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Direct synthesis of magnetite nanoparticles from iron(II) carboxymethylcellulose and their performance as NMR contrast agents
Journal of Magnetism and Magnetic Materials 397 (2016) 28–32
Contents lists available at ScienceDirect
Journal of Magnetism and Magnetic Materials journal homepage: www.elsevier.com/locate/jmmm
Direct synthesis of magnetite nanoparticles from iron(II) carboxymethylcellulose and their performance as NMR contrast agents Delmarcio Gomes da Silva a, Sergio Hiroshi Toma a, Fernando Menegatti de Melo a, Larissa Vieira C. Carvalho b, Alvicler Magalhães b, Edvaldo Sabadini b, Antônio Domingues dos Santos c, Koiti Araki a, e Henrique E. Toma a,n a
Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil Instituto de Química, Universidade Estadual de Campinas – UNICAMP, Campinas, SP, Brazil c Instituto de Física, Universidade de São Paulo, São Paulo, SP, Brazil b
art ic l e i nf o
a b s t r a c t
Article history: Received 1 June 2015 Received in revised form 4 August 2015 Accepted 20 August 2015 Available online 21 August 2015
Iron(II) carboxymethylcellulose (CMC) has been successfully employed in the synthesis of hydrophylic magnetite nanoparticles stabilized with a biopolymer coating, aiming applications in NMR imaging. The new method encompasses a convenient one-step synthetic procedure, allowing a good size control and yielding particles of about 10 nm (core size). In addition to the biocompatibility, the nanoparticles have promoted a drastic reduction in the transverse relaxation time (T2) of the water protons. The relaxivity rates have been investigated as a function of the nanoparticles concentration, showing a better performance in relation to the common NMR contrast agents available in the market. & 2015 Elsevier B.V. All rights reserved.
Keywords: NMR imaging Contrast agents Superparamagnetic nanoparticles Carboxymethylcelulose Relaxation time
1. Introduction Nanoparticles (NP), such as biofunctionalized magnetite species (MagNP), have furthered important advances in biomedicine. In special, the magnetite nanoparticles have been preferred because of their good compatibility with the biological environment [1], reflecting their iron oxide constitution also found in the ferritins. Functional MagNPs basically consist of two essential parts: a magnetic core and a surface coating. The magnetic core allows their manipulation by an applied magnetic field, while the surface coating is essential for the nanoparticles stability and performance. Recently, MagNPs have been used for diagnostic and therapeutic applications [2–4], as drug carriers, active elements in hyperthermia, and in biomedical analysis by nuclear magnetic resonance (NMR) [5,6]. As a matter of fact, MagNPs provide effective contrast agents in NMR imaging, reducing the proton relaxation times (T2) of the water molecules and promoting a darkening effect on the NMR image. For this reason, MagNPs are negative contrast agents. The possibility of modifying the MagNP surface with chemical and biological molecules can greatly n
Corresponding author. E-mail address: [email protected] (eH.E. Toma).
http://dx.doi.org/10.1016/j.jmmm.2015.08.092 0304-8853/& 2015 Elsevier B.V. All rights reserved.
improve their performance in terms of stability and biocompatibility. In our research group, functionalized MagNPs have been explored in biological catalysis and in environmental remediation, including an interesting process denoted magnetic nanohydrometallurgy [7]. This process exploits the magnetic properties and the chemical functionalization of the MagNPs for capturing metal ions, such as Cu2 þ , and then performing their in situ reduction at the electrode surface [8–11]. During the development of the present work, Sivakumar et al. [1] have published a remarkable paper on the biomedical applications of folate-tagged carboxymethylcelulose MagNPs, of about 150 nm. From the analysis of toxicological behavior of the MagNPs in three different cell types, they demonstrated a good biocompatibility between the CMC surface and the cellular environment. In a broad sense, CMC based nanomaterials have been successfully applied not only in chemotherapy [1], but also in environmental management for removing organic compounds [12]. It has also been employed in the creation of hybrid magnetic materials [13], and many other materials [14–22]. At the present time, the preparation of functionalized MagNPs involves multi-step procedures, usually based on the co-precipitation method [1,12,13,15]. In this work we are reporting an alternative, fast and convenient one-step method for the production of MagNPs functionalized with CMC (MagNP-CMC). This
D.G. da Silva et al. / Journal of Magnetism and Magnetic Materials 397 (2016) 28–32
method is based on the use of iron(II) carboxymethylcellulose complex as the starting material. Accordingly, the iron(II) complex with CMC seems to provide a suitable polymeric environment for nucleating the seeds, ensuring a good distribution and uniformity of size for the synthesized MagNPs. In addition, the formation of a stable CMC coating is naturally accomplished in the process. Besides the nanoparticles synthesis and characterization, we have also investigated their performance as contrast agents in NMR imaging (IMR), which is an essential tool in medicine. This technique explores the spin relaxation processes associated with the water molecules, in order to generate bi and trimensional images of the internal organisms. For this reason, we carried out a systematic study on the relaxation behavior of the CMC coated MagNPs in low field NMR, at several concentrations, and compared with the relaxivity parameters reported for the main commercial contrast agents. Such comparison revealed a convenient alternative agent, exhibiting excellent contrast performance, good biocompatibility and lower cost.
29
transferred to the reaction medium containing the Fe(II)–CMC complex, keeping the stirring rate (600 rpm) for 30 min. Finally, the suspension was cooled to room temperature, and the black magnetic nanoparticles were precipitated with acetone and confined at the bottom, with an external magnet. After removing the solution, the magnetically confined solid was redispersed in acetone, and the same procedure was repeated three times. The isolated solid was kept dried at room temperature.
3. Results and discussion A very simple way of obtaining MagNPs involves the direct reaction of iron(II) sulfate with nitrate ions in strongly alkaline medium. In this process, the initially formed iron(II) hydroxides get partially oxidized with the nitrate ions according to the reaction 12Fe2 þ þ23OH þNO3 -4Fe3O4 þNH3 þ10H2O
2. Materials and methods Carboxymethylcellulose sodium salt was obtained from Sigma Aldrich, with an average molecular weight of 250,000 g mol 1 and a degree of carboxylate substitution (DS) of 0.9. Ferrous sulfate (FeSO4 7H2O), potassium hydroxide (KOH) and potassium nitrate (KNO3) were obtained from Labsynth. All reactants were analytical grade, and used without any treatment. Nanoparticles size were analyzed by Dynamic Light Scattering (DLS), using a Nanotrac 252 model instrument, from Microtrac. Infrared Spectra were recorded on a Bruker FTIR, ALPHA model spectrophotomer. DTA/TGA (Thermogravimetric Analysis/Differential Thermal Analysis) measurements were carried out on a Shimadzu DTG 60 equipment, under an inert atmosphere. Scanning Probe Microscopy (SPM) images were obtained using a PicoSPM I equipment, with a PicoScan2100 controller, and a MACMode setup from Molecular Imaging. The hysteresis curves were obtained using a vibrating sample magnetometer manufactured by EG&G Princeton Applied Research-model 4500. Analysis by Transmission Electron Microscopy were carried out at our local Analytical Center, using a JEOL, model JEM 2100 equipment, operating with a LaB6 eletron emitting filament, allowing a maximum acceleration voltage of 200 kV. The scanning transmission image (STEM) was obtained with a HAADF detector (“High Angle Annular Dark Field”). Evaluation of the iron percentage in the nanoparticles was performed using an ICP OES equipment from Thermo Fisher Scientific, Cambridge, England, constituted by an iCAP 6300 Duo model. The NMR probe used in the analysis consisted of a Bruker Minispec mq20 spectrometer operating at 0.47 T. A 20 MHz frequency was used in the spin echo pulse sequence (CPMG). Magnetic resonance images were obtained using a 500 MHz machine, and a mic500S2/AS probe, attached to the Bruker advance III console.
The role of the nitrate ions in this process is quite unusual, since the reduction seems to proceed up to the formation of NH3, instead of N2. As a matter of fact, the formation of NH3 in this process can be readily perceived from its characteristic smell [23]. Because of its simplicity, this method has been successfully adopted in our experimental undergraduate courses, but leading always to very large nanoparticles ( 4400 nm), displaying strong magnetization response. For this reason, if CMC were added at the end of the process, the generated products would not be suitable for applications in nanobiomedicine. In contrast, by starting with the iron(II)–CMC complex, a controlled growth of nanoparticles can be performed, leading to smaller superparamagnetic nanoparticles. Presumably, the CMC matrix is providing a special polymeric environment for the nucleation process. After the synthesis, the size distribution of MagNP-CMC was probed by dynamic light scattering (DLS), as shown in Fig. 1, revealing 65% hydrodynamic radius distribution, in the range of 45– 75 nm, encompassing the nanoparticules core and the polymer coating, with a maximum distribution rate at 63 nm (35%). FTIR spectrum of MagNP-CMC, in KBr pellets, is shown in Fig. 2, in comparison with the spectrum of pure CMC. The sharp peak at 618 cm 1 coincides with the characteristic ν(Fe–O) stretching vibrational peak of the Fe3O4 core, but its shape reveals the presence of some SO42 ions employed in the process. The CMC peaks are consistent with the literature [24]. The most relevant peaks are associated with the carboxylate group, corresponding to the symmetric (νsymCOO) and asymmetric (νasymCOO) stretching vibrations observed at 1604 and 1422 cm 1 for free CMC and 1590 and 1417 cm 1 for MagNP-CMC, respectively. In general, the difference between the symmetric and asymmetric frequencies increases
2.1. Synthesis of magnetic nanoparticles (MagNP-CMC) Stock solution of CMC was prepared by solubilizing 7.5 g of the polymer in 1.0 L of distilled water. For the production of magnetic nanoparticles coated with CMC (MagNP-CMC), 200 ml of the stock solution was transferred to a flask (1 L) equipped with condenser and mechanical stirrer. The solution was heated to 85 °C under stirring (600 rpm) and then, a solution containing 6.8 mmol of FeSO4 7H2O in 80 ml of water was added to the reaction medium, generating the yellow Fe(II)-CMC complex. In a separate flask, 30 mmol of KOH and 0.35 mmol of KNO3 were solubilized in 20 ml of water and then heated to 85 °C. The hot solution was quickly
Fig. 1. Typical size distribution of MagNP-CMC probed by DLS, showing the predominant hydrodynamic radius around 63 nm, in aqueous solution.
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D.G. da Silva et al. / Journal of Magnetism and Magnetic Materials 397 (2016) 28–32
Fig. 2. FTIR spectra of CMC and MagNP-CMC species, in KBr pellets, and their corresponding assignments.
Fig. 4. TEM image of MagNP-CMC, showing the nanoparticle cores in the agglomerate.
when the carboxylate group is monocoordinated to a metal ion, and decreases substantially when it is coordinated in a chelate form [20]. In the case of CMC and MagNP-CMC, the negligible frequency difference in both cases indicated a weak interaction of the CMC carboxylate groups with the iron sites in the Fe3O4 core, rulling out any strong coordination bonding. The saturation magnetization of the MagNP-CMC was analyzed using a vibrating sample magnetometer (VSM). The magnetic susceptibility curves were measured at 20 kOe saturation field. The synthesized NP exhibited superparamagnetic regime and the saturation magnetization value (MSAT) was 14.08 emu g 1, as shown in Fig. 3. The low saturation magnetization value observed in the sample can be related to the high contribution mass, due to presence of the polymer (CMC) coating on the particles. Typical TEM images of MagNP-CMC can be seen in Fig. 4, exhibiting a typical aggregation of the nanoparticules probably induced by the sample preparation procedure. In spite of this, it is possible to identify the individual nanoparticles cores displaying polygonal shapes of 10.5 71.1 nm size. The presence of the CMC coating is barely discernable in the TEM image. The presence of the CMC coating can be better seen by comparing the tapping mode and the phase contrast AFM images in Fig. 5. Such a comparison allows to observed the presence of MagNP cores in the isolated nanoparticles surrounded by the CMC coating, also showing their pecular association in the agglomerates,
Fig. 5. AFM tapping mode (A) and phase constrast images (B) of MagNP-CMC, showing the presence of small nanoparticles and the composition of some aggregates, with the individual Fe3O4 cores surrounded by the CMC polymer.
3.1. MagNP-CMC as NMR contrast agent
Fig. 3. Magnetization curve for MagNP-CMC at room temperature, showing the superparamagnetic behavior, with a saturation magnetization of 14.08 emu g 1.
Contrast agents are species capable of modifying the relaxation time of the NMR proton signals in water. It should be noted that relaxation is a necessary process for allowing the nucleii spins to return to the ground state, and enabling further excitations as
D.G. da Silva et al. / Journal of Magnetism and Magnetic Materials 397 (2016) 28–32
31
Table 1 NMR analysis-values T2 (ms) obtained (24.1 °C) as a function of the concentration of the samples (ppm) prepared with MagNP-CMC dispersed in water (ppm). [MagNP-CMC]/ppm in water
Relaxation time – T2/ms
Water 2 5 10 20 50 100
2278 467 212 107 64.2 28.1 15.4
Fig. 7. Contrasting IMR images obtained with 10 and 100 ppm MagNP-CMC.
2 ppm of MagNP-CMC, can reduce the relaxation time of water protons by a factor of five. On the other extreme, 100 ppm of MagNP-CMC reduce T2 by 148 times. The parameter characterizing the ability of MagNP to reduce the relaxation times can be expressed by the relaxivity constant, R2. The relationship between relaxation time T2 and the nanoparticles concentration is given by Eq. (1)
1 1 = 0 + R2 [Fe3O4 ] T2 T2
Fig. 6. Plot of relaxivity vs the concentration of Fe3O4 for MagNP-CMC aqueous suspensions.
required for generating the NMR signals. This property is conveniently measured as the relaxivity parameter, or the specific relaxation time under controlled experimental conditions (concentration, temperature and magnetic field intensity). There are two types of relaxation processes, involving the spinnetwork (T1) and spin-spin (T2) mechanisms [25,26]. Accordingly, contrast agents such as gadolinium-DTPA complexes have been associated with the T1 relaxation mode, generating a positive contrast which increases the brightness of the image. In contrast, superparamagnetic nanoparticles act as negative contrast agents, promoting image darkening by reducing the T2 relaxation time [27–32]. In order to investigate the influence of MagNP-CMC on the T2 relaxation time of the water proton, a stock suspension of MagNPCMC was prepared and the accurate concentration of Fe3O4 (2.46 g L 1) determined by ICP OES. Then, systematic T2 relaxation measurements were carried out at several concentrations of MagNP-CMC varying from 2 to 100 ppm, (or mg L 1) in aqueous medium. The analyses were performed at room temperature (24.1 °C) and Table 1 shows the T2 values obtained for each concentration of MagNP-CMC. Surprisingly, even a minimum amount, such as
(1)
where R2 is the relaxivity of superparamagnetic nanoparticles. The relaxivity plot for MagNP-CMC is shown in Fig. 6. It is possible to observe a linear correlation between the reciprocal of T2 with respect to concentration, in accordance with Eq. (1). The calculated relaxivity for MagNP-CMC was 13274 mM 1 s 1. At high concentrations, e.g. 41 mM, MagNP-CMC can lead to detector saturation, and for this reason only diluted solutions have been considered for the relaxivity calculation. As a matter of fact, this effect becomes apparent in the last two measurements involving 50 and 100 ppm MagNP-CMC, by the small deviations observed from the linear plot. A comparison of the relaxivity values of MagNP-CMC and several commercial contrast agents can be found in Table 2. The comparison between the nanoparticles synthesized in this work and the commercial contrast agents (Table 2), considering a similar external magnetic field (0.47 T), reveals that MagNP-CMC is superior to Endorems, Feridexs and Sinerems. The MagNP-CMC performance is just slightly smaller than Resovists, however its facility of preparation and the low cost should be taken into consideration, reinforcing its candidacy as a viable contrast agent. The darkening effect (negative contrast) promoted by the superparamagnetic agents, can be better seen in the T2 weighted images generated by IMR, as illustrated in Fig. 7. The experimental arrangement consists of two concentric NMR tubes, with 10 mm (Tube 1) and 5 mm (tube 2) diameters. Tube 1 was filled with water, providing the background for comparison purposes, while tube 2 contained an aqueous suspension of magnetic nanoparticles (MagNP-CMC). For illustration purposes, only two experiments involving 10 ppm and 100 ppm MagNP-CMC, were
Table 2 Relaxivity values of MagNP-CMC and T2 commercial contrast agents. Sample
Use (agents)
Mean hydrodynamic diameter (nm)
Magnetic core size (nm)
Relaxivity R2 (mM 1 s 1)
MagNP-CMC Resovists (carbodextran SPIO SHU-555) [33] Endorems (dextran SPIO AMI-25) [33] Feridexs (dextran USPIO MION 46 L) [34] Sinerems (dextran SPIO) [33]
T2 T2 T2 T2 T2
63 62 80–150 72 20–40
10.5 4.2 4.8–5.6 – 4–6
132 (0.47 T) 151 (0.47 T) 98.3 (0.47 T) 100 (0.47 T) 53 (0.47 T)
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D.G. da Silva et al. / Journal of Magnetism and Magnetic Materials 397 (2016) 28–32
selected from the complete set of measurements employed in the R2 determination, As can be seen in Fig. 7, there is a large dark contrast for the suspension of 100 ppm, in relation to 10 ppm MagNP-CMC.
4. Conclusion The direct use of the iron(II) CMC complex provides a practical route for obtaining stable, biocompatible, hydrophilic magnetic colloids, with a lower cost, high yield and involving a single step process. The synthesized MagNP-CMC material exhibited excellent capability of reducing the T2 relaxation times of the protons NMR signals of water, showing a superior performance in relation to most similar commercial contrast agents.
Acknowledgements We greatly acknowledge the assistance of Drs. Sergio Romero, Marcelo Nakamura, Adir Jose Moreira and Raphael Alvim in the experimental part, and the support from FAPESP (Grant 2013/ 24725-4) and CNPq (Grant 482383/2013-5).
References [1] B. Sivakumar, R.G. Aswathy, Y. Nagaoka, M. Suzuki, T. Fukuda, Y. Yoshida, T. Maekawa, D.N. Sakthikumar, Langmuir 29 (10) (2013) 3453–3466. [2] H.B. Na, I.C. Song, T. Hyeon, Adv. Mater. 21 (21) (2009) 2133–2148. [3] L. Babes, B.T. Denizot, G. Tanguy, J.J. Le Jeune, P. Jallet, J. Colloid Interface Sci. 212 (2) (1999) 474–482. [4] J. Huang, X. Zhong, L. Wang, L. Yang, H. Mao, Theranostics 2 (1) (2012) 86. [5] M.E. Davis, Nat. Rev. Drug Discov. 7 (9) (2008) 771–782. [6] H. Shao, T.-J. Yoon, M. Liong, R. Weissleder, H. Lee, Beilstein J. Nanotechnol. 1 (1) (2010) 142–154. [7] H.E. Toma, Green Chem. 17 (4) (2015) 2027–2041.
[8] C.G. Netto, H.E. Toma, L.H. Andrade, J. Mol. Catal. B: Enzym. (2013) 71–9285 (2013) 71–92. [9] L.H. Andrade, L.P. Rebelo, C.G. Netto, H.E. Toma, J. Mol. Catal. B: Enzym. 66 (1) (2010) 55–62. [10] U. Condomitti, A. Zuin, M.A. Novak, K. Araki, H.E. Toma, Electrochem. Commun. 13 (1) (2011) 72–74. [11] M. Yamaura, R. Camilo, L. Sampaio, M. Macedo, M. Nakamura, H. Toma, J. Magn. Magn. Mater. 279 (2) (2004) 210–217. [12] N.A. Eltouny, P.A. Ariya, Indus. Eng. Chem. Res. 51 (39) (2012) 12787–12795. [13] J.F. Luna‐Martínez, E. Reyes‐Melo, V. González‐González, C. Guerrero‐Salazar, A. Torres‐Castro, S. Sepúlveda‐Guzmán, J. Appl. Polym. Sci. 127 (3) (2013) 2325–2331. [14] J. Virkutyte, R.S. Varma, Chem. Sci. 2 (5) (2011) 837–846. [15] P.R. Chang, J. Yu, X. Ma, D.P. Anderson, Carbohydr. Polym. 83 (2) (2011) 640–644. [16] S. Butun, F.G. Ince, H. Erdugan, N. Sahiner, Carbohydr. Polym. 86 (2) (2011) 636–643. [17] S. Kaihara, Y. Suzuki, K. Fujimoto, Colloids Surf. B: Biointerfaces 85 (2) (2011) 343–348. [18] J. Wotschadlo, T. Liebert, T. Heinze, K. Wagner, M. Schnabelrauch, S. Dutz, R. Müller, F. Steiniger, M. Schwalbe, T.C. Kroll, J. Magn. Magn. Mater. 321 (10) (2009) 1469–1473. [19] F. He, D. Zhao, Environ. Sci. Technol. 41 (17) (2007) 6216–6221. [20] S. Si, A. Kotal, T.K. Mandal, S. Giri, H. Nakamura, T. Kohara, Chem. Mater. 16 (18) (2004) 3489–3496. [21] T. Ohno, T. Wakabayashi, A. Takemura, J. Yoshida, A. Ito, M. Shinkai, H. Honda, T. Kobayashi, J. Neuro-Oncol. 56 (3) (2002) 233–239. [22] P. Sipos, Romanian Rep. Phys. 58 (3) (2006) 269. [23] T. Sugimoto, E. Matijević, J. Colloid Interface Sci. 74 (1) (1980) 227–243. [24] Z. Li, L. Wei, M. Gao, H. Lei, Adv. Mater. 17 (8) (2005) 1001–1005. [25] J. Jeener, B. Meier, P. Bachmann, R. Ernst, J. Chem. Phys. 71 (11) (1979) 4546–4553. [26] S. Macura, R. Ernst, Mol. Phys. 41 (1) (1980) 95–117. [27] J.W.M. Bulte, D.L. Kraitchman, NMR Biomed. 17 (7) (2004) 484–499. [28] A.K. Gupta, M. Gupta, Biomaterials 26 (18) (2005) 3995–4021. [29] T. Neuberger, B. Schopf, H. Hofmann, M. Hofmann, B. von Rechenberg, J. Magn. Magn. Mater. 293 (1) (2005) 483–496. [30] D.K. Kim, M. Mikhaylova, F.H. Wang, J. Kehr, B. Bjelke, Y. Zhang, T. Tsakalakos, M. Muhammed, Chem. Mater. 15 (23) (2003) 4343–4351. [31] P. Caravan, J.J. Ellison, T.J. McMurry, R.B. Lauffer, Chem. Rev. 99 (9) (1999) 2293–2352. [32] H.J. Weinmann, R.C. Brasch, W.R. Press, G.E. Wesbey, Am. J. Roentgenol. 142 (3) (1984) 619–624. [33] Y.-X.J. Wang, S.M. Hussain, G.P. Krestin, Eur. Radiol. 11 (11) (2001) 2319–2331. [34] T. Allkemper, C. Bremer, L. Matuszewski, W. Ebert, P. Reimer, Radiology 223 (2) (2002) 432–438.
Contents lists available at ScienceDirect
Journal of Magnetism and Magnetic Materials journal homepage: www.elsevier.com/locate/jmmm
Direct synthesis of magnetite nanoparticles from iron(II) carboxymethylcellulose and their performance as NMR contrast agents Delmarcio Gomes da Silva a, Sergio Hiroshi Toma a, Fernando Menegatti de Melo a, Larissa Vieira C. Carvalho b, Alvicler Magalhães b, Edvaldo Sabadini b, Antônio Domingues dos Santos c, Koiti Araki a, e Henrique E. Toma a,n a
Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil Instituto de Química, Universidade Estadual de Campinas – UNICAMP, Campinas, SP, Brazil c Instituto de Física, Universidade de São Paulo, São Paulo, SP, Brazil b
art ic l e i nf o
a b s t r a c t
Article history: Received 1 June 2015 Received in revised form 4 August 2015 Accepted 20 August 2015 Available online 21 August 2015
Iron(II) carboxymethylcellulose (CMC) has been successfully employed in the synthesis of hydrophylic magnetite nanoparticles stabilized with a biopolymer coating, aiming applications in NMR imaging. The new method encompasses a convenient one-step synthetic procedure, allowing a good size control and yielding particles of about 10 nm (core size). In addition to the biocompatibility, the nanoparticles have promoted a drastic reduction in the transverse relaxation time (T2) of the water protons. The relaxivity rates have been investigated as a function of the nanoparticles concentration, showing a better performance in relation to the common NMR contrast agents available in the market. & 2015 Elsevier B.V. All rights reserved.
Keywords: NMR imaging Contrast agents Superparamagnetic nanoparticles Carboxymethylcelulose Relaxation time
1. Introduction Nanoparticles (NP), such as biofunctionalized magnetite species (MagNP), have furthered important advances in biomedicine. In special, the magnetite nanoparticles have been preferred because of their good compatibility with the biological environment [1], reflecting their iron oxide constitution also found in the ferritins. Functional MagNPs basically consist of two essential parts: a magnetic core and a surface coating. The magnetic core allows their manipulation by an applied magnetic field, while the surface coating is essential for the nanoparticles stability and performance. Recently, MagNPs have been used for diagnostic and therapeutic applications [2–4], as drug carriers, active elements in hyperthermia, and in biomedical analysis by nuclear magnetic resonance (NMR) [5,6]. As a matter of fact, MagNPs provide effective contrast agents in NMR imaging, reducing the proton relaxation times (T2) of the water molecules and promoting a darkening effect on the NMR image. For this reason, MagNPs are negative contrast agents. The possibility of modifying the MagNP surface with chemical and biological molecules can greatly n
Corresponding author. E-mail address: [email protected] (eH.E. Toma).
http://dx.doi.org/10.1016/j.jmmm.2015.08.092 0304-8853/& 2015 Elsevier B.V. All rights reserved.
improve their performance in terms of stability and biocompatibility. In our research group, functionalized MagNPs have been explored in biological catalysis and in environmental remediation, including an interesting process denoted magnetic nanohydrometallurgy [7]. This process exploits the magnetic properties and the chemical functionalization of the MagNPs for capturing metal ions, such as Cu2 þ , and then performing their in situ reduction at the electrode surface [8–11]. During the development of the present work, Sivakumar et al. [1] have published a remarkable paper on the biomedical applications of folate-tagged carboxymethylcelulose MagNPs, of about 150 nm. From the analysis of toxicological behavior of the MagNPs in three different cell types, they demonstrated a good biocompatibility between the CMC surface and the cellular environment. In a broad sense, CMC based nanomaterials have been successfully applied not only in chemotherapy [1], but also in environmental management for removing organic compounds [12]. It has also been employed in the creation of hybrid magnetic materials [13], and many other materials [14–22]. At the present time, the preparation of functionalized MagNPs involves multi-step procedures, usually based on the co-precipitation method [1,12,13,15]. In this work we are reporting an alternative, fast and convenient one-step method for the production of MagNPs functionalized with CMC (MagNP-CMC). This
D.G. da Silva et al. / Journal of Magnetism and Magnetic Materials 397 (2016) 28–32
method is based on the use of iron(II) carboxymethylcellulose complex as the starting material. Accordingly, the iron(II) complex with CMC seems to provide a suitable polymeric environment for nucleating the seeds, ensuring a good distribution and uniformity of size for the synthesized MagNPs. In addition, the formation of a stable CMC coating is naturally accomplished in the process. Besides the nanoparticles synthesis and characterization, we have also investigated their performance as contrast agents in NMR imaging (IMR), which is an essential tool in medicine. This technique explores the spin relaxation processes associated with the water molecules, in order to generate bi and trimensional images of the internal organisms. For this reason, we carried out a systematic study on the relaxation behavior of the CMC coated MagNPs in low field NMR, at several concentrations, and compared with the relaxivity parameters reported for the main commercial contrast agents. Such comparison revealed a convenient alternative agent, exhibiting excellent contrast performance, good biocompatibility and lower cost.
29
transferred to the reaction medium containing the Fe(II)–CMC complex, keeping the stirring rate (600 rpm) for 30 min. Finally, the suspension was cooled to room temperature, and the black magnetic nanoparticles were precipitated with acetone and confined at the bottom, with an external magnet. After removing the solution, the magnetically confined solid was redispersed in acetone, and the same procedure was repeated three times. The isolated solid was kept dried at room temperature.
3. Results and discussion A very simple way of obtaining MagNPs involves the direct reaction of iron(II) sulfate with nitrate ions in strongly alkaline medium. In this process, the initially formed iron(II) hydroxides get partially oxidized with the nitrate ions according to the reaction 12Fe2 þ þ23OH þNO3 -4Fe3O4 þNH3 þ10H2O
2. Materials and methods Carboxymethylcellulose sodium salt was obtained from Sigma Aldrich, with an average molecular weight of 250,000 g mol 1 and a degree of carboxylate substitution (DS) of 0.9. Ferrous sulfate (FeSO4 7H2O), potassium hydroxide (KOH) and potassium nitrate (KNO3) were obtained from Labsynth. All reactants were analytical grade, and used without any treatment. Nanoparticles size were analyzed by Dynamic Light Scattering (DLS), using a Nanotrac 252 model instrument, from Microtrac. Infrared Spectra were recorded on a Bruker FTIR, ALPHA model spectrophotomer. DTA/TGA (Thermogravimetric Analysis/Differential Thermal Analysis) measurements were carried out on a Shimadzu DTG 60 equipment, under an inert atmosphere. Scanning Probe Microscopy (SPM) images were obtained using a PicoSPM I equipment, with a PicoScan2100 controller, and a MACMode setup from Molecular Imaging. The hysteresis curves were obtained using a vibrating sample magnetometer manufactured by EG&G Princeton Applied Research-model 4500. Analysis by Transmission Electron Microscopy were carried out at our local Analytical Center, using a JEOL, model JEM 2100 equipment, operating with a LaB6 eletron emitting filament, allowing a maximum acceleration voltage of 200 kV. The scanning transmission image (STEM) was obtained with a HAADF detector (“High Angle Annular Dark Field”). Evaluation of the iron percentage in the nanoparticles was performed using an ICP OES equipment from Thermo Fisher Scientific, Cambridge, England, constituted by an iCAP 6300 Duo model. The NMR probe used in the analysis consisted of a Bruker Minispec mq20 spectrometer operating at 0.47 T. A 20 MHz frequency was used in the spin echo pulse sequence (CPMG). Magnetic resonance images were obtained using a 500 MHz machine, and a mic500S2/AS probe, attached to the Bruker advance III console.
The role of the nitrate ions in this process is quite unusual, since the reduction seems to proceed up to the formation of NH3, instead of N2. As a matter of fact, the formation of NH3 in this process can be readily perceived from its characteristic smell [23]. Because of its simplicity, this method has been successfully adopted in our experimental undergraduate courses, but leading always to very large nanoparticles ( 4400 nm), displaying strong magnetization response. For this reason, if CMC were added at the end of the process, the generated products would not be suitable for applications in nanobiomedicine. In contrast, by starting with the iron(II)–CMC complex, a controlled growth of nanoparticles can be performed, leading to smaller superparamagnetic nanoparticles. Presumably, the CMC matrix is providing a special polymeric environment for the nucleation process. After the synthesis, the size distribution of MagNP-CMC was probed by dynamic light scattering (DLS), as shown in Fig. 1, revealing 65% hydrodynamic radius distribution, in the range of 45– 75 nm, encompassing the nanoparticules core and the polymer coating, with a maximum distribution rate at 63 nm (35%). FTIR spectrum of MagNP-CMC, in KBr pellets, is shown in Fig. 2, in comparison with the spectrum of pure CMC. The sharp peak at 618 cm 1 coincides with the characteristic ν(Fe–O) stretching vibrational peak of the Fe3O4 core, but its shape reveals the presence of some SO42 ions employed in the process. The CMC peaks are consistent with the literature [24]. The most relevant peaks are associated with the carboxylate group, corresponding to the symmetric (νsymCOO) and asymmetric (νasymCOO) stretching vibrations observed at 1604 and 1422 cm 1 for free CMC and 1590 and 1417 cm 1 for MagNP-CMC, respectively. In general, the difference between the symmetric and asymmetric frequencies increases
2.1. Synthesis of magnetic nanoparticles (MagNP-CMC) Stock solution of CMC was prepared by solubilizing 7.5 g of the polymer in 1.0 L of distilled water. For the production of magnetic nanoparticles coated with CMC (MagNP-CMC), 200 ml of the stock solution was transferred to a flask (1 L) equipped with condenser and mechanical stirrer. The solution was heated to 85 °C under stirring (600 rpm) and then, a solution containing 6.8 mmol of FeSO4 7H2O in 80 ml of water was added to the reaction medium, generating the yellow Fe(II)-CMC complex. In a separate flask, 30 mmol of KOH and 0.35 mmol of KNO3 were solubilized in 20 ml of water and then heated to 85 °C. The hot solution was quickly
Fig. 1. Typical size distribution of MagNP-CMC probed by DLS, showing the predominant hydrodynamic radius around 63 nm, in aqueous solution.
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Fig. 2. FTIR spectra of CMC and MagNP-CMC species, in KBr pellets, and their corresponding assignments.
Fig. 4. TEM image of MagNP-CMC, showing the nanoparticle cores in the agglomerate.
when the carboxylate group is monocoordinated to a metal ion, and decreases substantially when it is coordinated in a chelate form [20]. In the case of CMC and MagNP-CMC, the negligible frequency difference in both cases indicated a weak interaction of the CMC carboxylate groups with the iron sites in the Fe3O4 core, rulling out any strong coordination bonding. The saturation magnetization of the MagNP-CMC was analyzed using a vibrating sample magnetometer (VSM). The magnetic susceptibility curves were measured at 20 kOe saturation field. The synthesized NP exhibited superparamagnetic regime and the saturation magnetization value (MSAT) was 14.08 emu g 1, as shown in Fig. 3. The low saturation magnetization value observed in the sample can be related to the high contribution mass, due to presence of the polymer (CMC) coating on the particles. Typical TEM images of MagNP-CMC can be seen in Fig. 4, exhibiting a typical aggregation of the nanoparticules probably induced by the sample preparation procedure. In spite of this, it is possible to identify the individual nanoparticles cores displaying polygonal shapes of 10.5 71.1 nm size. The presence of the CMC coating is barely discernable in the TEM image. The presence of the CMC coating can be better seen by comparing the tapping mode and the phase contrast AFM images in Fig. 5. Such a comparison allows to observed the presence of MagNP cores in the isolated nanoparticles surrounded by the CMC coating, also showing their pecular association in the agglomerates,
Fig. 5. AFM tapping mode (A) and phase constrast images (B) of MagNP-CMC, showing the presence of small nanoparticles and the composition of some aggregates, with the individual Fe3O4 cores surrounded by the CMC polymer.
3.1. MagNP-CMC as NMR contrast agent
Fig. 3. Magnetization curve for MagNP-CMC at room temperature, showing the superparamagnetic behavior, with a saturation magnetization of 14.08 emu g 1.
Contrast agents are species capable of modifying the relaxation time of the NMR proton signals in water. It should be noted that relaxation is a necessary process for allowing the nucleii spins to return to the ground state, and enabling further excitations as
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Table 1 NMR analysis-values T2 (ms) obtained (24.1 °C) as a function of the concentration of the samples (ppm) prepared with MagNP-CMC dispersed in water (ppm). [MagNP-CMC]/ppm in water
Relaxation time – T2/ms
Water 2 5 10 20 50 100
2278 467 212 107 64.2 28.1 15.4
Fig. 7. Contrasting IMR images obtained with 10 and 100 ppm MagNP-CMC.
2 ppm of MagNP-CMC, can reduce the relaxation time of water protons by a factor of five. On the other extreme, 100 ppm of MagNP-CMC reduce T2 by 148 times. The parameter characterizing the ability of MagNP to reduce the relaxation times can be expressed by the relaxivity constant, R2. The relationship between relaxation time T2 and the nanoparticles concentration is given by Eq. (1)
1 1 = 0 + R2 [Fe3O4 ] T2 T2
Fig. 6. Plot of relaxivity vs the concentration of Fe3O4 for MagNP-CMC aqueous suspensions.
required for generating the NMR signals. This property is conveniently measured as the relaxivity parameter, or the specific relaxation time under controlled experimental conditions (concentration, temperature and magnetic field intensity). There are two types of relaxation processes, involving the spinnetwork (T1) and spin-spin (T2) mechanisms [25,26]. Accordingly, contrast agents such as gadolinium-DTPA complexes have been associated with the T1 relaxation mode, generating a positive contrast which increases the brightness of the image. In contrast, superparamagnetic nanoparticles act as negative contrast agents, promoting image darkening by reducing the T2 relaxation time [27–32]. In order to investigate the influence of MagNP-CMC on the T2 relaxation time of the water proton, a stock suspension of MagNPCMC was prepared and the accurate concentration of Fe3O4 (2.46 g L 1) determined by ICP OES. Then, systematic T2 relaxation measurements were carried out at several concentrations of MagNP-CMC varying from 2 to 100 ppm, (or mg L 1) in aqueous medium. The analyses were performed at room temperature (24.1 °C) and Table 1 shows the T2 values obtained for each concentration of MagNP-CMC. Surprisingly, even a minimum amount, such as
(1)
where R2 is the relaxivity of superparamagnetic nanoparticles. The relaxivity plot for MagNP-CMC is shown in Fig. 6. It is possible to observe a linear correlation between the reciprocal of T2 with respect to concentration, in accordance with Eq. (1). The calculated relaxivity for MagNP-CMC was 13274 mM 1 s 1. At high concentrations, e.g. 41 mM, MagNP-CMC can lead to detector saturation, and for this reason only diluted solutions have been considered for the relaxivity calculation. As a matter of fact, this effect becomes apparent in the last two measurements involving 50 and 100 ppm MagNP-CMC, by the small deviations observed from the linear plot. A comparison of the relaxivity values of MagNP-CMC and several commercial contrast agents can be found in Table 2. The comparison between the nanoparticles synthesized in this work and the commercial contrast agents (Table 2), considering a similar external magnetic field (0.47 T), reveals that MagNP-CMC is superior to Endorems, Feridexs and Sinerems. The MagNP-CMC performance is just slightly smaller than Resovists, however its facility of preparation and the low cost should be taken into consideration, reinforcing its candidacy as a viable contrast agent. The darkening effect (negative contrast) promoted by the superparamagnetic agents, can be better seen in the T2 weighted images generated by IMR, as illustrated in Fig. 7. The experimental arrangement consists of two concentric NMR tubes, with 10 mm (Tube 1) and 5 mm (tube 2) diameters. Tube 1 was filled with water, providing the background for comparison purposes, while tube 2 contained an aqueous suspension of magnetic nanoparticles (MagNP-CMC). For illustration purposes, only two experiments involving 10 ppm and 100 ppm MagNP-CMC, were
Table 2 Relaxivity values of MagNP-CMC and T2 commercial contrast agents. Sample
Use (agents)
Mean hydrodynamic diameter (nm)
Magnetic core size (nm)
Relaxivity R2 (mM 1 s 1)
MagNP-CMC Resovists (carbodextran SPIO SHU-555) [33] Endorems (dextran SPIO AMI-25) [33] Feridexs (dextran USPIO MION 46 L) [34] Sinerems (dextran SPIO) [33]
T2 T2 T2 T2 T2
63 62 80–150 72 20–40
10.5 4.2 4.8–5.6 – 4–6
132 (0.47 T) 151 (0.47 T) 98.3 (0.47 T) 100 (0.47 T) 53 (0.47 T)
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selected from the complete set of measurements employed in the R2 determination, As can be seen in Fig. 7, there is a large dark contrast for the suspension of 100 ppm, in relation to 10 ppm MagNP-CMC.
4. Conclusion The direct use of the iron(II) CMC complex provides a practical route for obtaining stable, biocompatible, hydrophilic magnetic colloids, with a lower cost, high yield and involving a single step process. The synthesized MagNP-CMC material exhibited excellent capability of reducing the T2 relaxation times of the protons NMR signals of water, showing a superior performance in relation to most similar commercial contrast agents.
Acknowledgements We greatly acknowledge the assistance of Drs. Sergio Romero, Marcelo Nakamura, Adir Jose Moreira and Raphael Alvim in the experimental part, and the support from FAPESP (Grant 2013/ 24725-4) and CNPq (Grant 482383/2013-5).
References [1] B. Sivakumar, R.G. Aswathy, Y. Nagaoka, M. Suzuki, T. Fukuda, Y. Yoshida, T. Maekawa, D.N. Sakthikumar, Langmuir 29 (10) (2013) 3453–3466. [2] H.B. Na, I.C. Song, T. Hyeon, Adv. Mater. 21 (21) (2009) 2133–2148. [3] L. Babes, B.T. Denizot, G. Tanguy, J.J. Le Jeune, P. Jallet, J. Colloid Interface Sci. 212 (2) (1999) 474–482. [4] J. Huang, X. Zhong, L. Wang, L. Yang, H. Mao, Theranostics 2 (1) (2012) 86. [5] M.E. Davis, Nat. Rev. Drug Discov. 7 (9) (2008) 771–782. [6] H. Shao, T.-J. Yoon, M. Liong, R. Weissleder, H. Lee, Beilstein J. Nanotechnol. 1 (1) (2010) 142–154. [7] H.E. Toma, Green Chem. 17 (4) (2015) 2027–2041.
[8] C.G. Netto, H.E. Toma, L.H. Andrade, J. Mol. Catal. B: Enzym. (2013) 71–9285 (2013) 71–92. [9] L.H. Andrade, L.P. Rebelo, C.G. Netto, H.E. Toma, J. Mol. Catal. B: Enzym. 66 (1) (2010) 55–62. [10] U. Condomitti, A. Zuin, M.A. Novak, K. Araki, H.E. Toma, Electrochem. Commun. 13 (1) (2011) 72–74. [11] M. Yamaura, R. Camilo, L. Sampaio, M. Macedo, M. Nakamura, H. Toma, J. Magn. Magn. Mater. 279 (2) (2004) 210–217. [12] N.A. Eltouny, P.A. Ariya, Indus. Eng. Chem. Res. 51 (39) (2012) 12787–12795. [13] J.F. Luna‐Martínez, E. Reyes‐Melo, V. González‐González, C. Guerrero‐Salazar, A. Torres‐Castro, S. Sepúlveda‐Guzmán, J. Appl. Polym. Sci. 127 (3) (2013) 2325–2331. [14] J. Virkutyte, R.S. Varma, Chem. Sci. 2 (5) (2011) 837–846. [15] P.R. Chang, J. Yu, X. Ma, D.P. Anderson, Carbohydr. Polym. 83 (2) (2011) 640–644. [16] S. Butun, F.G. Ince, H. Erdugan, N. Sahiner, Carbohydr. Polym. 86 (2) (2011) 636–643. [17] S. Kaihara, Y. Suzuki, K. Fujimoto, Colloids Surf. B: Biointerfaces 85 (2) (2011) 343–348. [18] J. Wotschadlo, T. Liebert, T. Heinze, K. Wagner, M. Schnabelrauch, S. Dutz, R. Müller, F. Steiniger, M. Schwalbe, T.C. Kroll, J. Magn. Magn. Mater. 321 (10) (2009) 1469–1473. [19] F. He, D. Zhao, Environ. Sci. Technol. 41 (17) (2007) 6216–6221. [20] S. Si, A. Kotal, T.K. Mandal, S. Giri, H. Nakamura, T. Kohara, Chem. Mater. 16 (18) (2004) 3489–3496. [21] T. Ohno, T. Wakabayashi, A. Takemura, J. Yoshida, A. Ito, M. Shinkai, H. Honda, T. Kobayashi, J. Neuro-Oncol. 56 (3) (2002) 233–239. [22] P. Sipos, Romanian Rep. Phys. 58 (3) (2006) 269. [23] T. Sugimoto, E. Matijević, J. Colloid Interface Sci. 74 (1) (1980) 227–243. [24] Z. Li, L. Wei, M. Gao, H. Lei, Adv. Mater. 17 (8) (2005) 1001–1005. [25] J. Jeener, B. Meier, P. Bachmann, R. Ernst, J. Chem. Phys. 71 (11) (1979) 4546–4553. [26] S. Macura, R. Ernst, Mol. Phys. 41 (1) (1980) 95–117. [27] J.W.M. Bulte, D.L. Kraitchman, NMR Biomed. 17 (7) (2004) 484–499. [28] A.K. Gupta, M. Gupta, Biomaterials 26 (18) (2005) 3995–4021. [29] T. Neuberger, B. Schopf, H. Hofmann, M. Hofmann, B. von Rechenberg, J. Magn. Magn. Mater. 293 (1) (2005) 483–496. [30] D.K. Kim, M. Mikhaylova, F.H. Wang, J. Kehr, B. Bjelke, Y. Zhang, T. Tsakalakos, M. Muhammed, Chem. Mater. 15 (23) (2003) 4343–4351. [31] P. Caravan, J.J. Ellison, T.J. McMurry, R.B. Lauffer, Chem. Rev. 99 (9) (1999) 2293–2352. [32] H.J. Weinmann, R.C. Brasch, W.R. Press, G.E. Wesbey, Am. J. Roentgenol. 142 (3) (1984) 619–624. [33] Y.-X.J. Wang, S.M. Hussain, G.P. Krestin, Eur. Radiol. 11 (11) (2001) 2319–2331. [34] T. Allkemper, C. Bremer, L. Matuszewski, W. Ebert, P. Reimer, Radiology 223 (2) (2002) 432–438.