Chapter 7 MAGNETIC RESONANCE IMAGING (MRI) STUDIES OF MODIFIED MESOPOROUS SILICA

192 Chapter 7 MAGNETIC RESONANCE IMAGING (MRI) STUDIES OF MODIFIED MESOPOROUS SILICA 7.1. Introduction Imaging of molecular and cellular level changes occurring during diseased conditions with high resolution and sensitivity is crucial for early diagnosis. Imaging also helps in monitoring the progress in the treatment of disorders. An ideal diagnostic tool must be able to provide accurate, reproducible information with high degree of precision and must be patient-friendly giving least discomfort. One of the major non-invasive techniques that are widely employed for clinical diagnosis is magnetic resonance imaging (MRI). The attributes of magnetic resonance imaging allow high-resolution anatomical and functional images of numerous organ systems to be obtained. MRI is also highly suited for molecular imaging applications, providing images of molecular pathways to and from cells and enabling visualization of biological events at the cellular and subcellular level [1]. MRI provides excellent soft-tissue contrast and resolution by taking advantage of the relaxation properties of hydrogen nuclei in the local tissue and the surrounding contrast medium [2]. The hydrogen nuclei present in water, produce a signal, which can be generated into an image. Other nuclei commonly found in the biomolecules (e.g., 31 P, 13 C, and 15 N) have also been used in some forms of MRI for signal detection from the biological system. One great advantage of MRI lies in the fact that it does not produce ionizing radiation in contrast to other diagnostic imaging tools like positron emission

193 tomography (PET) and computed tomography (CT). However, the sensitivity of MRI is lower than PET. The origin of MRI signals lies in the spin relaxation processes that occur in the nuclei irradiated with radio frequency waves in the presence of an external magnetic field. 7.1.1. Relaxation processes Images are primarily T1 or T2 weighted or a form of mixed contrast. The T1 and T2 relaxations are dependent on the local environment being imaged and contrast agents used. Figure 7.1 depicts the processes leading to T1, T2 and T2 * relaxations.

194 Figure 7.1: T1 & T2 relaxation mechanism 7.1.1.1. T1 Processes At equilibrium, the net magnetization vector lies along the direction of the applied magnetic field B o and is referred to as the equilibrium magnetization M o. In this configuration, the Z component of magnetization M Z equals M o. M Z is referred to as the longitudinal magnetization and there is no transverse (M X or M Y ) magnetization in this case. It is possible to change the net magnetization by exposing the nuclear spin system to a frequency equal to the energy difference between the spin states. If enough energy is

195 provided to the system, it is possible to saturate the spins and make M Z =0. The time constant, which describes how M Z returns to its equilibrium value, is called the spinlattice relaxation time (T1). The equation governing this behavior as a function of the repetition time t after its displacement is: M Z = M O 1 e where T1 is the time taken to reduce the difference between the longitudinal magnetization (M Z ) and its equilibrium value and t is repetition time or the interval between consecutive pulses. t T1 If the net magnetization is placed along the -Z axis, it will gradually return to its equilibrium position along the +Z axis at a rate governed by T1. The equation governing this behavior as a function of the repetition time t after its displacement is: M Z = M O 1 2e Again, the spin-lattice relaxation time (T1) is the time to reduce the difference between the longitudinal magnetization (M Z ) and its equilibrium value. t T1 7.1.1.2. T2 & T2* Processes In addition to the rotation, the net magnetization starts to dephase because each of the constituent spin packets experience a slightly different magnetic field and preceses at its own Larmor frequency. The longer the elapsed time, the greater will be the phase difference. The net magnetization vector is initially along +Y and is assumed to be an overlap of several thinner vectors from the individual spin packets. The time constant,

196 which describes the return to equilibrium of the transverse magnetization, M XY, is called the spin-spin relaxation time, T2. M XY = M XYO e t T 2 T2 is always less than or equal to T1. The net magnetization in the XY plane becomes zero and then the longitudinal magnetization increases until M o lies along Z. The transverse component rotates about the direction of applied magnetization and dephases. T1 governs the rate of recovery of the longitudinal magnetization. In summary, the spin-spin relaxation time, T2, is the time taken to reduce the transverse magnetization. Though Figure 7.1 shows the T2 and T1 processes separately for clarity, in practice, both processes occur simultaneously with the only restriction being that T2 is less than or equal to T1. Two factors that contribute to the decay of transverse magnetization are molecular interactions (said to lead to a pure T2 molecular effect) and variations in B o (said to lead to an inhomogeneous T2 effect). The combination of these two factors results in the decay of transverse magnetization. The combined time constant is called T2 star and is denoted as T2*. The relationship between the T2 from molecular processes and that from inhomogeneities in the magnetic field is as follows: 1 T2 * = 1 T2 + 1 T2 in hom o

197 7.1.2. Contrast agents In order to trace a molecular entity, one needs to design an imaging process involving a specific molecule giving both a disease alarm and a detectable signal in one imaging modality. There are a number of magnetic molecules which provide their own inherent signal in MRI. Oxygen and lactate are examples of these materials widely used in MRI. Alternatively, a magnetic contrast agent (signaling probe) can be attached to biomolecules to allow for molecular imaging. A magnetic contrast agent is a magnetic molecule that can enhance the resolution and/or sensitivity of the magnetic resonance (MR) image by altering the relaxation times. Table 7.1 presents the influence of the magnetic material on the relaxation and MRI parameters. Table 7.1: Influence of magnetic property of materials on the MRI parameters Paramagnetic Superparamagnetic Effect on T1 Effect on T2 Effect on signal intensity MR pulse sequence T1 weighted sequences T2 weighted sequences Examples Gd-DTPA (diethylene triamine pentaacetic acid) Iron oxide Thus the contrast agents when is introduced into the body can change the contrast between the tissues. Contrast agents are predominantly paramagnetic materials, but a few are ferromagnetic. The contrast mechanism is different for these two classes of materials.

198 7.1.3. Contrast mechanism 7.1.3.1. Paramagnetic contrast agents Paramagnetic contrast agents change the contrast by creating magnetic fields that vary with time which promote spin-lattice and spin-spin relaxation of the water molecules. The time varying magnetic fields come from both rotational motion of the contrast agent and electron spin flips associated with the unpaired electrons in the paramagnetic material in the contrast agent. A typical paramagnetic contrast system consists of a complex of a paramagnetic metal ion such as manganese (Mn +2 ) or gadolinium (Gd +3 ). Gadolinium is the most common metal ion used in paramagnetic contrast agents. It has an electron spin of 7/2 and hence seven unpaired electrons promoting spin relaxation due to flipping spins and rotational motion. Apart from Gd +3, the divalent manganese is the only other ion approved as a paramagnetic agent for clinical imaging. The commercially available manganese-based paramagnetic constrast agent is Mn-dipyridoxyldiphosphate [Mn(DPDP)] 4 (Telsascan, GE Healthcare, USA) for liver imaging. Mn 2+ has five unpaired electrons and fast electronic relaxation rate. Gadolinium (Gd) or manganese-based MRI contrast agents that possess paramagnetic property can enhance the MRI signal intensity by shortening the longitudinal relaxation time (T1) of the protons from surrounding water molecules. [3-5]. Hence these are widely used in clinical imaging as they offer a positive contrast and provide high resolution. 7.1.3.2. Ferromagnetic and superparamagnetic contrast agents A ferromagnetic material is one which can spontaneously magnetize and when the size of the ferromagnetic materials is reduced below 20 nm, they turn superparamagnetic. Supermagnetic materials get instantly demagnetized without any hysteresis unlike

199 ferromagnetic materials demagnetize extremely slowly over a period of several years. These contrast agents change the contrast by distorting the B o magnetic field around ferromagnetic material in the contrast agent. This changes T2* of the water molecules around the ferromagnetic contrast agent. Ferromagnetic contrast agents are typically iron nanoparticles attached to an organic substrate. The ferromagnetic and superparamagnetic nanoparticles form assemblies of many atoms into an organized nanostructure. These nanoparticles can shorten transverse relaxation time (T2) and give rise to hypointense images due to negative contrast [6]. 7.1.4. Chemical structure Unfortunately, many paramagnetic metal ions (M) are toxic. To lessen their toxicity, these metal ions are typically complexed with other molecules or ions called ligands (L) to prevent them from complexing with molecules in the body. The stability of the metalligand complex (ML) is given by the formation constant (K) for the reaction. K = [ ML] M [ ][] L here, M Metal ion L Ligand K Complex formation constant Ideally, it is preferred to have a K value that would enable a complex to remain stable in physiological conditions. Unfortunately some buffers that are used to achieve the desired ph can precipitate gadolinium and alter the equilibrium. More over the challenging task in molecular imaging is that the detection sensitivity of the events at the cellular level is

200 low. So, traditional gadolinium contrast agents are ineffective to receive greater sensitivity. Gadolinium based contrast agents exhibit micromolar sensitivity, whereas nanomolar sensitivity is required for effective diagnosis. The sensitivity of a magnetic contrast agent can be improved by enhancing the relaxation times. In general, a contrast agent that alters the longitudinal relaxivity (T1) will brighten the image while a material that alters the transverse relaxivity (T2) will darken the image. A key criterion in imaging organs of the body is the accumulation of the magnetic contrast agent specifically in the organ/tissue of interest. This can be accomplished by introducing a targeting ligand on the surface of the magnetic contrast agent. Table 7.2 summarizes the attempts reported already to improve organ specificity by chemical functionalization of the magnetic contrast agent. Table 7.2: Use of different contrast agents for various biological applications Molecular agent Specific applications References Transferrin conjugated USPIO Cancer cells [7] Iron oxide conjugated Herceptin-MAb Breast cancer [8] Folate-conjugated (dendrimer) NPs Ovarian cancer [9] Beta-Amyloid1-40 peptide targeted MION Alzheimer [10] Gd-DTPA-Aß targeting Peptide Alzheimer [11, 12] Dextran coated SPIO (Ferumoxide) Liver [13] Dextran coated USPIO (Ferumoxtran-10) Lymph node, liver, angiography [14] Citrate iron oxide (VSOP-C184) Angiography [15] More organ specific, safe, non-toxic and efficacious contrast agents with a higher biocompatibility, biodistribution and tissue clearance are continually being developed to keep up with demand. Nanostructured iron oxide has been shown to possess numerous biomedical applications owing to its nano dimensions and superparamagnetic property.

201 These include RNA fishing, magnetic field activated cell sorting (MACS), magnetic contrast agents, drug delivery vehicles and the induction of hyperthermia to treat cancer [16-19]. However, recent reports have also indicated a potential toxic effect of the iron oxide nanoparticles, which have been reported to increase inflammatory reactions and induce apoptosis [20, 21]. Moreover, the absence of functional groups makes it difficult to link recognition motifs to specifically target regions of interest. Hence, a material that will not trigger adverse reactions in biological systems, but at the same time possesses magnetic properties suitable for diagnostic and therapeutic applications and functionalities and that is amenable for modifications, would be of clinical use. Mesoporous silica offers a highly oriented matrix with tunable pore size and easily modifiable functional groups. We hypothesize that the introduction of superparamagnetic iron oxide nanoparticles into the mesopores of silica may serve to integrate the unique structural characteristics of mesoporous silica with the advantages of the superior magnetic properties of iron oxide nanoparticles, which could be used for numerous in vivo applications. Most importantly, the particle growth can effectively be confined to the channels of mesoporous silica, thus limiting the particle size to the nanoscale, which is essential to retain the superparamagnetic behavior of iron oxide. Yiu et al. have reported the synthesis of iron oxide silica nanoparticles (γ-fe 2 O 3 -maghaemite and α- Fe 2 O 3 -haematite) with the iron oxide inside the mesochannels of the silica. Internalization of these particles inside mesenchymal stem cells and human osteoblasts has also been reported [22]. Furthermore, iron metal silica and magnetic silica (Fe 3 O 4 SBA 15) have been synthesized by temperature programmed reduction [23]. The iron to silica ratio is a crucial factor in determining the magnetization property of the nanoparticles. Alam et al. have used mesoporous silica as a template to synthesize Fe 2 O 3

202 nanoparticles with uniform and tunable size by a nanosieve approach [24]. Yiu et al. have demonstrated that the iron oxide SBA 15 nanoparticles coated with cationic polyethylene imine could be used for magnetofection of DNA into cells [25]. Studies on the use of nanoparticles as magnetic contrast agents have been limited to iron oxide, gadolinium complexes encapsulated in nanoparticles and single metal nanoparticles [26]. The excellent tunability and magnetic properties of covalently linked iron oxide and SBA 15 (IO SBA 15) could make it a potential candidate for magnetic contrast applications. The aim of the present study was to synthesize IO SBA 15 through a modified thermal pre-synthesis technique without compromising on the textural and structural properties of the mesoporous framework. 7.2. Materials & Methods 7.2.1. Materials Agar was procured from HiMedia, USA for use in the phantom agar assay. The mesoporous samples synthesized as per the protocols described in the previous chapters were used for the MRI study. Phosphate buffered saline was prepared from 0.1 M of disodiumhydrogen phosphate and sodium dihydrogen phosphate with 0.9 % of NaCl. Deionized water was used in all experiments. 7.2.2. Preparation of phantom agar gels for MR imaging Suspensions of IO SW and Sp- IO SW in the concentration range of 0 0.24 mg ml -1 were prepared in phosphate buffered saline (PBS). A 2.5% (w/v) agar solution was prepared by heating 250 mg of agar in 10 ml of phosphate buffer at 80 C for 20 minutes. The phantom gels were prepared by mixing 160 µl of the agar solution with 840 µl of

203 mesoporous sample suspension to achieve the desired concentration. The suspension was pre-heated at 60 C to avoid gelation while mixing and the warm mixture was mixed thoroughly in 2 ml microcentrifuge tubes. An aliquot of 250 µl of this mixture was transferred quickly to a 300 µl microcentrifuge tube and allowed to cool to room temperature. 7.2.3. Measurements of MR imaging characteristics of magnetic nanoparticles in phantom assay All MRI experiments were carried out at 4.7 T (Bruker BIOSPEC) using a 72 mm diameter transmitter/receiver coil. Tubes containing IO SW and Sp-IO-SW gel were positioned near the iso-centre of the magnet. Shimming of the main magnetic field was carried out to achieve a homogeneous magnetic field over the sample volume. To estimate the transverse relaxation time (T2) for each sample, coronal image (slice thickness = 2 mm) was acquired at various echo times (TE) from 20 to 320 ms with a repetition time (TR) of 10,000 ms. Similarly, the T1 relaxation time for each sample was measured by varying TR between 54.6 and 10 000 ms while keeping TE constant at 10.9 ms. After acquiring the images, the magnitudes of image intensities were measured using manually drawn regions of interest (ROIs) for each sample. Standard software provided by the manufacturer was used for data acquisition, reconstruction and visualization/analysis of the images. Relaxation rates R1 (=1/T1) and R2 (=1/T2) were calculated by mono-exponential curve fitting of the signal intensity vs. time (TE or TR) data (using Origin 6 software). The following equations were used for curve fitting:

204 [ ] For Re laxation Rate, R 1 : y = A * 1 e R 1 (TR ) For Re laxation Rate, R 2 : y = A + C e R 2 (TE ) [ ] Relaxivity was calculated for gels with different concentration of IO SW and Sp-IO SW. T1 relaxivity, R1 (or T2 relaxivity, R2) was calculated as the slope from a plot of R1 (or R2) versus IO-SW (or Sp-IO-SW) concentration in gels to compare the formulations sensitivity for contrast enhancing properties.

205 7.3. Results & Discussion Figure 7.2 shows the magnetic resonance (MR) images of IO SW and Sp-IO-SW samples that exhibit a significant concentration dependent change in the signal intensities. 0.24 mg/ml Control 0.12 mg/ml 0.06 mg/ml A Signal Intensity 220000 200000 180000 160000 140000 120000 100000 80000 60000 C 0 mg/ml 0.015 mg/ml 0.03 mg/ml 0.06 mg/ml 0.12 mg/ml 0.24 mg/ml 40000 20000 0 50 100 150 200 250 300 TE (msec -1 ) 0.015 mg/ml 0.03 mg/ml B Signal Intensity 300000 250000 200000 150000 100000 D 0 mg/ml 0.015 mg/ml 0.03 mg/ml 0.06 mg/ml 0.12 mg/ml 0.24 mg/ml 0.24 mg/ml Control 50000 0 50 100 150 200 250 300 TE (msec) 0.12 mg/ml 0.06 mg/ml 0.015 mg/ml 0.03 mg/ml Relaxation Rate, R 2 (msec -1 ) 0.010 0.009 0.008 0.007 0.006 Spherical IO-SBA-15 IO-SBA-15 Sp-IO-SW IO-SW E 0.005 0.00 0.05 0.10 0.15 0.20 0.25 Concentration of sample (mg/ml) Figure 7.2: Magnetic resonance signal intensity [A & B] of T2 and [C & D] T2 weighted images of [A & C] Sp-IO SW and [B & D] IO SW

206 The transverse relaxivity R2 (1/T2) and longitudinal relaxivity R1 (1/T1) exhibit a linear change with increasing concentrations of IO SW and Sp-IO-SW (Figure 7.3). While both IO-SW and Sp-IO SW exhibit similar slopes, the Sp-IO SW exhibits a sharper increase in R2 compared with the rodshaped samples (Figure 7.3). The T2 relaxation process occurs because of the exchange of energy between protons in water molecules [27]. On application of an external magnetic field, the magnetic particles create an inhomogeneity in the magnetic field that affects the micro-environment and results in dephasing of the magnetic moments of protons, leading to T2 shortening. The presence of the micropores and mesoporous channels in the IO SW and Sp-IO-SW samples contributes to anisotropic diffusion of water along the channels [28]. This results in efficient relaxation of water near the magnetic Fe 2 O 3 nanoparticles. The higher surface area to volume ratio of IO SW and Sp-IO-SW also contributes to better exchange of water with the iron oxide nanoparticles that are inside the mesopores and on the surface of the pore walls. The close proximity of protons to the contrast agent leads to a reduction in the spin lattice relaxation time [29]. The smaller pores and hydrophilic OH groups present in the IO SW and Sp-IO-SW contribute to retaining the water molecules more effectively in close proximity to the Fe 2 O 3 inside the micropores and mesopores. The Sp-IO SW has a smaller pore size (7 nm) and even smaller pore volume (0.4209 cm 3 g -1 ) thereby enabling more close interactions of the protons with the iron oxide inside the pores.

207 A 0.24 mg/ml Control 0.12 mg/ml 0.06 mg/ml 0.12 mg/ml 0.06 mg/ml Signal Intensity 450000 400000 350000 300000 250000 200000 150000 100000 0 mg/ml 0.015 mg/ml 0.03 mg/ml 0.06 mg/ml 0.12 mg/ml 0.24 mg/ml C 50000 0 0 2000 4000 6000 8000 10000 Recovery time [msec] 0.015 mg/ml 0.03 mg/ml 0.015 mg/ml 0.03 mg/ml 500000 D 400000 0.24 mg/ml B Control B 0.24 mg/ml Control Signal Intensity 300000 200000 100000 0 mg/ml 0.015 mg/ml 0.03 mg/ml 0.06 mg/ml 0.12 mg/ml 0.24 mg/ml 0 0 2000 4000 6000 8000 10000 Recovery time [msec] 0.12 mg/ml 0.06 mg/ml 0.12 mg/ml 0.06 mg/ml 0.010 0.009 Spherical IO-SBA-15 IO-SBA-15 E 0.015 mg/ml 0.03 mg/ml 0.015 mg/ml 0.03 mg/ml Relaxation Rate, R 1 (msec -1 ) 0.008 0.007 0.006 0.005 0.004 Sp-IO-SW IO-SW 0.003 0.00 0.05 0.10 0.15 0.20 0.25 Concentration of sample (mg/ml) Figure 7.3: Magnetic resonance signal intensity of T1 [A & B] & [C & D] T1 weighted images of [A & C] Sp-IO SW and [B & D] IO SW

208 The Solomon Bloembergen Morgan theory provides three important requirements for highly sensitive magnetic nanoparticles and they are a large number of labile water molecules in the vicinity of the metal, optimum residence lifetime of water at the metal site and slow tumbling motion of the nanoparticle [30]. The mesoporous channel in the IO SW and Sp-IO-SW enables both fast diffusion of large quantities of water which is retained effectively in the pores owing to the hydrophilic pore walls as well as restricting the tumbling motion of the molecules due to the constraining environment inside the micropores and mesopores (Figure 7.4). Figure 7.4: Schematic representation of the iron incorporated mesoporous silica Therefore the IO SW and Sp-IO-SW has immense potential to act as a highly sensitive magnetic contrast agent.

209 The specific relaxivities of the IO SW and Sp-IO-SW samples are shown in Table 7.3. The R2 values for both IO-SW and Sp-IO SW samples are similar (15.9 s -1 mg -1 ml and 15.7 s -1 mg -1 ml respectively) at 4.7 T. The R1 values of Sp-IO SW are twice that of the rod-shaped IO-SW samples (24.4 s -1 mg -1 ml and 12.2 s -1 mg -1 ml respectively) at 4.7 T, indicating good T1 contrast. This may be attributed to the greater confinement of water and closer proximity of protons in the smaller pores of Sp-IO SW. Table 7.3: Specific relaxivities of Sp-IO-SW and IO-SW Sample T1 relaxivity T2 relaxivity (s -1 mg -1 R ml) (s -1 mg -1 ml) R Spherical IO SW 24.4 0.992 15.7 0.994 IO SW 12.2 0.997 15.9 0.996 R- Regression coefficient A comparison of the R2/R1 ratios reported in literature for some nanostructured material is given in Table 7.4. The R2/R1 values for both IO-SW and Sp-IO SW are 0.64 and 1.3, respectively at 4.7 T. A ratio close to 1 indicates that the material possesses an excellent positive contrast nature imparting good resolution and sensitivity to the image [28]. Thus, the IO-SW samples show excellent positive and negative contrast properties which may be attributed to the better pore order (Chapter 6) while the Sp-IO SW samples possess a good potential to be used as a positive contrast agent. These results also highlight the importance of highly oriented mesoporous framework for excellent T1 and T2 contrast properties.

210 28.1 [27] Table 7.4: Magnetic contrast agents and their relaxivity parameters Contrast agent Contrast behaviour R1 R2 R2/R1 References PVP-grafted MNPs 2.6 (mmol/l) 1 72.1 (mmol/l) 1 s 1 s 1 MSN-Gd (at 3 T) 28.8 65.5 mm -1 s -1 mm -1 s -1 2.3 [31] MSN-Gd (at 9.4 T) 10.2 Carboxydextran-coated SPIO Fe-Pt IO-SW Sp-IO-SW 110.8 mm -1 s -1 mm -1 s -1 10.9 [31] 19.4 185.8 mm -1 s -1 mm -1 s -1 9.6 [32] 5.7 396.1 mm -1 s -1 mm -1 s -1 69.5 [33] 12.2 15.9 mm -1 s -1 mm -1 s -1 1.3 Present work 24.4 15.7 mm -1 s -1 mm -1 s -1 0.6 Present work 7.4. Conclusion The magnetic mesoporous silica (IO-SW and Sp-IO-SW) exhibited excellent magnetic resonance imaging property. The R2/R1 ratios obtained for the rod-like highly ordered mesoporous silica sample are superior to those reported in literature for mesoporous materials. Also, the surface functional groups in the mesoporous silica can be covalently modified with specific targeting ligands to enable selective imaging of tissues with high sensitivity and resolution. This study has demonstrated for the first time the importance of pore order on the magnetic contrast properties. The versatility of mesoporous silica combined with the superparamagnetic property of iron oxide represents the next generation of multifunctional magnetic contrast agents.