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Current Applied Physics 12 (2012) 969e974

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Current Applied Physics

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Particle size dependence of relaxivity for silica-coated iron oxide nanoparticles

Tanveer Ahmad a, Hongsub Bae a, Ilsu Rhee a,*, Yongmin Chang b, Jaejun Lee b, Sungwook Hong c

aDepartment of Physics, Kyungpook National University, 1370 Sankyuk-Dong, Daegu 702 701, Republic of KoreabDepartment of Radiology, Kyungpook University Hospital, Daegu 700 422, Republic of KoreacDivision of Science Education, Daegu University, Gyeongsan 712 714 Republic of Korea

a r t i c l e i n f o

Article history:Received 26 October 2011Received in revised form25 November 2011Accepted 26 December 2011Available online 2 January 2012

Keywords:Iron oxide nanoparticleSilica coatingRelaxivityContrast agentMRI

* Corresponding author. Tel.: þ82 01055125324; faE-mail address: ilrhee@knu.ac.kr (I. Rhee).

1567-1739/$ e see front matter � 2012 Elsevier B.V.doi:10.1016/j.cap.2011.12.020

a b s t r a c t

We investigate the particle size dependence of the relaxivity of hydrogen protons in an aqueous solutionof iron oxide (Fe3O4) nanoparticles coated in silica for biocompatibility. The T1 and T2 relaxation times forvarious concentrations of silica-coated nanoparticles were determined by a magnetic resonance scanner.We find that the relaxivity increased linearly with increasing particle size. The T2 relaxivity (R2) is morethan 50 times larger than the T1 relaxivity (R1) for the nanoparticle contrast agent, which reflects the factthat the T2 relaxation is mainly influenced by outer sphere processes. The high R2/R1 ratio demonstratesthat silica-coated iron oxide nanoparticles may serve as a T2 contrast agents in magnetic resonanceimaging with high efficacy.

� 2012 Elsevier B.V. All rights reserved.

1. Introduction

Recently magnetic nanoparticles have received considerableattention because of their unique properties and potential appli-cations in areas such as magnetic guided drug delivery [1], specifictargeting and imaging of cancer cells [2], hyperthermia treatmentof solid tumors [3], and contrast enhancement agents in magneticresonance imaging (MRI) [4]. MRI has been one of the mostpowerful medical diagnostic tools due to its non-invasive natureand multidimensional tomographic capabilities along with its highspatial resolution. In MRI techniques, magnetic nanoparticles canbe utilized as magnetic probes which have a signal-enhancingcapability. However, lack of surface tenability is one of the majorobstacles to using these magnetic nanoparticles for biocompatibleapplications. Various techniques for coating magnetic nano-particles with biocompatible layers have been widely studied.Coating magnetic nanoparticles with organic shells, such as macrocyclic surfactants [5] and polymers [6], or with inorganic shells [7],can enhance their stability, dispersibility, and functionality of theotherwise bare magnetic nanoparticles. The coating of magneticnanoparticles with polymers [8] and silica shells [9] has beenextensively studied.

x: þ82 539521739.

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Silica is one of the earth’s most abundant compounds and iswidely employed in both basic research and industry. Silica is animportant shell-forming material, preventing the aggregation ofmagnetic cores in liquid media by screening the magnetic dipolarattraction among the nanoparticles [10]. A silica coating protectsnanoparticles from leaching in an acidic environment, enhancingtheir chemical and thermal stabilities. Furthermore, opticallytransparent silica is a versatile material for surface modification,which is due to the existence of abundant silanol groups on thesilica layer. Silanols can easily react with alcohols and silanecoupling agents to produce stable dispersion in non-aqueoussolvents. The most important role of the silica coating is toprovide a chemically inert surface for magnetic nanoparticles inbiological systems [11,12].

In general, two different approaches have been employed tosynthesize silica coating on magnetite nanoparticles. The firstmethod relies on the well-known Stöber process [13], mainly usedfor the synthesis of silica particles, which comprises hydrolysis andpolycondensation of tetraethoxysilane under alkaline conditionsin ethanol. This Stöber method is also used for obtaining coreeshellnanoparticles [14,15], which is a quite easy way to form the silicalayer by simply mixing nanoparticles, aqueous solution, andTEOS (tetraethyle orthosilicate) in alcohol. Apart from the technicalsimplicity of the method, the coreeshell nanoparticles aremulticore and highly polydisperse, which restricts their use forbiomedical applications. The other method is based on

T. Ahmad et al. / Current Applied Physics 12 (2012) 969e974970

microemulsion synthesis, in which micelles or reverse micelles areused as mini-reactors to control the nanoparticle size and silicacoating on the magnetic nanoparticles [16,17]. Smaller and moreuniform particles can be synthesized by using the microemulsionapproach with good control over the amount of iron oxide and theresultant magnetic properties [18]. The particle size can becontrolled by varying the relative concentrations of iron salts andthe relative amounts of solvent and surfactant used in the micro-emulsion technique [19].

In this paper, we report the particle size dependence of therelaxivity of hydrogen protons in an aqueous solution of silica-coated iron oxide nanoparticles. Through MR scans and animalexperimentation, we find that silica-coated iron oxide nano-particles function as highly effective T2 contrast agents in magneticresonance imaging.

2. Experimental

Silica-coated iron oxide (Fe3O4) nanoparticles were synthesizedby the reverse micelle method [9]. For the synthesis of particles ofsize 11.5 nm, an emulsion was prepared by adding 3.5 g of sodiumdodecylbenzenesulfonate (NaDBS, Aldrich) to 30 ml of xylene(isomers plus ethylbenzene, 98.5þ%) solution, and mixing wellwith sonication for 30 min. The salt solution was composed ofFeCl2$4H2O, Fe(NO3)3 $9H2O ([Fe3þ]/[Fe2þ]) ¼ 2 and water (ACS,reagent, Aldrich). Under vigorous stirring at 500 rpm, an iron saltsolution, composed of 0.2 M FeCl2$4H2O (99% Aldrich), 0.4 MFe(NO3)3 $9H2O (98%, Aldrich), and 2 ml of water (ACS, reagent,Aldrich), was added to the emulsion solution. For stabilization ofthe reverse micelle solution (water-in-oil phase), the emulsion wasstirred continuously for 16 h at room temperature. Then, afterstirring the solution at 500 rpm under continuous argon flow for1 h in order to homogenize it, the micelle solution was slowlyheated to 90 �C, and 1 ml of a hydrazine solution (34 wt-% water

Fig. 1. TEM images of silica-coated iron oxide nanoparticles. Each of the three samples (S1,have 100 nm scale bars, and those in the right column, “b”, have 20 nm scale bars.

solution) was injected into the solution. The resulting solution wasaged for 3 h and cooled down to 40 �C within 90 min. After cooling,4 ml of TEOS (tetraethyle orthosilicate, Aldrich) was injected intothe emulsion. While stirring at 500 rpm for 6 h, silica shells wereformed on the surface of the iron oxide (Fe3O4) nanoparticles byhydrolysis of TEOS molecules in the water region of the reversemicelles. The coated nanoparticles were separated by acetone andsubsequent centrifugations. The collected nanoparticles wereredispersed in water for the relaxivity measurements. Similarly, byincreasing or decreasing the concentration of iron salts and theamount of water, we synthesized 14 nm and 10 nm particles,respectively, keeping the other experimental procedures andconditions unchanged. These 10 nm, 11.5 nm, and 14 nm sampleswere labeled as S1, S2, and S3, respectively.

The particle size distribution and structure of the silica-coatednanoparticles were checked with a TEM (transmission electronmicroscope, H-7600, Hitachi Ltd.). For the relaxivity measurements,aqueous solutions of various nanoparticle concentrations wereprepared. The concentration of nanoparticles in the aqueous solu-tion was measured with an ICP (inductively coupled plasma)spectrophotometer (Perkin Elmer 7300 DV USA). The T1 and T2relaxation times of hydrogen protons in the aqueous solution of thecoated nanoparticles were measured using an MR scanner (1.5TScanner, GE Medical System), which was also used in the animalexperimentation stage.

3. Results and discussion

Fig. 1 shows TEM images of the silica-coated iron oxide (Fe3O4)nanoparticles. The coated nanoparticles are spherical, with averagediameters of 10 nm, 11.5 nm, and 14 nm for samples S1, S2, and S3,respectively.

MR images of aqueous solutions of various nanoparticleconcentrations were obtained, some of which are shown in Fig. 2.

S2, and S3) is shown at two different scales: Images in the left column, denoted by “a”,

Fig. 2. MR images for T2 measurements. The circular images in the picture are MRimages for aqueous samples of varying concentrations. The figure shows the T2-weighted images of three samples (S1, S2, and S3) of aqueous solutions of iron oxidenanoparticles. A dose-dependent decrease in signal intensity is seen for particles in thesolution from left to right. Imaging parameters for all the three samples wereTR ¼ 2000 ms, TE ¼ 10 ms, TI ¼ 0.0 ms. However, different FOV and matrix values wereused. For S1, the FOV was 105 � 140 and a 320 � 160 matrix was used; for S2, thevalues were FOV ¼ 130 � 259 and matrix size ¼ 320 � 192, and for S3,FOV ¼ 105 � 209, and matrix size ¼ 320 � 160.

T. Ahmad et al. / Current Applied Physics 12 (2012) 969e974 971

We used three samples, S1, S2, and S3, with varying concentrationsof nanoparticles. For S1, five samples of varying concentrationsfrom 0.125 mM to 1.25 mM were used. For S2, and S3, 10 and 11samples of varying concentrations from 0.06 mM to 1.2 mM andfrom 0.12 mM to 1.75 mM were used, respectively. It is worthnoting that for the same imaging parameters and for the sameconcentrations of 0.5, 0.7, and 1.2 mM, a size-dependent increase inthe signal intensity is seen between samples S1, S2, and S3.

For T1 measurements, the inversion recovery pulse sequencewas used. MR images for 35 different values of TI (time of inver-sion), ranging from 50 to 1750ms, were obtained for samples S1, S2,and S3. The signal intensities for 35 different TI values can be ob-tained from these MR images. By fitting the data to the intensityfunction of the MR signal,

I w�1� 2Mo e� t=T1

�(1)

we can obtain the T1 relaxation time. Fig. 3-(a) shows the plots ofsignal intensity according to TI for three different samples ofnanoparticles, S1, S2, and S3, all at the same concentration of0.5 mM. A size-dependent increase in signal intensity is observed.The T1 relaxation in the S3 sample is faster than that of the S2 andS1 samples.

Fig. 3. MR signal intensity as functions of (a) The tim

The CPMG (Carr-Purcell-Meiboon-Gill) pulse sequence withmultiple spin echoes was used for T2 measurements. For sample S1,MR images for 22 different values of TE, ranging from 10 to 850 ms,were obtained, while for samples S2 and S3, MR images for 30different TE values, ranging from 10 to 1500ms, were obtained. Thesignal intensity function for T2 relaxation,

I w Mo e� t=T2 (2)

was used to determine the T2 relaxation times. Fig. 3-(b) shows T2relaxation times for the S1, S2 and S3 samples of nanoparticles, atthe same concentration of 0.5 mM. A size-dependent increase insignal intensity is observed. The T2 relaxation in the S3 sample isfaster than that of the S2 and S1 samples.

Relaxivity is a measure of the ability of MRI contrast agents toincrease the relaxation of the surrounding nuclear spins (hydrogenprotons), which can then be used to improve contrast in MRimages. Relaxivity is expressed in units of s�1 per mM of nano-particles. The contribution of paramagnetic contrast agents to therelaxation of the nuclear spins is due to both the inner and outersphere processes. The inner sphere process arises from the chem-ical interchange interaction between the bound water of theparamagnetic agents and the surrounding free water, whicheventually increases the relaxation of nuclear spins (with a largereffect on T1). On the other hand, the outer sphere process occurswhen the paramagnetic agents diffuse through free water. In thisprocess, random fluctuations of paramagnetic agents create localmagnetic field inhomogeneity, thus increasing the relaxation ofnuclear spins (with a larger effect on T2) [20]. In the clinically-usedgadolinium-based contrast agents, gadolinium ions are formed aschelates. Thus, the bound water of the chelates can continuouslyinteract with the surrounding free water and increase the T1relaxation of nuclear spins. Most gadolinium chelate agents have aninner sphere effect that is larger than the outer sphere effect, andtherefore, they are used as T1 contrast agents. On the other hand,coated ferrite nanoparticle agents are completely surrounded bytheir coating material, and the chemical interchange interaction(inner sphere process) does not occur. However, the ferrite nano-particles have a much larger magnetic moment than gadoliniumions and produce larger magnetic field fluctuations (inhomoge-neity). Due to this property of magnetic nanoparticles, they areconsidered to be ideal T2 contrast agents.

The relaxivities of nuclear spins in the aqueous solution ofmagnetic nanoparticles can be expressed as [21],

1Tim

¼ 1Tiþ RiC: (3)

e of inversion (TI) and (b) The time of echo (TE).

Fig. 4. Plots of (a) 1/T1 and (b) 1/T2 versus concentration of silica-coated iron oxide nanoparticles in aqueous solution.

Fig. 5. Plots of (a) R1 and (b) R2 relaxivity versus particle size of silica-coated iron oxide nanoparticles for the S1, S2, and S3 samples.

T. Ahmad et al. / Current Applied Physics 12 (2012) 969e974972

where i ¼ 1 or 2, and 1/Ti represents the relaxivity of nuclear spinswith no nanoparticle contrast agent. Ri is the relaxivity of nuclearspins per ppm of nanoparticles, and C represents the concentrationof nanoparticles in the aqueous solution.

Fig. 4-(a) plots 1/T1 against particle concentration for aqueoussolutions of samples S1, S2, and S3 of silica-coated iron oxidenanoparticles. The slope of each line represents the T1 relaxivity R1for that sample. The T1 relaxivities for the samples of S1, S2, and S3were 1.67, 1.88, and 2.27 (mM-s)�1, respectively. It is observed thatthe T1 relaxivity for the 14 nm sample (S3) was much greater thanthose for the 11.5 nm (S2) and 10 nm (S1) samples.

Fig. 4-(b) shows the change in 1/T2 as a function of particleconcentration for S1, S2, and S3 samples of silica-coated iron oxide

Fig. 6. T2-weighted MR images of the abdomen of a rat (S1-a) before and (S1-b) 15 min aftersignal intensity at the marked position (Ｏ) was measured as Ib/Ia ¼ 0.53. In other words, t

nanoparticles. The slope of the lines represents the T2 relaxivity R2.The T2 relaxivities for the S1, S2, and S3 samples were 81.8, 112.8,and 128.2 (mM-s)�1, respectively. The T2 relaxivity for the nano-particle samples was increased with increasing particle size. Rela-tively higher values of the T2 relaxivity than the T1 relaxivity showthat the iron oxide nanoparticles are effective T2 contrast agents.

From Fig. 5, it is clear that the values of the R1 and R2 relaxivitiesincrease linearly with increasing particle size. This is due to the factthat larger iron oxide nanoparticles possess higher magnetizationvalues and exhibit stronger MR contrast effects [22]. The ratios R2/R1 for samples S1, S2, and S3were 49, 59, and 57, respectively. Thesenanoparticles showed a high R2/R1 ratio at a field strength of 1.5 T. Acontrast agent with large relaxivity can give the same contrast

the injection of 0.21 mg of iron oxide nanoparticle agents into the rat vein. The ratio ofhe presence of the particles resulted in a 47% decrease in signal intensity.

Fig. 7. T2-weighted MR images of the abdomen of a rat (S3-a) before and (S3-b) 15 min after the injection of 0.21 mg of iron oxide nanoparticle agents into the rat vein. The ratio ofsignal intensity at the marked position (Ｏ) was measured as Ib/Ia ¼ 0.25. In other words, the presence of the particles resulted in a 75% decrease in signal intensity.

T. Ahmad et al. / Current Applied Physics 12 (2012) 969e974 973

effect with a lower dose compared to a contrast agent with smallrelaxivity. The ratio R2/R1 is also an indicator of the efficiency of T2contrast agents. Therefore, the values of R2 and the R2/R1 ratio areimportant parameters in evaluating the magnetic particles for useasMR contrast agents [23]. A T1 contrast agent has relatively high R1and low R2 relaxivities, providing a small R2/R1 ratio [24]. On theother hand, for a T2 contrast agent, a higher R2/R1 ratio results inbetter contrast efficacy.

The relaxivities of our synthesized silica-coated nanoparticlesare compared to that of the commercial T2 contrast agent Resovist(Schering AG, Germany) [25]. The R2/R1 ratio for silica-coated ironoxide nanoparticles at 1.5 T shows a threefold increase over that ofResovist at 1.41 T. As the R2/R1 ratio is known to increase with fieldstrength, we anticipate that silica-coated nanoparticles could,therefore, be used as T2 contrast agents at clinically relevant fieldstrengths (0.5e3 T). Moreover, the size of clinical-use iron oxide-based nanoparticles is around 50e200 nm, which is comparativelylarger than that of our silica-coated iron oxide nanoparticles[26,27].

The T2 relaxation enhancement effect of our coated sample wasalso observed in an animal model. We obtained abdominal MRimages of two rats, each having a weight of 150 g, both with andwithout the injection of the aqueous solution of silica-coatednanoparticles. The rats each received 0.3 ml of the aqueous solu-tion of sample S1 or S3, which corresponds to 0.21 mg of the ironoxide.

The MR images were obtained using an MRI scanner (1.5 T MRScanner, GE Medical System). A T2 image with no contrast agentwas taken for reference. Then, after the injection of the contrastagent, the T2 images were taken every 5 min. Image S1-a fromFig. 6 and image S3-a from Fig. 7 show abdominal MR imagesprior to injection of the agent. Most parts of these images corre-spond to the liver. Image S1-b from Fig. 6 and image S3-b fromFig. 7 show images taken 15 min after the injection of the agent.In these figures, with the contrast agent in the system, the liverportion of the image was rendered visibly darker. This can beattributed to faster T2 relaxation of nuclear spins in the liver, dueto the uptake of iron oxide nanoparticles by Kupffer cells in theliver. However, hepatic tumors either do not have Kupffer cells orhave Kupffer cells with reduced activity. Therefore, the T2 relax-ation time for the cells of a lesion does not change, and the imagebrightness remains the same as before the injection. This allowsus to achieve improved contrast between the liver and the tumor.The signal intensity after the injection of the S1 agent at themarked position (Ｏ in image S1-b in Fig. 6) became 47% smallerthan the signal intensity at the same position before the injection(Ｏ in image S1-a in Fig. 6). Meanwhile, the signal intensity after

the injection of the S3 agent at the marked position (Ｏ in imageS3-b in Fig. 7) became 75% smaller than the signal intensity at thesame position before the injection (Ｏ in image S3-a in Fig. 7). Theresults of this animal experimentation support the relaxivity datathat our contrast agent of silica-coated nanoparticles can be usedas a T2 agent in MRI.

4. Conclusion

We synthesized iron oxide (Fe3O4) nanoparticles by the reversemicelle method, and coated them with biocompatible silica. Wesynthesized three different samples of silica-coated iron oxide bycontrolling the nanoparticle size in the reverse micelles. The coatednanoparticles were found to be spherical in TEM images witha coreeshell structure, and showed a uniform size distributionwithan average diameter of 10 nm,11.5 nm and 14 nm for samples S1, S2and S3, respectively. The T1 and T2 relaxation times of hydrogenprotons in the aqueous solutions of various concentrations of silica-coated nanoparticles were determined by a magnetic resonancescanner. We found that the relaxivity increased linearly withincreasing particle size, showing the nanoscale size dependence ofMR properties. We found that the T2 relaxivity (R2) is much largerthan the T1 relaxivity (R1) for the nanoparticle contrast agent,which reflects the fact that the T2 relaxation is mainly influenced byouter sphere processes. The R2/R1 ratio for the silica-coated ironoxide nanoparticles was greater than 50. Of note, the R2/R1 ratio forour nanoparticles was three times higher than that of a commercialcontrast agent (Resovist), showing their potential as a T2 contrastagent with high efficiency. Animal experimentation showed 47%and 75% decreases in signal intensity for the 10 and 14 nm silica-coated iron oxide nanoparticles, which supported the notion thatour silica-coated iron oxide nanoparticles may be used as T2contrast agents in magnetic resonance imaging with high efficacy.

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