Towards molecular imaging by means of MRI

Towards Molecular Imaging by Means of

MRI

Towards Molecular Imaging by Means of

MRI

Proefschrift

ter verkrijging van de graad van doctor aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus prof. dr. ir. J. T. Fokkema voorzitter van het College voor Promoties,

in het openbaar te verdedigen

op dinsdag 13 mei 2008 om 10.00 uur

door

Małgorzata NOREK

Magister Fizyki (Uniwersytet Jagielloński w Krakowie, Polen) Geboren te Bochnia (Polen)

Dit proefschrift is goedgekeurd door promotor: Prof. dr. R. A. Sheldon

Toegevoegd promotor: Dr. ir. J. A. Peters

Samenstelling promotiecommissie:

Rector Magnificus, voorzitter

Prof. dr. R. A. Sheldon Technische Universiteit Delft, promotor

Dr. ir. J. A. Peters Technische Universiteit Delft, toegevoegd promotor Prof. dr. C. F. G. C. Geraldes Universiteit van Coimbra, Portugal

Dr. U. Zeitler Radboud Universiteit van Nijmegen Prof. dr. H. Th. Wolterbeek Technische Universiteit Delft Prof. dr. Ir. H. van Bekkum Technische Universiteit Delft Prof. dr. W. R. Hagen Technische Universiteit Delft

Contents

1 General Introduction………...11

1.1 Contrast Agents for MRI………11

1.2 Theory ………13

1.2.1 Relaxation rate enhancement mechanisms………13

1.2.1.1 Longitudinal relaxation rate enhancement ………...15

1.2.1.2 Transverse relaxation rate enhancement………...16

1.3 Towards Dy(III) complexes as High Field Contrast Agents………...19

1.4 Dy(III) compounds as susceptibility contrast agents………...25

1.5 Dy(III) containing materials as potential contrast agents for molecular imaging……...30

1.6 Concluding remarks………....35

1.7 Outline of this thesis..……….36

References………...38

2 NMR Transversal Relaxivity of Suspensions of Lanthanide Oxide Nanoparticles…...43

2.1 Introduction……….44

2.2 Expermental Section………...45

2.3 Results and Discussion………47

2.3.1 Bulk magnetic susceptibility shifts………..48

2.3.2 Relaxation rates (R1, R2*, and R2)……….…...50

2.4 Conclusions ………61

3 Tuning of the Size of Dy2O3 Nanoparticles for Optimal Performance as an MRI

Contrast Agents…..……….71

3.1 Introduction………...72

3.2 Experimental Section………..74

3.3 Results and Discussion………76

3.4 Conclusions……….87

Appendix 2………..……….………...90

References………...93

4 The Dependence of the Magnetization of Dy2O3 Nanoparticles on the Temperature. Effects of Particle Size and Coating………….……….…………..97

4.1 Introduction………...98

4.2 Experimental Section………100

4.3 Results and Discussion……….101

4.3.1 M (B) as a function of temperature and particle size……….101

4.3.2 Effect of particle coating on M (B)..…….……….………109

4.4 Conclusions………...111

References………113

5 NMR Transversal Relaxivity of Aqueous Suspensions of Particles of Ln3+-Based Zeolite Type Materials………..115

5. 1 Introduction………..116

5.2 Experimental Section………119

5.3 Results and Discussion………..…120

5.3.1 Characterization of the Ln-Av-9 particles………..120

5.3.3 Transverse relaxation rates………123

5.4 Conclusions………...131

References……….134

6 1H Relaxivity of Water in Aqueous Suspensions of Gd3+ loaded NaY Nanozeolites and AlTUD-1 Mesoporous Material; the Influence of Si/Al Ratio and Pore Size…………..137

6.1 Introduction………...138

6.2 Experimental Section………140

6.3 Results and Discussion……….142

6.3.1 Characterization of the Gd3+ loaded materials………...142

6.3.2 Relaxivity measurements………...146 6.4 Conclusions………...153 References……….155 Summary...159 Samenvatting………163 Aknowledgements………167 List of Publications….………..169 Curriculum Vitae………..171

Chapter 1. General Introduction

1.1 Contrast Agents for MRI

Contrast agents (CAs) for Magnetic Resonance Imaging (MRI) can be classified as positive or negative depending on whether they give rise to a brightening or a darkening in the MR image. Positive CAs have a predominant enhancement effect on T1, whereas negative CAs affect predominantly T2. To date, the majority of the commercially available positive CAs are Gd(III) chelates, whereas iron oxide particles are applied as negative CAs. 1,2

The success of Gd3+ in MRI applications can be traced back to its isotropic electronic ground state (8S7/2). Its half filled f-orbital with seven electrons has no net orbital momentum, resulting in near zero spin-orbit interaction. Consequently, the electronic relaxation time Tie is relatively long, around 104 -105 orders of magnitude longer than the Tie of the other paramagnetic lanthanides, leading to comparably large effects on both longitudinal and transverse proton relaxations at relatively low field (<1.5 T).3 Maximal longitudinal relaxivities are usually reached for compounds with slow molecular tumbling at about 0.2 T. Above 1 T, the water proton relaxivities decrease: the longitudinal one towards zero, and the transverse one, because of so-called “secular” term, towards some non-zero value (depending on the relaxation mechanism).4

By contrast, the electronic ground state of Dy(III) (6H15/2) is highly anisotropic. Its nine f-electrons distribute among the seven f-orbitals with a large spin-orbit interaction, and as a result the electronic relaxation times are very short ( ~10-13 s). Because of their low value of

Tie, Dy(III) compounds affect very little proton relaxation processes. As a result of the anisotropy of the ground state of Dy, a Larmor frequency difference, ∆ω, exists between water coordinated to Dy(III) and the bulk water.5 The modulation of ∆ω by the water

exchange process is an important contribution to the transverse relaxivity, which is proportional to the strength of the external magnetic field.

At present there is a trend towards higher magnetic fields (> 1.5 T) in MRI, which is dictated by the desire to improve the spatial resolution of the images, to increase the contrast to noise ratio, and to shorten the measuring time. In general, Gd(III) and Dy(III) based CAs are complementary, since the former have their optimal performance in fields up to approximately 1.5 T, whereas the latter seem to possess all important features to provide high water proton relaxivity enhancement at magnetic fields > 2.4 T. As a result recently, a revival of the interest of Dy(III) based contrast agents has occurred.

Because of the high magnetic moment, Dy(III) complexes have been proposed in the 1990s as so-called susceptibility contrast agents for T2- and T2*- weighted imaging. The susceptibility induced relaxation mechanism does not require a direct interaction of the paramagnetic ions with water protons, but its efficiency is strongly dependent on the concentration of paramagnetic entities: relatively high amounts of CAs (> 0.3 mmol/kg) have to be delivered in order to achieve sufficient contrast.6,7 Another important requirement for susceptibility CAs to be efficient is that they should be compartmentalized.8-10 When homogeneously distributed, these compounds are not able to form the strong local field gradient needed for the shortening of the transverse relaxation time T2 in the absence of any contact or dipolar interactions. Water protons diffusing through this strong field gradient lose their phase coherence, and as an effect T2 is shortened. Dy(III) complexes, especially the non-ionic Dy-DTPA-BMA (see Figure 1.1) have been applied in, for example, the imaging of cerebral perfusion and delineation of ischemic regions in myocardium.11,12 An important advantage over Gd(III) complexes (apart from a higher magnetic moment) is the short T1e of Dy, which does not introduce any disturbing factors in the interpretation of the T2- and T2*- weighted images, therefore providing a selective negative contrast enhancement. It was found,

for instance, that the long T1e of Gd seriously complicates the calculation of regional cerebral blood volumes from MR images made after administration of Gd(III)-based CAs.13,14

N N N NH HN O O O O O O _O O O H H CH3 H₃C Dy-DTPA-BMA

Figure 1.1 Chemical structure of Dy-DTPA-BMA.

In this chapter, we review the literature on theory of the relaxation rate enhancements by Dy(III) compounds and their potential applications as CAs at relatively high magnetic field (>1.5 T) and as susceptibility CAs. Materials containing a high payload of Dy(III) as potential CAs in molecular imaging by means of MRI will be also discussed.

1.2 Theory

1.2.1 Relaxation rate enhancement mechanisms

Upon addition of a lanthanide containing compound or material to an aqueous medium, the relaxation rates of the water nuclei are enhanced. This effect arises via four mechanisms: the diamagnetic (Ridia), the dipolar (RiD), the contact (RiC), and the Curie mechanisms (Riχ) (Eq 1.1).

Ri = Ridia + RiD + RiC + Riχ i = 1,2 Eq 1.1 The diamagnetic contribution to the relaxation rate enhancement is usually negligible. The dipolar contribution is an effect via space and is the result of dipolar coupling between the electron spin of the unpaired electrons of the lanthanide ion and the nuclear spins. It can

an effect that is transmitted through chemical bonds and it results from a scalar coupling between the unpaired electrons and the nuclear spins.16 A contribution, which is insignificant for Gd(III), but which plays an important role in the relaxation enhancement of water nuclei in the presence of Dy(III), is the so called Curie spin relaxation. It arises from the dipolar interaction of nuclei with the thermal average of the electron spin polarization (“Curie spin”).17,18 It can become an important contributor to the nuclear relaxivity only when the electronic relaxation time T1e of the paramagnetic ion in question is short enough to allow for spins to come back to their thermal equilibrium before the molecule changes its position. In other words, during the time T1e the molecule must be virtually stable, which is equivalent to the condition: τR>> T1e,where τR is the rotational correlation time. Under this condition, the relaxation process induced by the “Curie moment” is unaffected by the electronic relaxation time. For small complexes τR is ~ 10-10 s and for dysprosium T1e ~ 10-13 s, so the above prerequisite is met well. At room temperature, the induced magnetization can be expressed by Eq 1.2. 2 2 _{( 1)} 3 B C g J J B kT μ μ = + Eq 1.2 Here μB is the Bohr magneton, g the Landé factor (for Dy g=1.333), J the total spin quantum number (for Dy J=15/2), B the external magnetic field strength, k the Boltzmann constant, and T the temperature. For physiological temperatures, it can be deduced from Eq 1.2 that the Curie mechanism can play a role for the paramagnetic ions characterized by large J , and at relatively high magnetic fields. Among paramagnetic ions Dy(III) possesses the highest J.

B

The overall relaxation process is modulated by chemical exchange, which is given by the Swift-Connick theory and has been described in detail by Vander Elst. et al. 19,20 (see below).

1.2.1.1 Longitudinal relaxation rate enhancement

The proton paramagnetic longitudinal relaxation rate of water is the sum of inner (R1IS) and outer (R1OS) sphere contributions. The former is given by Eq 1.3:

1 1 1 IS M M R fq T τ = + Eq 1.3 R1IS depends on the molar ratio of Dy(III) and water (f), on the number of water molecules coordinated to the paramagnetic ion (q), on the residence time of the coordinated water molecules exchanging with the bulk (τM), and on the relaxation rate of the bound nuclei (R1M =1/T1M ). The R1M relaxation rate is composed of contributions from the contact (R1C), the dipolar (R1D), and the Curie mechanisms (R1χ). For lanthanide ions other than Gd(III), the contact term is much smaller than the dipolar and Curie ones, and can thus be neglected.21 So at the end R1M =R1D + R1χ, which terms are expressed by Eq 1.4 and 1.5.

2 2 2 0 1 1 6 2 2 2 2 1 2 3 7 2 1 15 4 1 1 C C D I eff I C S C R r μ _{γ μ} τ π ω τ ⎡ ⎤ ⎛ ⎞ = _{⎜ ⎟ ⎢} + + ⎝ ⎠ _{⎣ ⎦} 2 τ ω τ + _⎥ Eq 1.4 2 2 2 0 1 6 2 2 3 2 1 5 4 1 CC I C I CC R r χ μ _{γ μ} τ π ω ⎡ ⎤ ⎛ ⎞ = _{⎜ ⎟ ⎢} + ⎝ ⎠ _⎣ τ _⎦⎥ Eq 1.5 Here μ0 is the vacuum permeability, γI the gyromagnetic ratio of the 1H nucleus, μeff =μBg√J(J+1), the effective magnetic moment of the lanthanide ion (for Dy μeff=10.6μB), r the distance between Dy(III) and the water proton, ωI, ωS are the angular precession frequencies of the proton and an electron respectively, τCiis the correlation time modulating the dipolar interaction (τCi-1= τR-1 + τM-1 + Tie-1), and, τCCthe correlation time for the Curie contribution (τCC-1= τR-1 + τM-1).

For Dy(III) complexes, the correlation time τCi is dominated by the short electronic relaxation times (τCi-1= Tie-1) and, therefore, the dipolar interaction is very low. Since τM is usually significantly longer than τR, the Curie mechanism is mostly influenced by the latter

In the fast exchange limit, when τM <<T1M , the inner sphere longitudinal relaxivity

R1IS is dominated by the relaxation rate of water coordinated to the metal ion, R1M (R1IS=fqR1M). For the opposite, the slow exchange limit, when τM >>T1M , R1IS is proportional to the water exchange rate (R1IS=fq/τM) and, therefore, the relaxivity can be significantly quenched for large values of τM. 22,23

The outer sphere longitudinal relaxation, R1OS, also consists of a dipolar term (R1DOS) modulated by the electronic relaxation times and a Curie term(R1χOS), modulated by the translational correlation time τd (τd=a2/D; a is the distance of the closest approach, D the relative diffusion constant). These two contributions are given by Eq 1.6 and 1.7, respectively.24 2 2 2 2 0 1 32 [3 ( , , ) 7 ( , , )] 135000 4 OS D I B A D I d ie D S d ie M R g N j T j T aD μ π _{γ μ ω τ ω τ} π ⎛ ⎞ ⎛ ⎞ =_{⎜ ⎟⎜ ⎟} × + ⎝ ⎠⎝ ⎠ Eq 1.6 2 2 2 2 2 0 1 32 [ ( , ) 45000 4 OS I B A C I d M R g N aD χ μ π ] j_χ γ μ μ ω π ⎛ ⎞ = _{⎜ ⎟} ⎝ ⎠ × τ Eq 1.7

Here, NA is the Avogadro number; M is the molar concentration of the paramagnetic center, and jD and jχ are the spectral density functions for dipolar and Curie interaction, respectively. Summarizing, as long as the condition τM <<T1M holds, the total longitudinal relaxivity will be negligible at low magnetic fields (B < 1.5 T) since it is governed by the short Tie, whereas it will increase at higher fields due to the contribution of the Curie mechanism, until the ωI2τR2 andωI2τd2 dispersions occur.

1.2.1.2 Transverse relaxation rate enhancement

The inner sphere term of the transverse relaxivity is given by Eq 1.8:

2 2 2 2 2 2 2 1 1 1 1 1 IS M M M M T T R fq T ω τ τ ω τ + + Δ = ⎛ ⎞ + + Δ ⎜ ⎟ ⎝ ⎠ Eq 1.8

where 1/T2M = R2M is the transverse relaxation rate of the coordinated water and Δω is the chemical shift difference between Dy-bound and free water (in rad s-1). In general, R2M has three contributors: dipolar R2D, Curie dipolar R2χ, and Curie contact R2C, which are given by Eq 1.9-1.11: 2 2 2 0 1 2 6 1 2 2 1 2 3 7 1 1 4 15 4 1 1 C C D I eff C I C S C R r μ _{γ μ τ} τ π ω τ ⎡ ⎤ ⎛ ⎞ = _{⎜ ⎟ ⎢} + + + ⎝ ⎠ _{⎣ ⎦} 2 2 2 τ ω τ + _{⎥ Eq 1.9} 2 2 2 0 2 6 3 1 1 4 5 4 1 CC I C CC I CC R r χ μ _{γ μ τ} π ω ⎡ ⎤ ⎛ ⎞ = _{⎜ ⎟ ⎢} + + ⎝ ⎠ _⎣ 2 2 _⎦ τ τ ⎥ Eq 1.10 2 2 4 3 C cont M R = Δω τ Eq 1.11

and R2M=R2D +R2χ +R2C. All symbols in Eq 1.9-1.11 are defined above and the induced contact shift, Δωcont, is given by Eq 1.12, where A/_his the Dy-1H hyperfine coupling constant.

( 1) 3 μ ω + Δ = h B cont g J J B A kT Eq 1.12

In the fast exchange limit, where τM <<T2M and when τM<<∆ω-1, Eq 1.8 can be simplified to: 2 2 2 1 IS M M R fq T τ ω ⎛ ⎞ ≅ _⎜ + Δ ⎝ ⎠⎟ Eq 1.13 When the water exchange slows down to such an extent that the condition τM ≥ ∆ω-1 can be met, R2IS will be equal to fqR2M at low magnetic fields, since then ∆ω is very low and can be neglected, but at intermediate and high fields Eq 1.8 becomes:

2 2 ₁ 2 IS M M R fq τ ω ₂ τ ω Δ ≅ + Δ Eq 1.14 From the above considerations, two important conclusions can be derived:

1) At a low magnetic field, when τM ∆ω2<<1/T2M and τM 2∆ω2<<1, the inner sphere transverse relaxivity will be completely governed by the transverse relaxivity of the coordinated water

R2M (similarly to R1IS in the fast exchange limit):

2

IS

2M

R = fqR Eq 1.15 2) At intermediate and high magnetic fields, the relaxivity will be dominated by the water exchange mechanism and then two cases can be distinguished:

a. τM ∆ω2>>1/T2M, whereas τM 2∆ω2<<1: the inner sphere transverse relaxivity is proportional to the water exchange time τM ,and to the ∆ω2:

2 2

IS

M

R = fqτ Δω Eq 1.16 b. τM ∆ω2>>1/T2M, but τM 2∆ω2>>1: this regime may occur at strong magnetic fields, where

∆ω2 is very large. Under those conditions, R2IS is independent on ∆ω, and decreases with increasing water residence times (Eq 1.17).

2 IS M fq R τ = Eq 1.17

It should be noted that Eq 1.17 also holds for the slow exchange limit, when τM >>T2M, and

R2IS = R1IS in this case.

Simulations using the equations above show that the optimum in curves of R1IS as function of

Figure 1.2 Effect of τM on the proton transverse relaxivity of a Dy-complex at 310 K (Tie = 0.15 ps, τR = 65 ps, q = 1, and ∆ω= 1.33x105 expressed here in [rad·s-1·T-1]. Reprinted with permission from [20]. Copyright (2002) Wiley-Liss, Inc.

As in the case of longitudinal relaxation rate, the transverse relaxivity has also an outer sphere contribution (R2OS), consisting of a dipolar term (R2DOS) modulated by Tie, and a Curie term (R2χOS) modulated by τd (see Eq 1.18 and 1.19).

2 2 2 2 0 2 32 [1.5 ( , , ) 2 (0, , ) 6.5 ( , , ) 135000 4 OS D I B A D I d ie D d ie D S M ] d ie R g N j T j T j T aD μ π _{γ μ ω τ τ ω τ} π ⎛ ⎞ = _{⎜ ⎟} × + + ⎝ ⎠ Eq 1.18 2 2 2 2 2 0 2 32 [1.5 ( , ) 2 (0, )] 45000 4 OS I B A C I d d M R g N j aD χ χ μ π jχ γ μ μ ω τ π ⎛ ⎞ = _{⎜ ⎟} × ⎝ ⎠ + τ Eq 1.19

1.3 Towards Dy(III) complexes as High Field Contrast Agents

The Curie contribution to longitudinal relaxation of water was observed for the first time by Bertini et al. 25 These authors investigated lanthanide(III) aqua complexes through the water proton relaxation between 0.01 and 600 MHz. It was pointed out that the high ground state degeneracy and large spin-orbit interactions in the Ln(III) ions (except Gd(III)) should be

The longitudinal water relaxivity rB1B (rB1 Bis the relaxation rate expressed in sP

-1

P

per mM concentration of paramagnetic ions) in the presence of Dy complexes, although it increases with the magnetic field strength due to the Curie mechanism (Eq 1.5), is still significantly lower than the rB1B value of commercial Gd(III) complexes. Therefore, the attention was

concentrated on the rB2B relaxivity of Dy(III) compounds.

A first systematic study of transverse relaxation rB2 B(sP

-1

P

mMP

-1

P

) of paramagnetic ions and chelates in the field range 0.04-1.5 T, was performed by Vymazal. et al.P

26

P

For the Dy(III) chelates Dy-DTPA-BMA and Dy-PL-DTPA (PL: poly-L-lysine, see Figure 1.3), the measured relaxivity was very small in this field range, but at the higher end of this range a slight increase of rB2B relaxivity was observed, which was explained by the dephasing effect of water

protons in the presence of the average electron magnetization, which is equivalent to Curie-induced relaxation process. Bulte et al. have presented an interesting study on the relaxivity of a generation 5 ammonia-core PAMAM dendrimer linked to the bifunctional dysprosium ligand p-SCN-Bz-DOTA (see Figure 1.3) in the same magnetic field range.P

27

P

Figure 1.3 Chemical structures of the ligands PL-DTPA and PAMAM- p-SCN-Bz-DOTA.

Thanks to its high molecular weight (21.6 kDa), this compound has a longer blood half- life than low-molecular weight Dy-DOTA, or Dy-DTPA chelates. Furthermore, as can be seen from the measured relaxivity of all three compounds, Dy-DOTA-PAMAM has significantly higher rB2B values than Dy-DOTA and Dy-DTPA. The magnitude of rB2 Bwas found to increase

with the magnetic field and upon decreasing the temperature, which indicates that the Curie mechanism dominates under those conditions (see Figure 1.4). The relatively high rB2B values of

Dy-DOTA-PAMAM compared to that for low molecular weight Dy-chelates can be ascribed to its longer τBRB correlation time, which is the main carrier for “Curie spin” contribution.

H₃N CH CH2 CH₂ CH2 CH2 NHR H N CH CH2 CH₂ CH2 CH2 NHR C O H N CH CH2 CH₂ CH2 CH2 NHR COO n R = N N N COO COO COO OOC C O PAMAM dendimer G=5 NHR 76 NH₂ 20 PL-DTPA PAMAM-p-SCN-Bz-DOTA N N N N COO COO OOC OOC HN S R = PAMAM dendrimer

Figure 1.4 Relaxivities of Dy-PAMAM- p-SCN-Bz-DOTA as a function of field strength.

The rB2B data are shown at 276 (▲), 283 (▼), 293 (●), and 310 (O O) K with solid lines (with

dashed lines the rB1B data are shown). Reprinted with permission from [27]. Copyright (1998)

Lippincot, Williams & Wilkins.

A study of Caravan et al., also at fields between 0.05 and 1.5 T, demonstrates that the transverse relaxation enhancement of bulk water is predominantly an inner-sphere effect.P

28

P

They compared the relaxivity of two Dy-complexes, one with one water molecule in the first coordination sphere of the metal ion (Dy-L1) and a second one with no coordinated water molecule (Dy-L2) (see Figure 1.5). The Gd(III) complex of L1 is a contrast agent for blood pool imaging, also known as MS-325.P

29

P

The second complex, the relaxivity of which is totally governed by the outer sphere effect, did not show any significant influence on the water P

1

P

H relaxation rate. Upon non-covalent binding of human serum albumin (HSA) to Dy-L1, its value of rB2B was found to increase with a factor 3-8 compared with rB2 Bin the presence of

dipolar term to RB2M B(Eq 1.10). Attention was also paid to the importance of the water

residence time, τBMB, which, independently from the Curie dipolar term RB2χB, increases both RB2M

B

(through RB2CB)BBdominating in the low magnetic field range, and the τBM B∆ωP

2

P

term (see Eq 1.13)

dominating in intermediate and high fields (as long as τBMB <<TB1MB ). P

17

P

O studies were performed to determine τBMB for the Dy-L1 complex, and it was found to be 3.2 x 10P

-8

P

s at room temperature. For these values of τBMB and the field B=1.4 T the inner sphere contribution to rB2B is

negligible.

Figure 1.5 Chemical structures of Dy-L1 and Dy-L2.

The residence time of exchanging coordinated water molecules, τBMB is the most

powerful modulator of the proton transverse relaxation in intermediate and high fields. It was shown that for a series of Dy(III) complexes characterized by similar rotational correlation times, τBRB, those with the longest τBM Bgave the largest transverse relaxation rate enhancements.P

30

P

Aime et al., in a relaxometric study of some Dy-DOTA complexes, have demonstrated that the design of efficient negative contrast agents requires the fine tuning of both τBM B and ∆ω.P

31 P N N N O O O O O O _O O O H H N N N O O O O O O N O O _O O O O O O O P O O O Dy-L1 Dy-L2 O P O O O

long τBMB can be disadvantageous, since then the condition τBM PB

2

PBB∆ωP

2

P

>>1 can be met (see Eq 1.17). In this case the RB2PB

IS

P

relaxivity is quenched by the long water residence time. The effect of τBMB on rB2B was investigated by Vander Elst. et al.P

32

P

The Dy-DOTA-4AmCE complex (see Figure 1.6), characterized by a very slow water exchange (τBMB is of the order of

8 μs at 310 K and 21 μs at 298 K), was compared with several Dy-DTPA derivatives, characterized by τBMB values in the ns range.

N N N N O H N COOEt O H N COOEt O H N EtOOC O H N EtOOC

Figure 1.6 Chemical structure of DOTA-4AmCE.

While Dy-DOTA-4AmCE gives better proton relaxivity at low and intermediate fields up to approximately 2.4 T, it becomes significantly less efficient than Dy complexes of DTPA derivatives at higher fields. Thus it was concluded that to optimize the transverse relaxivity at intermediate fields (0.5-2.4 T), Dy-complexes with long water residence times are needed (τBM

B

> 1 μs), whereas at fields higher than 2.4 T, complexes with τBMB values ranging between 0.1 μs

and 1 μs are optimal. Figure 1.7 displays simulated Nuclear Magnetic Relaxation Dispersion (NMRD) profiles as a function of τBMB. It illustrates, that upon increase of τBMB, the relaxivities

level off at a level, which increases at increasing Larmor frequencies. Therefore, to design optimal high field TB2B contrast agents, fine-tuning of the residence time of water protons is

Figure 1.7 Effect of τBMB on the rB2B NMRD profiles of a Dy-complex at 310 K (TBieB, τBRB, q, and ∆ωP

P

arePPthe same as in Figure 1.2). Reprinted with permission from [20]. Copyright (2002)

Wiley-Liss Inc.

1.4 Dy(III) compounds as susceptibility contrast agents

As mentioned in the introduction, susceptibility-induced relaxation is an effect due the loss of phase coherence of water protons diffusing in strong field gradients and it does not require a direct interaction of the paramagnetic ion with a water proton. Many factors are affecting the rate of the loss of transverse magnetization: magnitudes and spacing of the inhomogeneities, the rate of the water diffusion, the magnetic field strength, the echo time applied etc.P

33

P

Both spin-echo (SE) and fast scan gradient echo (GRE) pulse sequences can be used to observe the effects of magnetic susceptibility-induced signal changes. When susceptibility agents are used in conjunction with ultra-fast imaging techniques, functional tissue perfusion maps can be generated.P

34,35

P

In black blood Magnetic Resonance Angiography (MRA), which is the recognized tool for the evaluation of vascular diseases, intravascular susceptibility-based

contrast agents can significantly improve the accuracy of the assessment of arterial lumen size and vessel wall morphology.P

36

P

Dy(III) complexes are very suitable as negative contrast agents since they are all characterized by a large magnetic moment and by having a small effect on TB1B.P

37

P

The requirement of high dosages of CAs to cause sufficient negative contrast enhancement has led to a search for complexes with low osmolality. Up to now, the most important example is the Dy complex of DTPA-bis(methylamide) (DTPA-BMA, Figure 1.1). The ligand contains only three anionic carboxylate binding sites and, consequently, a neutral species is formed upon complexation with Dy(III). Since the resulting complex does not require counterions, it is non-ionic, which give rise to low osmolal solutions and higher stability constants.TP

38,39

PT

A study of the response of different cardiovascular parameters in rats upon injection of three equimolar dosages of ionic dysprosium diethylenetriamine pentaacetic acid dimeglumine ((NMG)B2BDyDTPA) or non-ionic DTPA-BMA CAs, showed superiority of the latter.

Dy-DTPA-BMA, administered even at a dose as high as 0.5 mmol/kg, caused no appreciable homodynamic alterations. This positive response was related to low osmolalities of the injected solutions.P

40

P

Dy-DTPA-BMA has been widely used in the imaging of myocardial and brain perfusion. The use of contrast media in MR imaging of the central nervous system is well established.P

41

P

Susceptibility CAs are advantageous over TB1B CAs because their effect and

kinetics do not depend on a breakdown of the blood-brain barrier.P

42

P

In normal brain the agents are confined in the intravascular space causing field gradients at the interface of the capillary space and surrounding tissue. When breakdown of the blood-brain barrier occurs, the CAs may accumulate within cerebral tissue. In this way the susceptibility effect is enhanced, while dipolar interaction is lost. Hence, abnormalities in the blood-brain barrier do not cause as

much local signal intensity change during measurements of cerebral perfusion by means of susceptibility CAs as would be the case with TB1B–weighted CAs.

Relaxometric properties and biokinetics of susceptibility agents can be related to important physiological parameters such as cerebral blood volume and cerebral blood flow.P

43

P

With the fast gradient-echo pulse sequence, signal intensity versus time data, based on the first pass effect of the contrast agent through the microvasculature, can be converted to concentration versus time data. Calculations of the changes of signal intensities over time for each voxel can then be used to produce high-resolution, regional blood volume images.P

44

P

It has been shown that the magnetic susceptibility agent Dy-DTA-BMA provides excellent gray/white matter contrast and information about cerebral blood flow.P

45

P

It also significantly shortens the time of detection of cerebral ischemic regions that resulted from stroke-induced perfusion deficits. Perfusion deficits were detected as a relative hyperintensity region in ischemic tissues relative to normally-perfused cerebral tissues.P

46

P

It is worth mentioning that the Dy-DTPA-BMA CA has shown to be equally effective as iron oxide superparamagnetic particles for the diagnosis of perfusion deficits, although the same susceptibility effect could be achieved only at much higher doses: 0.5 mmol/kg of Dy-DTPA-BMA as compared with 0.06 mmolFe/kg iron oxide particles.TP

47

PT

The primary goal of cardiac MRI contrast enhancement is to better define the status of myocardial perfusion. Accurate measurement of the size of the ischemic region provides an important index for the prognosis of the patient with ischemic heart disease. Contrast differences between acutely infarcted and normal myocardium can be significantly improved by using the Dy-DTPA-BMA agent. In normal myocardium, compartmentalization occurs because the contrast agent is excluded from the intracellular space. In ischemic or infarcted myocardium, the signal intensity is unaltered after administration of the CA. Therefore, the

the agent is largely excluded from the ischemic zone and homogeneously distributed in the intracellular space.P

48,49

P

In animals with coronary artery occlusion, the Dy-DTPA-BMA CA can effectively erase the signal from normally perfused myocardium on immediate post contrast images, leaving the region of ischemia as an easily recognized zone of higher signal intensity.P

50,51

P

Acquisition times with echo-planar imaging are in the range of 30-50 ms and can be gated to a specific phase of the cardiac cycle. Monitoring of the first-pass kinetics, assuming that the contrast media molecules remain primarily in the intravascular compartment, provides a means to estimate regional myocardial perfusion. Contrast-enhanced MR perfusion imaging can demonstrate the presence and relative severity of hypoperfused myocardium.P

52,53

P

It has also been shown that the abnormal myocardial areas after coronary occlusion delineated by Dy-DTPA-BMA are in closer relationship to the areas of deficient myocardial perfusion (as determined by P

201

P

thallium autoradiography) than that defined by TB1B

-enhanching contrast media.P

54

P

Preliminary human studies with non-ionic Dy-DTPA-BMA in conjunction with fast GRE and SE pulse sequences appeared to be promising in the imaging of myocardium perfusion. The fast GRE sequence was found to be more adequate in depicting the susceptibility effect during first pass of a contrast agent. SE imaging requires a longer acquisition time and, therefore, is not able to complete imaging when the agent is at its maximum concentration. The doses of the agent causing a sufficient decrease in signal intensity were 0.4 and 0.6 mmol/kg, which have a sufficient safety index (lethal dose in relation to effective dose) of 56 and 36, respectively. No adverse effects were observed and no substantial changes in heart rate or systolic blood pressure were reported.P

55

P

Electrocardiographically-gated, susceptibility-weighted echo-planar imaging (with 0.25 mmol/kg of Dy-DTPA-BMA) was also employed in imaging perfusion deficits in ischemic heart. Attention has been paid to saturation effects which may mask a relative

reduced response in ischemic segments, and other susceptibility artifacts have been analyzed.P

56

P

An excellent infarct visualization can be achieved by using a double-contrast technique, combining a positive and a negative CA.P

57,58

P

In TB1B-weighted images, a reperfused

infarction is depicted as a hyper-intense zone due to the increase in the fraction of tissue water that is accessible to the positive contrast agents.TP

59,60

PT

Because the magnitude of the Dy(III)-induced signal loss depends on the microheterogeneity of its distribution (exclusion from intracellular space), a Dy-based contrast agent will loose its potency to enhance the proton relaxation in the injured myocardium, where the loss of myocardial cell integrity appears. Therefore, the signal intensity of that zone remains unchanged upon administration of the CA. In normal myocardium, the presence of Dy(III) will cause significant darkening of the image. As a result, upon injection of both positive and negative CAs, the contrast between infarcted and non-infarcted zones becomes much more pronounced, providing a better delineation of the ischemic regions. In fact, this was confirmed in experiments on animals which were exposed to the injection of Dy-DTPA-BMA, and its congener Gd-DTPA-BMA before MRI analysis.P

61-63

P

The double-contrast effect has demonstrated its usefulness in the identification of the cell areas with reduced membrane integrity.P

64

P

Blood samples with varying hematocrit levels, containing either intact or lysed cells, were investigated. While in the samples with lysed cells the Dy-DTPA-BMA-induced susceptibility effect was negligible, this effect dominated in the intact cells due to compartmentalization of the CA inside cells. The difference between compartmentalized and non-compartmentalized regions upon applying a combination of Dy- and Gd-DTPA-BMA in MRI, appeared to be helpful in the evaluation of pathological changes of tubular necrosis in rat kidney.P

65

P

size, as measured by MRI, was identical to the size found at a postmortem examination within the limits of accuracy.P

66

P

A similar effect has been reported in a study on rat intestine: administration of both Gd- and Dy-DTPA-BMA improved the contrast between ischemic and normal intestine in TB1B/TB2B-weighted images.TP

67

PT

The double-contrast technique can of course be applied by using different positive and negative CAs. For example manganese compounds as positive CAs were administered together with barium compounds as negative CAs for imaging the gastrointestinal track, or the Cr(III) chelate of HIDA (N-hydroxy iminodiacetic acid) in combination with superparamagnetic Fe oxide were successfully applied in MRI of the bile duct and the bladder.P

68,69

P

In general, the synergistic effect of combining dipolar relaxation and susceptibility induced relaxation seems to offer MR images of significantly increased quality as compared to single, TB1B- or TB2B-enhanced MR images.

1.5 Dy(III) containing materials as potential contrast agents for molecular imaging

Recent developments in biochemical research have provided detailed knowledge of the structures of many receptors and understanding of mechanisms of the interaction involved in molecular recognition processes.P

70

P

Thanks to that, the design of contrast agents for imaging of cellular molecular events involved in normal and pathologic processes, has become feasible. Although MRI offers much higher spatial resolution than other imaging modalities like Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), its use in molecular imaging is strongly hampered by low sensitivity: a relatively high concentration of CAs has to be delivered in order to achieve the desired contrast (10P

-5

P

M).P

71

P

The design of materials loaded with high amounts of paramagnetic ions seems to be a promising way to overcome this problem. Since, magnetic entities containing a high payload of Ln(III) are able to produce large field gradients, they are extremely efficient in proton TB2

B

shortening; compartmentalization is not needed anymore. These phenomena can offer new applications in susceptibility-induced contrast enhancement.

An interesting example of this new approach is the application of red blood cells

(RBC) loaded with Dy-DTPA-BMA.P

73

P

The gradient echo transverse RB2B* relaxivity for

different tissues was measured as a function of RBCs concentration in vivo, and the dependency was found to be linear within a given tissue type (liver, spleen, kidney, skeletal muscle). However, the slope (k) of curves of RB2B* versus the concentration of the susceptibility

agent in the tissue varied significantly between different tissues. This is relevant in strategies to map regional blood volumes by MRI; here, usually k is assumed to be the same for all tissues. The differences in k values were explained by variations in the micro vascular architecture over the various tissues, which in turn influence the spatial distribution of intravascular CAs. Dy(III)-bearing RBCs can be considered as potential CAs for imaging of perfusion and tissue blood volume with some advantages over other agents like: long half-life, and circulation in the intravascular space with little sequestration.P

72

P

The role of dysprosium sequestration on the transverse relaxivity was analyzed as well. As can be seen in Figure 1.8, it is obvious that the susceptibility effect works only in the case of intact RBCs and is lost in lysed cells due to homogeneous distribution of the Dy(III) chelate in the latter case.P

Figure 1.8 Transverse relaxation effects of dysprosium sequestration within red cells. The

slope of RB2B vs [Dy] was 41.3+/-2.6 sP

-1

P

mMP

-1

P

for intact cells; after lysis it dropped to 1.0+/-0.25 sP -1 P mMP -1 P

(left panel). The slope of RB2B* vs [Dy] in the red cell mixture was 106+/-1.8 sP

-1 P mMP -1 P

for intact cells; after cell lysis it dropped to 1.5+/-0.4 sP

-1

P

mMP

-1

P

(right panel). Reprinted with permission from [73]. Copyright (2000) Wiley-Liss, Inc.

Starch particles (SP) consisting of epichlorohydrin cross-linked hydrolyzed potato starch labeled with Dy-diethylenetriamine pentaacetic acid (Dy-DTPA), with a mean diameter of about 2.5 μm, have also been envisaged as a potential negative contrast agents for liver (see Figure 1.9).P

75

P

Despite of the highly packing with paramagnetic ions, the starch particles follow the Curie low, indicating the absence of magnetic interaction between the ions. Dy-DTPA-SP reduced the liver MRI signal intensity in SE and GRE images and can be considered as large magnetized particles due to magnetization of the paramagnetic ions. Magnetization arises from the alignment of the magnetic moments of the Dy(III) ions upon placement of the particle in a static magnetic field. As usual, the susceptibility effect results from the diffusion of water protons in the outer sphere environment. The observation of the

accumulation of particles in the lungs and potential particle degradation were pointed out as a biological limitation for their use as CAs. P

76 P HOOC N N N HOOC COOH COOH COOH

Figure 1.9 Schematic structure of Dy-DTPA labeled starch particle and the chemical structure

of DTPA.

Water-soluble endohedral metallofullerenols (Ln@CB82B(OH)BnB) with encapsulated

lanthanide ions have been proposed for use as CAs in MRI. The Ln(III) ions in the fullerenols cages do not contact directly with water. Among them Dy-metallofullerenols demonstrated a relatively large effect on the transverse proton relaxivity which is increasing with the external magnetic field. This effect was ascribed to the outer sphere Curie mechanism.P

77

P

A series of aluminium-free zeolite-type silicates (Ln-AV-9) with lanthanide ions incorporated in the framework have promising properties for application as negative CAs at high magnetic fields (see Figure 1.10).TP

78

PT

These particles with an average size 5-10 μm, are characterized by negligible rB1B since, as in the case of Ln@CB82B(OH)BnB, water molecules have no

DyDTPA DyDTPA DyDTPA DyDTPA DyDTPA DyDTPA DyDTPA DTPA

diffusion of water is very slow precluding the propagation of any possible relaxivity process taking place in the interior of the zeolites. However Ln-AV-9, containing a large amount of Ln(III) per particle, shortens significantly TB2PB

* P . The largest rB2PB * P = 1/ TB2PB * P (406 sP -1 P mMP -1 P at B=7 T and room temperature) was registered for Dy-AV-9. Detailed transversal relaxivity analysis of this material will be given in chapter 5.

Figure 1.10 Structure of Ln-AV-9 materials. Reprinted with permission from [78]. Copyright

(2005) The Royal Society of Chemistry.

Recently a series of lanthanide oxide nanoparticles (LnB2BOB3B) have been investigated.P

79

P

These particles have a very high density of Ln(III) ions (around 10P

6

P

Ln(III) ions per particle) and their magnetic properties make them good candidates for RB2B*- and RB2B-weighted imaging.

Thus after coating and attachment of targeting vectors, they may have potential as CAs in molecular imaging with MRI. Among the lanthanide oxide particles, dysprosium oxide nanoparticles show the highest transverse relaxivity (both RB2B and RB2B*). The transverse

relaxation rates RB2 Bwere measured by means of the Carr-Purcell-Meiboom-Gill pulse sequence

(CPMG) and appeared to be strongly dependent on the time interval between two refocusing pulses, τBCPB. RB2B(τBCPB) curves saturated at values which were always 5-7 times smaller than

to B and to μBeffPB 2 P .P 79 P

The detailed physical characterization of the relaxivity properties of this material will be provided in chapters 2 and 3.

1. 6 Concluding remarks

Dysprosium compounds present a broad range of potential applications: at low magnetic fields as susceptibility-induced contrast agent, at high fields both as susceptibility and relaxivity-induced contrast agents. The compartmentalization of Dy-complexes is a prerequisite to work as susceptibility induced CAs. However, compartmentalization is not needed with particles containing a high payload of dysprosium, which after surface modification (coating with biocompatible materials) and targeting towards specific cell receptors, can be delivered to the site of interest causing large local signal loss. Many of these particles demonstrate promising physico-chemical properties for use as efficient CAs in MRI.

The problem how to make them biocompatible (reduce biological toxicity, minimizing or delaying the particle uptake by the mononuclear phagocyte system, etc.) remains a challenging task. Many factors can influence the behavior of magnetic entities in a biological environment, but the main are the size of the particles, the surface charge density and the hydrophilicity/ hydrophobicity balance. In general, the smaller, the more neutral and the more hydrophilic a particle is, the longer is its plasma half-life.

Dy-complexes, thanks to extremely short electronic relaxation, can be efficient “relaxers” at intermediate and high magnetic fields. Short electronic relaxation times are due to the highly anisotropic ground state of the Dy(III) ion, which also gives rise to a chemical shift difference between the water bound to the ion and the bulk water, ∆ω. The latter is modulated by the residence time of the water molecule in the inner sphere of the Dy(III) ion,

the relaxivity is quenched by the small water exchange rate. Therefore, it is important, when designing the Dy-complexes for TB2B-weighted imaging, to pay attention mostly to two

parameters: ∆ω, which increases with the external magnetic field and τBMB, which should be

compromised with the value of ∆ω to keep the transverse relaxivity optimal. The rotational correlation time, τBRB, is responsible for the “Curie spin”-induced relaxivity, RBiχB, which can be

important at low magnetic fields (where usually condition τBM B∆ωP

2

P

<<1/TB2MB holds), or for high

molecular-weight compounds.

1.7 Outline of this thesis

The work presented in this thesis is focused on the design of highly efficient contrast agents for molecular imaging by means of MRI based on the detailed physical characterization of the given material. Specifically, attention is paid on the development of CA’s for magnetic fields higher than 1 Tesla.

In Chapter 2 the transversal relaxivity rB2B of aqueous suspensions of lanthanide oxide

nanoparticles is investigated. These nanoparticles are characterized by a very efficient TB2B

shortening of water protons, which increases with the magnetic field, B, and the square of the effective magnetic moment of the lanthanide of interest. A significant difference between rB2B*

and rB2B, as determined by the classical CPMG pulse sequence, was explained by the presence

of xanthan gum, which forms a thick layer on the particle’s surface and was used as an emulsifier to prevent particles agglomeration in magnetic field. A semi-empirical model was developed to rationalize the relaxation behavior in these systems.

In Chapter 3, the dysprosium oxide nanoparticles are further investigated. The attention is focused to the effect of the size of the particles and the strength of external magnetic field, B, on the rB2B relxivity. The optimal particle size for each magnetic field is determined. The

particles surface and the strength of the local field inhomogeneities caused by the single magnetic perturber.

In Chapter 4, magnetization measurements of the samples obtained by lyophylization of a colloidal solution of DyB2BOB3 Bnanoparticles (rBpB: 1 and 50 nm) in water and dextran, and at

temperatures varying between 1.4 K and 298 K, is presented. Two components were distinguished in the magnetization curves: one associated with the simple paramagnetic behavior of the core dysprosium ions, and another, ascribed to the canting DyP

3+

P

at the particles surface. The nanosize effects of the DyB2BOB3B material on M (B) curves are discussed.

In Chapter 5, a detailed analysis of transversal relaxation of an aqueous suspension of the mesoporous material Ln-AV-9 is given. The relaxivity, as measured by the CPMG method, is always much smaller than the corresponding value of rB2B* (up to 20 times in the case of

Dy-AV-9), because of the big size of the magnetic entities (5 – 20 μm). A simple model is developed to describe the system, taking into account the residual diffusion effects in the static dephasing regime (SDR).

In Chapter 6, the longitudinal water relaxivity rB1B is investigated in the presence of GdP

3+

P

loaded nanozeolites NaY. The main interest is the influence of the pore size and the Si/Al ratio on rB1B. With respect to the former parameter, the mesoporous material Al-TUD1, loaded

with GdP

3+

P

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(79)T Norek, M.; Roch, A.; Muller, R. N.; Gillis, P.; Pereira, G. A.; Geraldes, C. F. G. C.;

Chapter 2. NMR Transversal Relaxivity of Suspensions of Lanthanide Oxide Nanoparticles

Abstract

Aqueous suspensions of paramagnetic lanthanide oxide nanoparticles have been studied by NMR relaxometry. The observed RB2B* relaxivities are explained by the static dephasing regime

(SDR) theory. The corresponding RB2B relaxivities are considerably smaller and are strongly

dependent on the interval between the two refocusing pulses. The experimental data are rationalized by assuming the value of the diffusion correlation time, τBDB, to be very long in a

layer with adsorbed xanthan on the particle's surface. In this layer, the refocusing pulses are fully effective and RB2B ≈ 0. Outside this layer, the diffusion model for weakly magnetized

particles was applied. From the fit of the experimental relaxation data with this model, both the particle radii (rBpB) and the radii of the spheres, within which the refocusing pulses are fully

effective (rBdiffB), were estimated. The values of rBpB obtained are in agreement with those

determined by dynamic light scattering. Because the value of rBdiffB depends on the external

magnetic field B and on the magnetic moment of the lanthanide of interest (μBeffPB

2

P

), the RB2B

relaxivity was found to be proportional to B and to μBeffPB

2

P

2.1 Introduction

During the last decades, the rapid progress in biochemical research has provided detailed insight into molecular recognition processes. These developments enable the design of contrast agents (CAs) for molecular imagingP

1

P

with medical diagnostic techniques including Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT) and Magnetic Resonance Imaging (MRI). MRI has a significantly higher spatial resolution (µm) than radiodiagnostic techniques (mm), but its use as a tool for the investigation of cellular molecular events in normal and pathological processes is hampered by its low sensitivity: a relatively large local concentration of CA is required (about 10P

-5

P

M) to achieve the desired contrast enhancement.P

2,3

P

Other imaging modalities such as PET, SPECT (10P -11 P – 10P -12 P

M) and optical fluorescence imaging (10P

-15

P

– 10P

-17

P

M) are much more adequate in this respect.P

4

P

A possible approach to overcome the problems related with the low sensitivity of MRI is to apply vectorized CAs, which would bring a high payload of paramagnetic compound to the site of interest. For lanthanide ion based contrast agents, this was realized in various ways and different materials have been proposed including: Gd-loaded apoferritinP

5

P

, which allows the visualization of hepatocytes when the number of Gd-complexes per cell is about 4×10P

7

P

, perfluorocarbon nanoparticles, which contain around 94200 GdP

3+

P

ions per particle providing extremely high relaxivity per particle and which have been already successfully used in molecular imaging of angiogenesis.P

6-10

Alternatively, this may be achieved with superparamagnetic (SPM) particles, single domain ferromagnets possessing a very high magnetic moment (around 10P

4 P μBBB).P 11,12 P SPM particles have a much smaller effect on the TB1B water proton relaxation time than on the TB2B. Their

relaxivity can be well described by the quantum mechanical outer-sphere theory. Because of their small size (20-60 nm in diameter), the extreme motional narrowing conditions are

satisfied, which state that water diffusion between SPM particles is rapid with respect to the difference in resonance frequencies of the various sites. In this regime the TB2B* relaxation time

is predicted to be equal to TB2B. When iron-oxide particles are compartmentalized within cells,

the internal magnetization of the compartment due to their presence has to be taken into account. In this situation the motional narrowing assumption breaks down, which results in

RB2B* ( = 1/TB2*) to be larger than RB B2B. Consequently, RB2B*-weighted MRI images are potentially

the most sensitive to the presence of cellularly compartmentalized magnetized particles.P

13-15

P

Nanozeolites present another approach. GdP

3+

P

exchanged zeolite NaY nanoparticles of an average size of 80 nm, contain about 40,000 GdP

3+

P

ions per particle. The longitudinal relaxivity

rB1B (rB1 Bis the relaxation rate expressed in sP

-1

P

mMP

-1

P

Gd) is limited by the water exchange between the interior of the zeolite and the bulk.P

16

P

It was observed that rB2B relaxivity is

independent of the pore structure of the zeolite and that it increases with the external field strength.P

17

P

In materials like Ln-AV-9, which have LnP

3+

P

ions incorporated in the zeolite framework, direct interaction between LnP

3+

P

ions and water molecules is impossible. As a result, they have a very low rB1B relaxivity, but at the same time they have very strong impact on

the TB2B relaxation.P

18

In this paper we present a study on lanthanide oxide (LnB2BOB3B) nanoparticles. These particles

have a very high density of LnP

3+

P

ions, and their magnetic properties make them good candidates for RB2B-weighted imaging, and therefore, after coating and attachment of targeting

vectors, they may have potential as CAs in molecular imaging with MRI.

2.2 Experimental Section

The lanthanide oxide nanoparticles were purchased from Aldrich and had a diameter of less than 40 nm as determined with XRD by the supplier.

Water proton transverse relaxation times, TB2B, were measured at 20, 60 MHz (Mini-spec

PC120 and PC160, respectively, spin analyzers obtained from Bruker), 200 MHz (on a Bruker Avance-200 console connected to a 200 MHz cryomagnet), 300 MHz (Varian-INOVA spectrometer), 400 MHz (Varian VXR-400 S spectrometer) and 500 MHz (Varian Unity 500 spectrometer), using the Carr-Purcell-Meiboom-Gill pulse sequence (CPMG). The values of

TB2B* were evaluated from the linewidths. All experimental values of relaxation rates were

corrected for diamagnetic contributions using a solution of 1 wt% of xanthan in water.

The LnB2BOB3B suspensions for relaxometric studies were prepared by mixing the solid particles

with doubly distilled water containing 1 wt% of xanthan gum as a surfactant and dispersing them in an ultrasonic bath for 5 min.

The self-diffusion coefficient of the samples was measured on the 200 MHz spectrometer equipped with a variable-temperature high-resolution diffusion probe. A PGMSE pulse sequence was used for the determination of the diffusion constants. The temperature was maintained by water circulation in the gradient coil. The calibration of the gradients was performed on pure HB2BO.

High-resolution transmission electron microscopy (HRTEM) was performed on a Jeol JEM-2010 electron microscope operated at 200 kV.

The Dynamic Light Scattering (DLS) was performed with a DLS/SLS/ALV-5000 apparatus using a 35 mW HeNe laser with a wavelength of 633 nm. The intensity autocorrelation function was measured at 90º and analyzed with the CONTIN method. All samples were placed in an ultrasonic bath and were centrifuged prior to the DLS measurements in order to remove dust and other contaminants.