www.advmat.de
COMMUNICATION
www.MaterialsViews.com
Anchoring Group Effects of Surface Ligands on Magnetic Properties of Fe3O4 Nanoparticles: Towards High Performance MRI Contrast Agents Jianfeng Zeng, Lihong Jing, Yi Hou, Mingxia Jiao, Ruirui Qiao, Qiaojuan Jia, Chunyan Liu, Fang Fang, Hao Lei, and Mingyuan Gao* Due to strong size-related effects, inorganic nanocrystals present unique physical properties, e.g., fluorescence of semiconductor nanoparticles,[1] surface plasmon resonance of metal particles,[2] and superparamagnetism of magnetic nanoparticles.[3] Apart from the particle size-related effects, the large surface-to-volume ratio also gives rise to remarkable surface effects on the physiochemical properties of the inorganic nanocrystals.[4–6] Surface ligand is in fact indispensable in the solution synthesis of high quality nanoparticles not only for increasing the compatibility of the underlying nanoparticles with dispersion media, but also for preventing the particles from uncontrollable growth. Therefore, the physical effects directly associated with particle surface ligand are important subjects for achieving advanced materials. With respect to applications, the magnetic properties to metal oxide nanoparticles are the same important as the fluorescent properties to quantum dots (QDs). The effects of surface ligand on the optical properties of QDs have received intensive studies over the past decades.[4,7–9] Nevertheless, the impacts of surface ligand on the magnetic properties of metal oxide nanocrystals are much less studied so far.[5,10] One of the biggest challenges is to vary the surface coating structure without altering the size and dispersibility of the nanoparticles. Magnetic resonance imaging (MRI) contrast agents represent one of the major applications for magnetic nanoparticles.[11–16] To disclose the impacts of surface ligand on the magnetic properties associated with MRI applications, two differently sized Fe3O4 nanoparticles capped by polyethylene glycol (PEG) ligands through different anchoring groups were prepared. The anchoring group effects on the relaxometric properties of the PEGylated Fe3O4 nanoparticles were discussed in combination
J. F. Zeng, Dr. L. H. Jing, Prof. Y. Hou, M. X. Jiao, R. R. Qiao, Dr. C. Y. Liu, Prof. M. Y. Gao Institute of Chemistry Chinese Academy of Sciences Bei Yi Jie 2, Zhong Guan Cun, Beijing 100190, China E-mail: [email protected] Dr. Q. J. Jia School of Chemistry and Environment South China Normal University Guangzhou 510006, China Prof. H. Lei, Dr. F. Fang Wuhan Institute of Physics and Mathematics Chinese Academy of Sciences Wuhan 430071, China
DOI: 10.1002/adma.201304744
2694
wileyonlinelibrary.com
with theoretical analysis. In addition, careful animal experiments were carried out for showing the outstanding performance of the PEGylated Fe3O4 particles with optimized structures as a dual-modal MRI contrast agent in sensitive detection of tumors in vivo. The hydrophobic Fe3O4 particles with average sizes of 3.6 nm and 10.9 nm were prepared through conventional thermal decomposition method by using oleic acid and oleylamine as surface ligands.[17,18] Since diphosphate (DP), hydroxamate (HX), and catechol (CC) groups possess higher binding affinities to Fe3+ than the anchoring groups of the initial hydrophobic ligands, PEG2000 ligands bearing DP group, HX group, and CC group, denoted as DP-PEG, HX-PEG, and CC-PEG, respectively, were designed and used to replace the hydrophobic ligands so as to achieve water-soluble Fe3O4 nanoparticles. The chemical structures of the PEG ligands are provided in the left panel of Figure 1. The transmission electron microscopy (TEM) images of the PEGylated particles are presented in the right panel of Figure 1. Careful statistical studies reveal that the ligand exchange did not alter the size or the size distribution profiles of both nanoparticles as shown in Figure S1 in Supporting Information (SI). Further dynamic light scattering (DLS) results shown in Figure S2 reveal that 3.6 nm and 10.9 nm Fe3O4 nanoparticles in cyclohexane present single scattering peak locating at 6.1 nm and 12.6 nm, respectively, before ligand exchange. After ligand exchange, the hydrodynamic size of the PEGylated nanoparticles in water apparently increases, while the size distribution profiles remain nearly unchanged in comparison with those of the hydrophobic counterparts. In addition, the hydrodynamic sizes and size distribution profiles are nearly independent of anchoring group with respect to particles of both sizes, which makes it possible to further compare their MRI contrast enhancement effect solely associated with the surface ligand. Although similar efforts have previously been devoted, the large difference in the size and size distribution among the resulting particles makes it difficult to extract reliable information solely associated with the ligand effects.[19] As shown in the insets of Figure 1, the solutions containing PEGylated Fe3O4 nanoparticles with the same core size and Fe concentration exhibit different degrees of brown colors, especially for 3.6 nm particles, indicating that the chelating effect of the anchoring groups alter the d-d transitions in Fe3O4 nanoparticles by different degrees. The MRI contrast enhancement effects of the aforementioned PEGylated Fe3O4 nanoparticles were evaluated on a clinical 3 T MRI scanner at room temperature. Figure 2 shows six sets of the T1- and T2-weighted MR images of aqueous
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, 26, 2694–2698
www.advmat.de www.MaterialsViews.com
solutions containing 3.6 nm or 10.9 nm Fe3O4 nanoparticles capped by PEGs through different anchoring groups. As expected, 10.9 nm Fe3O4 nanoparticles present stronger T2 effect and no obvious T1 effect. But quite unexpectedly, 3.6 nm Fe3O4 nanoparticles show strong T2 effect apart from obvious T1 effect. In fact, recent investigations have demonstrated that magnetic iron oxide nanoparticles below 5 nm are potentially useful as T1 contrast agent,[10,20–24] because the paramagnetism becomes dominant for small particles due to the increased degree of spin disorders on the particle surface.[3] Nevertheless, how to effectively enhance the T1 effect by reducing the particle size without depressing the T2 effect remains an open question.
3
r1 =
128π 2γ I2M n ⎛ 1 ⎞ 2 ⎜⎝ 1 + L/ a ⎟⎠ MS τ D J A 405ρ
r2 =
256π 2γ I2M n ⎛ 1 ⎞ MS2τ D ⎜ 1215ρ ⎝ 1 + L / a ⎟⎠
(
2ω Iτ D
)
COMMUNICATION
Figure 1. Left panel: chemical structures of the PEGs used for exchanging the hydrophobic ligands of 3.6 nm Fe3O4 nanoparticle (S) and 10.9 nm Fe3O4 nanoparticle (L); Right panel: TEM images of the resulting PEGylated particles (the embeded scale bars correspond to 50 nm); Insets: photographs of aqueous solutions of the PEGylated Fe3O4 particles with an equal Fe concentration of 20 mM.
The imaging results in Figure 2 further reveal that 3.6 nm Fe3O4 particles capped by CC-PEG and HX-PEG present stronger T2 and T1 effects than 3.6 nm particle capped by DP-PEG, especially when [Fe] is higher than 0.4 mmol/L. This anchoring group effect can also be observed from 10.9 nm Fe3O4 nanoparticles, but relatively weak. To further quantify the results presented in Figure 2, the experimentally determined longitudinal and transverse relaxation rates were simulated through linear regression fitting as provided in Figure S3. The resulting concentration-independent relaxivities, i.e., longitudinal relaxivity (r1) and transverse relaxivity (r2), are provided in Table 1. It is quite conclusive that the anchoring group has stronger impacts on r2 and r2/r1 ratio. In general, the HX-PEG and CC-PEG give rise to higher r2 values and r2/r1 ratios than the DP-PEG, irrespective of the particle size, rather in consistence with the T2-weighted imaging results shown in Figure 2. Nevertheless, there is no direct correlation between the anchoring groups and r1 values for both sizes of particles. According to theory, the relaxation enhancement generally follows the inner-sphere and outer-sphere mechanisms.[25] The inner-sphere proton relaxation involves water molecules directly bonding to paramagnetic ions, while the outer-sphere relaxation is associated with water molecules beyond the directly bonding ones. We assume that the experimentally observed proton relaxation is mainly dominated by the outer-sphere mechanism due to the highly hydrophilic nature of the PEG coatings, according to the boundary conditions associated with high magnetic field, r1 and r2 can then be given by[26–29] (1)
3
(2)
The assumptions and detailed processes for deriving Equation (1) and (2) are provided in SI. In the above equations, γI represents the proton gyromagnetic ratio, Mn and ρ are the molar mass and density of Fe3O4, respectively, a is the radius of Fe3O4 particles extracted from TEM measurements, and MS is the saturation magnetization of the Fe3O4 core particles, L is the water impermeable thickness of surface coating layer, r is the effective radius of particles (r = a + L), D is the water diffusion coefficient, τD is the translational diffusion time (τD = r2/D), and JA represents the Ayant’s spectral density function that can be given by 5z z 2 + 8 8 JA (z) = z 2 z3 4 z 4 z 5 z 6 1+ z + + + + + 2 6 81 81 648 1+
Figure 2. T1-weighted and T2-weighted MR images of aqueous solutions containing 3.6 nm or 10.9 nm Fe3O4 nanoparticles capped by different PEG ligands.
Adv. Mater. 2014, 26, 2694–2698
(3)
where z = 2ω Iτ D . By assuming that the water impermeable thicknesses of the PEGylated nanoparticles are independent of the anchoring
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
2695
www.advmat.de
COMMUNICATION
www.MaterialsViews.com Table 1. Relaxivities (mM−1s−1) and r2/r1 ratios of the PEGylated Fe3O4 nanoparticles. 3.6 nm Fe3O4 particles Sample S@DP-PEG
r1
r2/r1
Sample
r2
r1
r2/r1
24.6
3.21
7.69
L@DP-PEG
79.1
3.24
24.4
S@HX-PEG
48.8
4.20
11.6
L@HX-PEG
92.1
3.12
29.6
S@CC-PEG
44.8
3.47
12.9
L@CC-PEG
89.7
2.48
36.2
groups, according to Equation (1) and (2), the relaxivities of the PEGylated Fe3O4 nanoparticles are mainly determined by a, MS, and τD. The influence of a can be ruled out as the PEGylation process did not alter the particle size. Therefore, the impacts of MS and τD are discussed below. The MS values of the PEGylated Fe3O4 particles together with those of the hydrophobic mother particles are provided in Table S1. In comparison with the corresponding hydrophobic counterparts, the PEGylated particles present largely decreased MS values, suggesting that the stronger binding affinity of the anchoring groups largely increases the spin disorder degrees. More careful observations reveal that the MS values follow an order of hydroxamate > catechol > diphosphate. According to literature, the first step binding constants (lg K1) of catechol and hydroxamate group for Fe3+ are 20.01 and 11.42,[30,31] respectively, and the pKSP of FePO4 is of 21.88.[32] Therefore, the binding affinities of PEG ligands to Fe3+ follow an order of diphosphate > catechol > hydroxamate, which suggests that MS is inversely correlated to the binding affinity of the anchoring group. This conclusion can find a stronger support if comparing 3.6 nm Fe3O4 particle capped by HX-PEG with that capped by CC-PEG. As shown in Table S2, these two samples have very comparable organic content, i.e., 40.1% vs. 40.9%, but the former particle presents a much higher MS value than the latter particle, i.e., 22.9 vs. 16.3 emu per gram of Fe3O4, as shown in Table S1. The translational diffusion time (τD) for water molecules to pass through PEG coating is obviously determined by the PEG density on particle surface. The thermal gravimetric analysis (TGA) reveal that the anchoring groups give rise to different surface PEG densities following the order of binding affinities, as shown in Table S2. However, r2 values follow a reversed order for both sizes of particles. As r2 is a function of the translational diffusion time (τD) and will increase with τD, it can be deduced that r2 is primarily dominated by MS rather than τD according to the current results. According to Equation (1), r1 is proportional to the square of MS and τ D J A ( 2ω Iτ D ) . The latter presents a non-monotonic variation against τD, as shown in Figure 3. Since r1 is only slightly varied by the anchoring groups, contrasting to r2 according to the experimental results shown in Table 1, it can be deduced that the variation of r1 induced by MS is largely canceled by that introduced by τDJA. Apart from the concentration-independent relaxivities, the r2/r1 ratio is also an important parameter widely used for ascertaining the suitability of Fe3O4 nanoparticles as T1 or T2 contrast agents.[18–23] By combining Equation (1) and (2), the r2/r1 ratio can be given by r2 = r1 3 J A
2696
10.9 nm Fe3O4 particles
r2
(
2 2ω Iτ D
wileyonlinelibrary.com
)
(4)
Figure 3. Plots of τDJA and r2/r1 ratio versus τD at given magnetic field of 3T.
Equation (4) indicates that r2/r1 ratio is only the function of τD, while the proton Larmor frequency ωI is fixed at 1.2773 × 108 Hz by 3 T external magnetic field. Since r2/r1 ratio monotonically increases against τD, as shown in Figure 3, according to Equation (4) the DP-PEG coated Fe3O4 nanoparticles should theoretically show higher r2/r1 ratio than CC-PEG and HX-PEG coated ones due to its higher surface PEG density. But quite to the contrary, as shown in Table 1, for both sizes of Fe3O4 nanoparticles the CC-PEG and HX-PEG give rise to much higher r2/r1 ratios than the DP-PEG, which strongly suggests that some important factors are missing in the proposed relaxometric mechanisms. In principle, the transverse relaxation originates from the dephasing of coherent proton spins, which is induced by the fluctuating dipolar interaction between the proton spin and the superparamagnetic particle’s global magnetic moment. Therefore, any factor influencing the fluctuation of magnetic field can affect the transverse relaxation. It is known that when an atom is placed in a magnetic field, its electrons circulate about the direction of the applied magnetic field. The circulation can give rise to a small magnetic field opposite to the externally applied field and thus introduce magnetic field inhomogeneity locally, but normally neglectable. Nevertheless, the circulation of π electrons can create greatly enhanced extra magnetic fields. In this context, the catechol and hydroxamate groups consisting of π–π conjugation and p-π conjugation, respectively, will lead to increased inhomogeneity to the local magnetic field around the underlying Fe3O4 nanoparticles than the unconjugated diphosphate group, and thus more effectively accelerate the transverse relaxation of water protons nearby, which explains that r2/r1 ratios follow an order of π–π conjugation
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, 26, 2694–2698
www.advmat.de www.MaterialsViews.com
Adv. Mater. 2014, 26, 2694–2698
COMMUNICATION
(catechol) > p-π conjugation (hydroxamate) > non-conjugation (diphosphate). As a matter of fact, the anchoring group effect on the transverse relaxivity is also reflected by r2 values provided in Table 1. For example, the r2 values of CC-PEG capped nanoparticles of both sizes are close to those of HX-PEG coated ones although the former particles have smaller saturation magnetizations as shown in Table S1. This can be explained by the fact that the conjugated structure of catechol involving benzene is much larger than that of hydroxamate group possessing p-π conjugation, and further suggests that the chemical structure of the anchoring group plays a remarkable role for enhancing T2 effect of Fe3O4 particles, which consequently allows 3.6 nm Fe3O4 nanoparticles capped by CC-PEG or HX-PEG to act not only as T1 contrast agent but also as T2 contrast agent. To further show the feasibility of using Fe3O4 nanoparticles as high performance T1/T2 dual-modality contrast agents, the HX-PEG coated 3.6 nm particle was adopted in the following animal experiments for detecting tumors in vivo upon the enhanced permeability and retention effect associated with the tumors. Two sets of T1-weighted and T2-weighted MR images, acquired simultaneously before and at different time points after intravenous injection of the nanoparticles into a tumorbearing mouse, are provided in the upper panel of Figure 4. The quantitative temporal variations of R1 and R2 values of the same tumor region given in lower panel of Figure 4 reveal that both R1 and R2 values increase synchronizingly and then gradually decrease. The maximal ΔR1 of 33% and ΔR2 of 54% were reached approximately 4 h postinjection. These variations are clearly reflected by the color-coded tumor imaged through both modalities, which manifests the excellent performance of the current particles as T1/T2 dual-modality MRI probe. Theoretically, ΔR2/ΔR1 ratio should be independent of Fe3O4 concentration. However, the experimentally determined ΔR2/ΔR1 ratio is not a constant against time. It firstly increases and then decreases as shown in Figure S4. This is because the accumulation of the particles within tumor will lead to particle aggregation that gives rise to increased r2/r1 and ΔR2/ΔR1 as well. As particles are gradually washed out, ΔR2/ΔR1 decreases again. In literature, great efforts have been spent to suppress the r2/r1 ratio down to at least 5 in order to enable small iron oxide particles as T1 contrast agent.[10,20–24] Nevertheless, the above results indicate that the r2/r1 ratio as the commonly used criterion for judging the suitability of iron oxide particles as T1 contrast agent may need to be reconsidered, because 3.6 nm Fe3O4 particle coated by HX-PEG presents a r2/r1 ratio higher than 11. In summary, to disclose the effects of the anchoring group of surface ligands on the magnetic properties of Fe3O4 nanoparticles, PEG2000 ligands bearing anchoring groups such as diphosphate, hydroxamate, and catechol, were designed and used to replace the hydrophobic ligands of 3.6 nm and 10.9 nm Fe3O4 nanoparticles. Towards MRI applications, dynamic light scattering studies were carried out for ruling out the influence of particle aggregation on the relaxometric behavior of the PEGylated particles. Then, MRI contrast enhancement properties of the resultant nanoparticles were carefully compared and the related theories were simplified to facilitate the discussion about the impacts of the anchoring group on saturation magnetization (MS), transverse relaxivity (r2), longitudinal relaxivity
Figure 4. Upper panel: transverse T1-weighted and T2-weighted MR images of nude mouse bearing a subcutenously transplanted tumor acquired before (Pre) and at 1, 4, and 8 h postinjection of HX-PEG coated 3.6 nm Fe3O4 nanoparticle through tail vein, the tumor site is color-coded for better showing the T1/T2 contrast enhancement effects; Lower panel: relative R1 and R2 values of the tumor site extracted at different time points postinjection.
(r1), and r2/r1 ratio. It has been demonstrated that the binding affinity of the surface ligand is strongly correlated with MS of the Fe3O4 particles, irrespective of the particle size. In addition, the chemical structure of the anchoring groups has remarkable effects on r2 and r2/r1 ratio. In particular, the conjugated structure in the anchoring group can largely enhance T2 effect by increasing inhomogeneity of the local magnetic field. In conclusion, the current investigations have for the first time, in combination with theoretical analysis, systematically uncovered the impacts of anchoring group of organic ligands on the magnetic properties especially the relaxometric properties of Fe3O4 nanoparticles, and thus provide a new strategy to tailor magnetic nanoparticles for achieving high performance MRI contrast agents potentially useful for versatile biomedical applications.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
2697
www.advmat.de
COMMUNICATION
www.MaterialsViews.com
Acknowledgements The authors thank the National Basic Research Program of China (2011CB935800) and NSFC (81090271, 21003135, 21021003) for financial support. The authors are grateful to Dr. Kan Liu from Cancer Hospital of Chinese Academy of Medical Sciences for relaxivity measurements. Received: September 21, 2013 Revised: January 13, 2014 Published online: February 24, 2014 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
2698
A. P. Alivisatos, Science 1996, 271, 933. K. A. Willets, R. P. Van Duyne, Annu. Rev. Phys. Chem. 2007, 58, 267. R. H. Kodama, J. Magn. Magn. Mater. 1999, 200, 359. M. Y. Gao, S. Kirstein, H. Möhwald, A. L. Rogach, A. Kornowski, A. Eychmüller, H. Weller, J. Phys. Chem. B 1998, 102, 8360. H. Duan, M. Kuang, X. Wang, Y. A. Wang, H. Mao, S. Nie, J. Phys. Chem. C 2008, 112, 8127. S. K. Ghosh, S. Nath, S. Kundu, K. Esumi, T. Pal, J. Phys. Chem. B 2004, 108, 13963. G. Kalyuzhny, R. W. Murray, J. Phys. Chem. B 2005, 109, 7012. M. Soreni-Harari, N. Yaacobi-Gross, D. Steiner, A. Aharoni, U. Banin, O. Millo, N. Tessler, Nano Lett. 2008, 8, 678. S. Kilina, S. Ivanov, S. Tretiak, J. Am. Chem. Soc. 2009, 131, 7717. U. I. Tromsdorf, O. T. Bruns, S. C. Salmen, U. Beisiegel, H. Weller, Nano Lett. 2009, 9, 4434. J. H. Lee, Y. M. Huh, Y. Jun, J. Seo, J. Jang, H. T. Song, S. Kim, E. J. Cho, H. G. Yoon, J. S. Suh, J. Cheon, Nat. Med. 2007, 13, 95. R. R. Qiao, C. H. Yang, M. Y. Gao, J. Mater. Chem. 2009, 19, 6274. R. R. Qiao, J. F. Zeng, Q. J. Jia, J. Du, L. Shen, M. Y. Gao, Acta Phys.Chim. Sin. 2012, 28, 993. Z. Li, H. Chen, H. B. Bao, M. Y. Gao, Chem. Mater. 2004, 16, 1391.
wileyonlinelibrary.com
[15] Z. Li, L. Wei, M. Y. Gao, H. Lei, Adv. Mater. 2005, 17, 1001. [16] F. Q. Hu, L. Wei, Z. Zhou, Y. L. Ran, Z. Li, M. Y. Gao, Adv. Mater. 2006, 18, 2553. [17] S. H. Sun, H. Zeng, J. Am. Chem. Soc. 2002, 124, 8204. [18] J. Park, K. An, Y. Hwang, J.-G. Park, H.-J. Noh, J.-Y. Kim, J.-H. Park, N.-M. Hwang, T. Hyeon, Nat. Mater. 2004, 3, 891. [19] E. D. Smolensky, H. Y. E. Park, T. S. Berquo, V. C. Pierre, Contrast Media Mol. I. 2011, 6, 189. [20] E. Taboada, E. Rodríguez, A. Roig, J. Oró, A. Roch, R. N. Muller, Langmuir 2007, 23, 4583. [21] B. H. Kim, N. Lee, H. Kim, K. An, Y. I. Park, Y. Choi, K. Shin, Y. Lee, S. G. Kwon, H. B. Na, J.-G. Park, T.-Y. Ahn, Y.-W. Kim, W. K. Moon, S. H. Choi, T. Hyeon, J. Am. Chem. Soc. 2011, 133, 12624. [22] F. Q. Hu, Q. J. Jia, Y. L. Li, M. Y. Gao, Nanotechnology 2011, 22, 245604. [23] L. Sandiford, A. Phinikaridou, A. Protti, L. K. Meszaros, X. Cui, Y. Yan, G. Frodsham, P. A. Williamson, N. Gaddum, R. M. Botnar, P. J. Blower, M. A. Green, R. T. M. de Rosales, ACS Nano 2013, 7, 500. [24] Z. Li, P. W. Yi, Q. Sun, H. Lei, H. L. Zhao, Z. H. Zhu, S. C. Smith, M. B. Lan, G. Q. Lu, Adv. Funct. Mater. 2012, 22, 2387. [25] P. Caravan, J. J. Ellison, T. J. McMurry, R. B. Lauffer, Chem. Rev. 1999, 99, 2293. [26] S. H. Koenig, K. E. Kellar, Magn. Reson. Med. 1995, 34, 227. [27] R. A. Brooks, F. Moiny, P. Gillis, Magn. Reson. Med. 2001, 45, 1014. [28] R. N. Muller, L. Vander Elst, A. Roch, J. A. Peters, E. Csajbok, P. Gillis, Y. Gossuin, Adv. Inorg. Chem. 2005, 57, 239. [29] S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. Vander Elst, R. N. Muller, Chem. Rev. 2008, 108, 2064. [30] G. Crisponi, M. Remelli, Coord. Chem. Rev. 2008, 252, 1225. [31] G. Crisponi, V. M. Nurchi, R. Silvagni, G. Faa, Polyhedron 1999, 18, 3219. [32] S. Kagaya, Y. Araki, N. Hirai, K. Hasegawa, Talanta 2005, 67, 90.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, 26, 2694–2698
COMMUNICATION
www.MaterialsViews.com
Anchoring Group Effects of Surface Ligands on Magnetic Properties of Fe3O4 Nanoparticles: Towards High Performance MRI Contrast Agents Jianfeng Zeng, Lihong Jing, Yi Hou, Mingxia Jiao, Ruirui Qiao, Qiaojuan Jia, Chunyan Liu, Fang Fang, Hao Lei, and Mingyuan Gao* Due to strong size-related effects, inorganic nanocrystals present unique physical properties, e.g., fluorescence of semiconductor nanoparticles,[1] surface plasmon resonance of metal particles,[2] and superparamagnetism of magnetic nanoparticles.[3] Apart from the particle size-related effects, the large surface-to-volume ratio also gives rise to remarkable surface effects on the physiochemical properties of the inorganic nanocrystals.[4–6] Surface ligand is in fact indispensable in the solution synthesis of high quality nanoparticles not only for increasing the compatibility of the underlying nanoparticles with dispersion media, but also for preventing the particles from uncontrollable growth. Therefore, the physical effects directly associated with particle surface ligand are important subjects for achieving advanced materials. With respect to applications, the magnetic properties to metal oxide nanoparticles are the same important as the fluorescent properties to quantum dots (QDs). The effects of surface ligand on the optical properties of QDs have received intensive studies over the past decades.[4,7–9] Nevertheless, the impacts of surface ligand on the magnetic properties of metal oxide nanocrystals are much less studied so far.[5,10] One of the biggest challenges is to vary the surface coating structure without altering the size and dispersibility of the nanoparticles. Magnetic resonance imaging (MRI) contrast agents represent one of the major applications for magnetic nanoparticles.[11–16] To disclose the impacts of surface ligand on the magnetic properties associated with MRI applications, two differently sized Fe3O4 nanoparticles capped by polyethylene glycol (PEG) ligands through different anchoring groups were prepared. The anchoring group effects on the relaxometric properties of the PEGylated Fe3O4 nanoparticles were discussed in combination
J. F. Zeng, Dr. L. H. Jing, Prof. Y. Hou, M. X. Jiao, R. R. Qiao, Dr. C. Y. Liu, Prof. M. Y. Gao Institute of Chemistry Chinese Academy of Sciences Bei Yi Jie 2, Zhong Guan Cun, Beijing 100190, China E-mail: [email protected] Dr. Q. J. Jia School of Chemistry and Environment South China Normal University Guangzhou 510006, China Prof. H. Lei, Dr. F. Fang Wuhan Institute of Physics and Mathematics Chinese Academy of Sciences Wuhan 430071, China
DOI: 10.1002/adma.201304744
2694
wileyonlinelibrary.com
with theoretical analysis. In addition, careful animal experiments were carried out for showing the outstanding performance of the PEGylated Fe3O4 particles with optimized structures as a dual-modal MRI contrast agent in sensitive detection of tumors in vivo. The hydrophobic Fe3O4 particles with average sizes of 3.6 nm and 10.9 nm were prepared through conventional thermal decomposition method by using oleic acid and oleylamine as surface ligands.[17,18] Since diphosphate (DP), hydroxamate (HX), and catechol (CC) groups possess higher binding affinities to Fe3+ than the anchoring groups of the initial hydrophobic ligands, PEG2000 ligands bearing DP group, HX group, and CC group, denoted as DP-PEG, HX-PEG, and CC-PEG, respectively, were designed and used to replace the hydrophobic ligands so as to achieve water-soluble Fe3O4 nanoparticles. The chemical structures of the PEG ligands are provided in the left panel of Figure 1. The transmission electron microscopy (TEM) images of the PEGylated particles are presented in the right panel of Figure 1. Careful statistical studies reveal that the ligand exchange did not alter the size or the size distribution profiles of both nanoparticles as shown in Figure S1 in Supporting Information (SI). Further dynamic light scattering (DLS) results shown in Figure S2 reveal that 3.6 nm and 10.9 nm Fe3O4 nanoparticles in cyclohexane present single scattering peak locating at 6.1 nm and 12.6 nm, respectively, before ligand exchange. After ligand exchange, the hydrodynamic size of the PEGylated nanoparticles in water apparently increases, while the size distribution profiles remain nearly unchanged in comparison with those of the hydrophobic counterparts. In addition, the hydrodynamic sizes and size distribution profiles are nearly independent of anchoring group with respect to particles of both sizes, which makes it possible to further compare their MRI contrast enhancement effect solely associated with the surface ligand. Although similar efforts have previously been devoted, the large difference in the size and size distribution among the resulting particles makes it difficult to extract reliable information solely associated with the ligand effects.[19] As shown in the insets of Figure 1, the solutions containing PEGylated Fe3O4 nanoparticles with the same core size and Fe concentration exhibit different degrees of brown colors, especially for 3.6 nm particles, indicating that the chelating effect of the anchoring groups alter the d-d transitions in Fe3O4 nanoparticles by different degrees. The MRI contrast enhancement effects of the aforementioned PEGylated Fe3O4 nanoparticles were evaluated on a clinical 3 T MRI scanner at room temperature. Figure 2 shows six sets of the T1- and T2-weighted MR images of aqueous
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, 26, 2694–2698
www.advmat.de www.MaterialsViews.com
solutions containing 3.6 nm or 10.9 nm Fe3O4 nanoparticles capped by PEGs through different anchoring groups. As expected, 10.9 nm Fe3O4 nanoparticles present stronger T2 effect and no obvious T1 effect. But quite unexpectedly, 3.6 nm Fe3O4 nanoparticles show strong T2 effect apart from obvious T1 effect. In fact, recent investigations have demonstrated that magnetic iron oxide nanoparticles below 5 nm are potentially useful as T1 contrast agent,[10,20–24] because the paramagnetism becomes dominant for small particles due to the increased degree of spin disorders on the particle surface.[3] Nevertheless, how to effectively enhance the T1 effect by reducing the particle size without depressing the T2 effect remains an open question.
3
r1 =
128π 2γ I2M n ⎛ 1 ⎞ 2 ⎜⎝ 1 + L/ a ⎟⎠ MS τ D J A 405ρ
r2 =
256π 2γ I2M n ⎛ 1 ⎞ MS2τ D ⎜ 1215ρ ⎝ 1 + L / a ⎟⎠
(
2ω Iτ D
)
COMMUNICATION
Figure 1. Left panel: chemical structures of the PEGs used for exchanging the hydrophobic ligands of 3.6 nm Fe3O4 nanoparticle (S) and 10.9 nm Fe3O4 nanoparticle (L); Right panel: TEM images of the resulting PEGylated particles (the embeded scale bars correspond to 50 nm); Insets: photographs of aqueous solutions of the PEGylated Fe3O4 particles with an equal Fe concentration of 20 mM.
The imaging results in Figure 2 further reveal that 3.6 nm Fe3O4 particles capped by CC-PEG and HX-PEG present stronger T2 and T1 effects than 3.6 nm particle capped by DP-PEG, especially when [Fe] is higher than 0.4 mmol/L. This anchoring group effect can also be observed from 10.9 nm Fe3O4 nanoparticles, but relatively weak. To further quantify the results presented in Figure 2, the experimentally determined longitudinal and transverse relaxation rates were simulated through linear regression fitting as provided in Figure S3. The resulting concentration-independent relaxivities, i.e., longitudinal relaxivity (r1) and transverse relaxivity (r2), are provided in Table 1. It is quite conclusive that the anchoring group has stronger impacts on r2 and r2/r1 ratio. In general, the HX-PEG and CC-PEG give rise to higher r2 values and r2/r1 ratios than the DP-PEG, irrespective of the particle size, rather in consistence with the T2-weighted imaging results shown in Figure 2. Nevertheless, there is no direct correlation between the anchoring groups and r1 values for both sizes of particles. According to theory, the relaxation enhancement generally follows the inner-sphere and outer-sphere mechanisms.[25] The inner-sphere proton relaxation involves water molecules directly bonding to paramagnetic ions, while the outer-sphere relaxation is associated with water molecules beyond the directly bonding ones. We assume that the experimentally observed proton relaxation is mainly dominated by the outer-sphere mechanism due to the highly hydrophilic nature of the PEG coatings, according to the boundary conditions associated with high magnetic field, r1 and r2 can then be given by[26–29] (1)
3
(2)
The assumptions and detailed processes for deriving Equation (1) and (2) are provided in SI. In the above equations, γI represents the proton gyromagnetic ratio, Mn and ρ are the molar mass and density of Fe3O4, respectively, a is the radius of Fe3O4 particles extracted from TEM measurements, and MS is the saturation magnetization of the Fe3O4 core particles, L is the water impermeable thickness of surface coating layer, r is the effective radius of particles (r = a + L), D is the water diffusion coefficient, τD is the translational diffusion time (τD = r2/D), and JA represents the Ayant’s spectral density function that can be given by 5z z 2 + 8 8 JA (z) = z 2 z3 4 z 4 z 5 z 6 1+ z + + + + + 2 6 81 81 648 1+
Figure 2. T1-weighted and T2-weighted MR images of aqueous solutions containing 3.6 nm or 10.9 nm Fe3O4 nanoparticles capped by different PEG ligands.
Adv. Mater. 2014, 26, 2694–2698
(3)
where z = 2ω Iτ D . By assuming that the water impermeable thicknesses of the PEGylated nanoparticles are independent of the anchoring
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
2695
www.advmat.de
COMMUNICATION
www.MaterialsViews.com Table 1. Relaxivities (mM−1s−1) and r2/r1 ratios of the PEGylated Fe3O4 nanoparticles. 3.6 nm Fe3O4 particles Sample S@DP-PEG
r1
r2/r1
Sample
r2
r1
r2/r1
24.6
3.21
7.69
L@DP-PEG
79.1
3.24
24.4
S@HX-PEG
48.8
4.20
11.6
L@HX-PEG
92.1
3.12
29.6
S@CC-PEG
44.8
3.47
12.9
L@CC-PEG
89.7
2.48
36.2
groups, according to Equation (1) and (2), the relaxivities of the PEGylated Fe3O4 nanoparticles are mainly determined by a, MS, and τD. The influence of a can be ruled out as the PEGylation process did not alter the particle size. Therefore, the impacts of MS and τD are discussed below. The MS values of the PEGylated Fe3O4 particles together with those of the hydrophobic mother particles are provided in Table S1. In comparison with the corresponding hydrophobic counterparts, the PEGylated particles present largely decreased MS values, suggesting that the stronger binding affinity of the anchoring groups largely increases the spin disorder degrees. More careful observations reveal that the MS values follow an order of hydroxamate > catechol > diphosphate. According to literature, the first step binding constants (lg K1) of catechol and hydroxamate group for Fe3+ are 20.01 and 11.42,[30,31] respectively, and the pKSP of FePO4 is of 21.88.[32] Therefore, the binding affinities of PEG ligands to Fe3+ follow an order of diphosphate > catechol > hydroxamate, which suggests that MS is inversely correlated to the binding affinity of the anchoring group. This conclusion can find a stronger support if comparing 3.6 nm Fe3O4 particle capped by HX-PEG with that capped by CC-PEG. As shown in Table S2, these two samples have very comparable organic content, i.e., 40.1% vs. 40.9%, but the former particle presents a much higher MS value than the latter particle, i.e., 22.9 vs. 16.3 emu per gram of Fe3O4, as shown in Table S1. The translational diffusion time (τD) for water molecules to pass through PEG coating is obviously determined by the PEG density on particle surface. The thermal gravimetric analysis (TGA) reveal that the anchoring groups give rise to different surface PEG densities following the order of binding affinities, as shown in Table S2. However, r2 values follow a reversed order for both sizes of particles. As r2 is a function of the translational diffusion time (τD) and will increase with τD, it can be deduced that r2 is primarily dominated by MS rather than τD according to the current results. According to Equation (1), r1 is proportional to the square of MS and τ D J A ( 2ω Iτ D ) . The latter presents a non-monotonic variation against τD, as shown in Figure 3. Since r1 is only slightly varied by the anchoring groups, contrasting to r2 according to the experimental results shown in Table 1, it can be deduced that the variation of r1 induced by MS is largely canceled by that introduced by τDJA. Apart from the concentration-independent relaxivities, the r2/r1 ratio is also an important parameter widely used for ascertaining the suitability of Fe3O4 nanoparticles as T1 or T2 contrast agents.[18–23] By combining Equation (1) and (2), the r2/r1 ratio can be given by r2 = r1 3 J A
2696
10.9 nm Fe3O4 particles
r2
(
2 2ω Iτ D
wileyonlinelibrary.com
)
(4)
Figure 3. Plots of τDJA and r2/r1 ratio versus τD at given magnetic field of 3T.
Equation (4) indicates that r2/r1 ratio is only the function of τD, while the proton Larmor frequency ωI is fixed at 1.2773 × 108 Hz by 3 T external magnetic field. Since r2/r1 ratio monotonically increases against τD, as shown in Figure 3, according to Equation (4) the DP-PEG coated Fe3O4 nanoparticles should theoretically show higher r2/r1 ratio than CC-PEG and HX-PEG coated ones due to its higher surface PEG density. But quite to the contrary, as shown in Table 1, for both sizes of Fe3O4 nanoparticles the CC-PEG and HX-PEG give rise to much higher r2/r1 ratios than the DP-PEG, which strongly suggests that some important factors are missing in the proposed relaxometric mechanisms. In principle, the transverse relaxation originates from the dephasing of coherent proton spins, which is induced by the fluctuating dipolar interaction between the proton spin and the superparamagnetic particle’s global magnetic moment. Therefore, any factor influencing the fluctuation of magnetic field can affect the transverse relaxation. It is known that when an atom is placed in a magnetic field, its electrons circulate about the direction of the applied magnetic field. The circulation can give rise to a small magnetic field opposite to the externally applied field and thus introduce magnetic field inhomogeneity locally, but normally neglectable. Nevertheless, the circulation of π electrons can create greatly enhanced extra magnetic fields. In this context, the catechol and hydroxamate groups consisting of π–π conjugation and p-π conjugation, respectively, will lead to increased inhomogeneity to the local magnetic field around the underlying Fe3O4 nanoparticles than the unconjugated diphosphate group, and thus more effectively accelerate the transverse relaxation of water protons nearby, which explains that r2/r1 ratios follow an order of π–π conjugation
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, 26, 2694–2698
www.advmat.de www.MaterialsViews.com
Adv. Mater. 2014, 26, 2694–2698
COMMUNICATION
(catechol) > p-π conjugation (hydroxamate) > non-conjugation (diphosphate). As a matter of fact, the anchoring group effect on the transverse relaxivity is also reflected by r2 values provided in Table 1. For example, the r2 values of CC-PEG capped nanoparticles of both sizes are close to those of HX-PEG coated ones although the former particles have smaller saturation magnetizations as shown in Table S1. This can be explained by the fact that the conjugated structure of catechol involving benzene is much larger than that of hydroxamate group possessing p-π conjugation, and further suggests that the chemical structure of the anchoring group plays a remarkable role for enhancing T2 effect of Fe3O4 particles, which consequently allows 3.6 nm Fe3O4 nanoparticles capped by CC-PEG or HX-PEG to act not only as T1 contrast agent but also as T2 contrast agent. To further show the feasibility of using Fe3O4 nanoparticles as high performance T1/T2 dual-modality contrast agents, the HX-PEG coated 3.6 nm particle was adopted in the following animal experiments for detecting tumors in vivo upon the enhanced permeability and retention effect associated with the tumors. Two sets of T1-weighted and T2-weighted MR images, acquired simultaneously before and at different time points after intravenous injection of the nanoparticles into a tumorbearing mouse, are provided in the upper panel of Figure 4. The quantitative temporal variations of R1 and R2 values of the same tumor region given in lower panel of Figure 4 reveal that both R1 and R2 values increase synchronizingly and then gradually decrease. The maximal ΔR1 of 33% and ΔR2 of 54% were reached approximately 4 h postinjection. These variations are clearly reflected by the color-coded tumor imaged through both modalities, which manifests the excellent performance of the current particles as T1/T2 dual-modality MRI probe. Theoretically, ΔR2/ΔR1 ratio should be independent of Fe3O4 concentration. However, the experimentally determined ΔR2/ΔR1 ratio is not a constant against time. It firstly increases and then decreases as shown in Figure S4. This is because the accumulation of the particles within tumor will lead to particle aggregation that gives rise to increased r2/r1 and ΔR2/ΔR1 as well. As particles are gradually washed out, ΔR2/ΔR1 decreases again. In literature, great efforts have been spent to suppress the r2/r1 ratio down to at least 5 in order to enable small iron oxide particles as T1 contrast agent.[10,20–24] Nevertheless, the above results indicate that the r2/r1 ratio as the commonly used criterion for judging the suitability of iron oxide particles as T1 contrast agent may need to be reconsidered, because 3.6 nm Fe3O4 particle coated by HX-PEG presents a r2/r1 ratio higher than 11. In summary, to disclose the effects of the anchoring group of surface ligands on the magnetic properties of Fe3O4 nanoparticles, PEG2000 ligands bearing anchoring groups such as diphosphate, hydroxamate, and catechol, were designed and used to replace the hydrophobic ligands of 3.6 nm and 10.9 nm Fe3O4 nanoparticles. Towards MRI applications, dynamic light scattering studies were carried out for ruling out the influence of particle aggregation on the relaxometric behavior of the PEGylated particles. Then, MRI contrast enhancement properties of the resultant nanoparticles were carefully compared and the related theories were simplified to facilitate the discussion about the impacts of the anchoring group on saturation magnetization (MS), transverse relaxivity (r2), longitudinal relaxivity
Figure 4. Upper panel: transverse T1-weighted and T2-weighted MR images of nude mouse bearing a subcutenously transplanted tumor acquired before (Pre) and at 1, 4, and 8 h postinjection of HX-PEG coated 3.6 nm Fe3O4 nanoparticle through tail vein, the tumor site is color-coded for better showing the T1/T2 contrast enhancement effects; Lower panel: relative R1 and R2 values of the tumor site extracted at different time points postinjection.
(r1), and r2/r1 ratio. It has been demonstrated that the binding affinity of the surface ligand is strongly correlated with MS of the Fe3O4 particles, irrespective of the particle size. In addition, the chemical structure of the anchoring groups has remarkable effects on r2 and r2/r1 ratio. In particular, the conjugated structure in the anchoring group can largely enhance T2 effect by increasing inhomogeneity of the local magnetic field. In conclusion, the current investigations have for the first time, in combination with theoretical analysis, systematically uncovered the impacts of anchoring group of organic ligands on the magnetic properties especially the relaxometric properties of Fe3O4 nanoparticles, and thus provide a new strategy to tailor magnetic nanoparticles for achieving high performance MRI contrast agents potentially useful for versatile biomedical applications.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
2697
www.advmat.de
COMMUNICATION
www.MaterialsViews.com
Acknowledgements The authors thank the National Basic Research Program of China (2011CB935800) and NSFC (81090271, 21003135, 21021003) for financial support. The authors are grateful to Dr. Kan Liu from Cancer Hospital of Chinese Academy of Medical Sciences for relaxivity measurements. Received: September 21, 2013 Revised: January 13, 2014 Published online: February 24, 2014 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
2698
A. P. Alivisatos, Science 1996, 271, 933. K. A. Willets, R. P. Van Duyne, Annu. Rev. Phys. Chem. 2007, 58, 267. R. H. Kodama, J. Magn. Magn. Mater. 1999, 200, 359. M. Y. Gao, S. Kirstein, H. Möhwald, A. L. Rogach, A. Kornowski, A. Eychmüller, H. Weller, J. Phys. Chem. B 1998, 102, 8360. H. Duan, M. Kuang, X. Wang, Y. A. Wang, H. Mao, S. Nie, J. Phys. Chem. C 2008, 112, 8127. S. K. Ghosh, S. Nath, S. Kundu, K. Esumi, T. Pal, J. Phys. Chem. B 2004, 108, 13963. G. Kalyuzhny, R. W. Murray, J. Phys. Chem. B 2005, 109, 7012. M. Soreni-Harari, N. Yaacobi-Gross, D. Steiner, A. Aharoni, U. Banin, O. Millo, N. Tessler, Nano Lett. 2008, 8, 678. S. Kilina, S. Ivanov, S. Tretiak, J. Am. Chem. Soc. 2009, 131, 7717. U. I. Tromsdorf, O. T. Bruns, S. C. Salmen, U. Beisiegel, H. Weller, Nano Lett. 2009, 9, 4434. J. H. Lee, Y. M. Huh, Y. Jun, J. Seo, J. Jang, H. T. Song, S. Kim, E. J. Cho, H. G. Yoon, J. S. Suh, J. Cheon, Nat. Med. 2007, 13, 95. R. R. Qiao, C. H. Yang, M. Y. Gao, J. Mater. Chem. 2009, 19, 6274. R. R. Qiao, J. F. Zeng, Q. J. Jia, J. Du, L. Shen, M. Y. Gao, Acta Phys.Chim. Sin. 2012, 28, 993. Z. Li, H. Chen, H. B. Bao, M. Y. Gao, Chem. Mater. 2004, 16, 1391.
wileyonlinelibrary.com
[15] Z. Li, L. Wei, M. Y. Gao, H. Lei, Adv. Mater. 2005, 17, 1001. [16] F. Q. Hu, L. Wei, Z. Zhou, Y. L. Ran, Z. Li, M. Y. Gao, Adv. Mater. 2006, 18, 2553. [17] S. H. Sun, H. Zeng, J. Am. Chem. Soc. 2002, 124, 8204. [18] J. Park, K. An, Y. Hwang, J.-G. Park, H.-J. Noh, J.-Y. Kim, J.-H. Park, N.-M. Hwang, T. Hyeon, Nat. Mater. 2004, 3, 891. [19] E. D. Smolensky, H. Y. E. Park, T. S. Berquo, V. C. Pierre, Contrast Media Mol. I. 2011, 6, 189. [20] E. Taboada, E. Rodríguez, A. Roig, J. Oró, A. Roch, R. N. Muller, Langmuir 2007, 23, 4583. [21] B. H. Kim, N. Lee, H. Kim, K. An, Y. I. Park, Y. Choi, K. Shin, Y. Lee, S. G. Kwon, H. B. Na, J.-G. Park, T.-Y. Ahn, Y.-W. Kim, W. K. Moon, S. H. Choi, T. Hyeon, J. Am. Chem. Soc. 2011, 133, 12624. [22] F. Q. Hu, Q. J. Jia, Y. L. Li, M. Y. Gao, Nanotechnology 2011, 22, 245604. [23] L. Sandiford, A. Phinikaridou, A. Protti, L. K. Meszaros, X. Cui, Y. Yan, G. Frodsham, P. A. Williamson, N. Gaddum, R. M. Botnar, P. J. Blower, M. A. Green, R. T. M. de Rosales, ACS Nano 2013, 7, 500. [24] Z. Li, P. W. Yi, Q. Sun, H. Lei, H. L. Zhao, Z. H. Zhu, S. C. Smith, M. B. Lan, G. Q. Lu, Adv. Funct. Mater. 2012, 22, 2387. [25] P. Caravan, J. J. Ellison, T. J. McMurry, R. B. Lauffer, Chem. Rev. 1999, 99, 2293. [26] S. H. Koenig, K. E. Kellar, Magn. Reson. Med. 1995, 34, 227. [27] R. A. Brooks, F. Moiny, P. Gillis, Magn. Reson. Med. 2001, 45, 1014. [28] R. N. Muller, L. Vander Elst, A. Roch, J. A. Peters, E. Csajbok, P. Gillis, Y. Gossuin, Adv. Inorg. Chem. 2005, 57, 239. [29] S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. Vander Elst, R. N. Muller, Chem. Rev. 2008, 108, 2064. [30] G. Crisponi, M. Remelli, Coord. Chem. Rev. 2008, 252, 1225. [31] G. Crisponi, V. M. Nurchi, R. Silvagni, G. Faa, Polyhedron 1999, 18, 3219. [32] S. Kagaya, Y. Araki, N. Hirai, K. Hasegawa, Talanta 2005, 67, 90.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, 26, 2694–2698
