layer. Dextrin is a polymer of ␣-(1 → 4) d-glucose that is produced by partial hydrolysis of starch. Dextrin was used in food, medicine and industry, because of its biocompatible and biodegradable characteristics [49,50]. FTIR studies showed that the surface of ZCFNPs is coated with a hydrophilic dextrin layer that is significant for the stability of ZCFNPs in human plasma. It also has an impact on interactions of ZCFNPs with biomolecules and cells. Dextrin bears hydroxyl functional groups and its interaction with proteins via hydrogen bonds can be predicted. Also, dextrin can shield the electrostatic interactions between ZCFNPs [51]. Although the presence of dextrin as a non-magnetic material on the ZCFNPs surface reduces the magnetization property of the dextrin-coated ZCFNPs, they retain their utility as a MRI contrast agent. In vitro, the value of r2∗ was higher than r2 , confirming the effectiveness of dextrin-coated ZCFNPs to create a huge contrast in the T2∗ -weighted images. In vivo, these NPs induced a negative contrast in T2 - and T2∗ -weighted images. In addition, calculation of the decrement in SIs in the tumor area indicates that the NPs have grater impact on T2∗ -weighted images.
5. Conclusion In summary, monodispersed dextrin-coated ZCFNPs were synthesized by a co-precipitation method. The size of NPs was 3.9 (±0.9, n = 200) nm with a narrow size distribution and a spherical morphology. FTIR studies confirmed the presence of dextrin on the surface of NPs. The magnetic property, and efficiency of dextrincoated ZCFNPs in MRI were evaluated both in vitro and in vivo. The NPs showed superparamagnetic behavior with contrast enhancement in T2∗ -weighted images. The dextrin-coated ZCFNPs have a great potential in MRI, and the present synthesis method and evaluation approaches of the NPs are extendable as a new route to other magnetic nanoparticles. Acknowledgments We would like to thank the Research Council of Shiraz University of Medical Sciences (8884), and the Iran National Science Foundation (INSF) for supporting this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2015.03.021.
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