¹⁹F MRI Probes with Tunable Chemical Switches

Abstract

Activatable ¹⁹F MRI small molecule probes have been developed to detect calcium ion, pH change, enzyme activity etc. However, small molecule based probes could not be applicable to in vivo applications owing to low sensitivity. Though PFC encapsulated nanoparticle are highly sensitive, activatable PFC encapsulated nanoparticles (switching OFF/ON-type probes) have not been reported. Thus, activatable PFC nanoparticles are highly desirable in order to realize various applications.

This thesis describes herein the development of switching OFF/ON type nanoparticles probes for detecting biological environment and biological functions. To develop activatable ¹⁹F MRI probes, the author utilized FLAME as highly sensitive contrast agent and PRE effect as modulation of ¹⁹FNMR/MRI signals. The PRE effects of Gd³⁺ complexes was efficient for decreasing the ¹⁹F NMR/MRI signals of fluorine compounds in FLAME. Based on this finding, the author attempted to develop an activatable ¹⁹F MRI probes (switching OFF/ON type probes) for the detection of reducing environment.

1 Magnetic Resonance Imaging

MRI is the imaging technique based on nuclear magnetic resonance (NMR) phenomena. MRI offers high resolution, deep tissue imaging, and no radiation exposure (Louie et al. 2000). To acquire high contrast images, contrast agents such as Gd³⁺ complexes and superparamagnetic iron oxide nanoparticle (SPIO) are widely used in the field of clinical and research (Fig. 7.1) (Lee et al. 2008). Gd³⁺ complexes shorten the longitudinal relaxation time (T ₁), results in enhancement of MRI signals. SPIO shorten the tranverse relaxation time (T ₂), results in attenuation of MRI signal intensities. Figure 7.2 shows the switching OFF/ON type probes based on Gd³⁺ complexes and SPIO (Perez et al. 2002). However, ¹H MRI often suffers from high background signals derived from water and lipid etc. Therefore, there is a limitation of monitoring of biological signals.

Fig. 7.1

(a) Clinically utilized T ₁ contrast agent, Dotarem^®, and T ₁ relaxation. (b) Clinically utilized T ₂ contrast agent, Resovist, and T ₂ relaxation

Fig. 7.2

The switching OFF/ON type probes based on (a) Gd³⁺ complexes and (b) SPIO

Recently, heteronuclear MRI has been attracted considerable attentions as the alternative ¹H MRI. Several non proton MRI such as ¹³C, ¹⁵N, ¹⁹F, ²⁹Si, ³¹P, and ¹²⁹Xe has been utilized in biological analysis (Table 7.1) (Cassidy et al. 2013). Among these non proton MRI, ¹⁹F MRI has considerable attentions, because fluorine has a 100% natural abundance and a high gyromagnetic ratio (Ahrens et al. 2005). In our bodies, there are a large amount of fluorine atoms in bones and teeth and almost no fluorine atoms in tissues. However, these fluorine atoms are immobilized in a solid state, exhibits very short T ₂ which results in invisible MRI. Therefore, the ¹⁹F MRI can acquire the image without the background signals.

Table 7.1 NMR observable nucleus and the sensitivity

Toward this ends, ¹⁹F MRI contrast agents (always ON type probes) have been utilized in visualization of foci, and cell tracker (Ahrens et al. 2005; Thurecht et al. 2010; Srinivas et al. 2007). In particular, perfluorocarbon (PFC) encapsulated nanoemulsions have attracted significant attention as highly sensitive ¹⁹F MRI contrast agents (Srinivas et al. 2010), and have been utilized as a cell tracker, and oxygen delivery. Recently, several activatable ¹⁹F MRI probes (switching OFF/ON type probes) have also been developed. However, there are only a few examples of in vivo applications owing to the low sensitivity of such probes.

2 Perfluorocarbon Encapsulated in Silica Nanoparticle (FLAME)

In the author’s research group, novel unique shape nanomaterials, which are perfluoro-15-crown-5 ether (PFCE)-encapsulated silica nanoparticles, FLAMEs (FLuorine Accumulated silica nanoparticle for MRI contrast Enhancement), were developed (Fig. 7.3) (Matsushita et al. 2014). FLAMEs are composed of a liquid PFCE, which shows the high molecular mobility to achieve the long T ₂, and a silica shell, which can be easily surface-modified for various functionalization. Although Ahrens et al. reported lipid-based PFCE nanoemulsions as ¹⁹F MRI contrast agents for immune cell tracking (Ahrens et al. 2005; Srinivas et al. 2007), the chemical modification of the lipid emulsion surface is limited due to the unstablity in organic solvents. In contrast, the silica shell fulfills the many demands such as high hydrophilicity, high stability in both aqueous and organic solutions, and chemically surface-modifiable property. In fact, various surface functionalization of FLAMEs was achieved and the functionalized FLAMEs were useful for monitoring a reporter protein expression in living cells and in vivo detection of a tumor. These biological applications represent only a fraction of the forthcoming applications.

Fig. 7.3

Illustration and transmission electron microscope image of FLAME. The molecular motion of PFC is highly retained and thus the sensitivity of the nanoparticles is high sensitive

3 Paramagnetic Relaxation Enhancement (PRE) Effect

There are three types of paramagnetic effects: paramagnetic relaxation enhancement (PRE) effect, pseudocontact shifts (PCSs), and residual dipolar couplings (RDCs) (Clore and Iwahara 2009). Since PCSs and RDCs are observed only in anisotropic electron systems, only PRE is effective in the case of SPIO and Gd³⁺ complexes (Keizer et al. 2007). The PRE decreases the spin-spin relaxation time (T ₂) and results in the broadening of the NMR signals and the decrease of the MRI signals. There are two types of the relaxation mechanism of PRE effect. One is PRE through dipole-dipole interaction and the other is PRE through Curie-spin relaxation. The PRE effect of Gd³⁺ complexes is occurred through dipole-dipole interaction. The transverse (Γ₂) PRE rates of Gd³⁺ are described by the Solomon–Bloembergen (SB) equations (Solomon 1955; Bloembergen and Morgan 1961; Lipari and Szabo 1982):

$$ {\varGamma}_2=\frac{1}{15}{\left(\frac{\mu_0}{4\pi}\right)}^2{\gamma}_{\mathrm{I}}^2{g}^2{\mu}_{\mathrm{B}}^2S\left(S+1\right)\left\{4{J}_{\mathrm{SB}}(0)+3{J}_{\mathrm{SB}}\left({\omega}_{\mathrm{I}}\right)\right\} $$

where μ ₀ is the permeability of free space, μ _B is the magnetic moment of the free electron, γ_I the fluorine gyromagnetic ratio, g is the electron g-factor, S is the electron spin quantum number, and ω _I/2π is the Larmor frequency of the fluorine compound. J _SB(ω) is the spectral density function;

$$ {J}_{\mathrm{SB}}\left(\omega \right)={r}^{-6}\frac{\tau_C}{1+{\left(\omega {\tau}_C\right)}^2} $$

τ _C is the correlation time, defined as (τ_r ⁻¹ + τ_s ⁻¹)⁻¹. τ_r is the rotational correlation time of the molecule, and τ _s is the effective electron relaxation time.

In contrast, Curie-spin relaxation arises from dipole-dipole interaction between a observable nuclide and the magnetization of the electron. The PRE effect of SPIOs is governed by Curie-spin relaxations owing to their high magnetic susceptibility. The Γ₂ PRE rates of Curie-spin relaxation are given by (Bertinin et al. 2002):

$$ {\varGamma}_2=\frac{1}{5}{\left(\frac{\mu_0}{4\pi}\right)}^2\frac{\omega_I{g}^4{\mu}_{\mathrm{B}}^4{S}^2{\left(S+1\right)}^2}{{\left(3{k}_BT\right)}^2{r}^6}\left(4{\tau}_r+\frac{3{\tau}_r}{1+{\omega_I}^2{\tau_r}^2}\right) $$

where k _B is the Boltzmann constant, T is temperature.

In both cases, PRE effect is effective over short distance due to its r ⁻⁶ dependency, where r is the distance between NMR-observable nuclei and a paramagnetic center. When the T ₂ relaxivity of SPIO is compared with that of Gd³⁺ complexes, SPIOs have higher T ₂ relaxivity than Gd³⁺ complexes (Table 7.2). Thus, SPIO is efficient for decreasing the ¹⁹F NMR/MRI signals of PFCE near the FLAME core compared with Gd³⁺ complexes.

Table 7.2 Relaxivities (mM⁻¹ s⁻¹) of paramagnetic contrast agents in H₂O at 37 °C (Rohrer et al. 2005)

4 Gadolinium Based-¹⁹F MRI Nanoprobe for Monitoring Reducing Environment

PRE effect is effective over short distance due to its r ⁻⁶ dependency, where r is the distance between NMR-observable nuclei and a paramagnetic center (Clore and Iwahara 2009; Iwahara and Clore 2006). The author’s research group has employed PRE effect to develop activatable ¹⁹F MRI small molecule probes for detection of enzyme activity (Mizukami et al. 2008). The probes consist of fluorine compound, enzyme substrate, and Gd³⁺ complex. Gd³⁺ complex was conjugated with fluorine compounds through enzyme substrate. The distance between fluorine compound and Gd³⁺ complex was approximately 2.2 nm, determined by molecular mechanic method. Since PRE effect is effective at such close distance, ¹⁹F NMR/MRI signal of the probes were decreased. Upon addition of enzyme, Gd³⁺ complexes were away from fluorine compounds, which results in high ¹⁹F NMR/MRI signal enhancements.

In the case of FLAME, most of PFCE compounds are more than 50 Å away from the surface-modified Gd³⁺ complexes due to the thickness of the silica shell. Thus, it was assumed that the PRE effect might not sufficiently attenuate the ¹⁹F NMR/MRI signals of FLAME.

The authors first confirmed whether the PRE of the Gd³⁺ complexes on the FLAME surface was effective. Different concentration of Gd³⁺ diethylenetriaminepentaacetate (DTPA) complexes were attached to FLAME to yield FLAME-DTPA-Gd1–2 (Scheme 7.1). The ¹⁹F NMR spectrum of FLAME-DTPA without Gd³⁺ exhibited a sharp, single peak (T ₂ = 420 ms). Meanwhile, that of FLAME-DTPA-Gd became a broader peak as Gd³⁺ concentration increased (Fig. 7.4a). The T ₂ of FLAME-DTPA-Gds decreased in Gd³⁺ concentration dependent manner (T ₂ = 68, 40 ms for FLAME-DTPA-Gd1, 2 respectively). Although the ¹⁹F MRI signal of FLAME-DTPA were observed due to the long T ₂, that of FLAME-DTPA-Gd was decreased with Gd³⁺ concentration increasing (Fig. 7.4b). These results indicated that the ¹⁹F NMR/MRI signals of PFCE in FLAME were affected by the PRE from the surface-modified Gd³⁺ complexes. Therefore, the author expected that activatable ¹⁹F MRI probes with high ¹⁹F MRI signal enhancement would be achieved by introducing a cleavable linker between FLAME and the surface-modified Gd³⁺ complexes.

Scheme 7.1

Preparation of FLAME-DTPA-Gd. (a) diethylenetriaminepentaacetic acid dianhydride, TEA, DMF; (b) GdCl₃·6H₂O, methanol

Fig. 7.4

¹⁹F NMR spectra and ¹⁹F MRI phantom images of FLAME-DTPA and FLAME-DTPA-Gd. For ¹⁹F NMR, C _PFCE = 0.6 mM, and the accumulation time was 1 min 22 s. For ¹⁹F MRI (Rapid Acquisition with the Refocused Echoes (RARE) method): T _R was 3000 ms. T _E,eff was 12 ms. The NEX was 64. The acquisition time was 12 min 48 s

This result was explained by the molecular mobility on the NMR/MRI measurement time scale. Iwahara et al. reported that the PRE effect was efficient in spite of the long average distance, when NMR-observable nuclei can occasionally enter the effective range of the PRE effect (Lee et al. 2008). The long T ₂ indicates that the PFCE in FLAME maintains high molecular mobility even in the nanoparticle structure (Matsushita et al. 2014). Although the PFCE at the center of the FLAME core is about 250 Å away from the surface Gd³⁺ complexes (where PRE is not efficient), the fluorine compounds can access the inner shell of FLAME on the measurement time scale. Near the inner shell, although the contribution of one Gd³⁺ complex to the PRE effect is small, the PRE effect from multiple surface Gd³⁺ complexes is combined, and thus the T ₂ of PFCE is efficiently decreased (Fig. 7.5). Although Grüll et al. observed the PRE of PFCE in Gd³⁺-modified nanoemulsions, where the distance between the Gd³⁺ complexes and the fluorine core was less than 22 Å (De Vries et al. 2014), we confirmed that the PRE was effective as such distance for the first time.

Fig. 7.5

Proposed relaxation mechanism of fluorine compounds in FLAME

Next, the authors designed activatable FLAMEs, FLAME-SS-Gd³⁺ (FSG), to image reducing environments. Gd³⁺ complexes were attached to the FLAME surface via disulfide linkers to reduce the T ₂ of the fluorine compounds by the PRE effect, which attenuates the ¹⁹F NMR/MRI signals (Fig. 7.6). When the disulfide of FSG was reduced, the Gd³⁺ complexes were cleaved from the FLAME surface. Then, the T ₂ of the encapsulated PFCE would be elongated and the ¹⁹F NMR/MRI signal intensity would increase.

Fig. 7.6

Design of activatable FLAME, FLAME-SS-Gd³⁺ (FSG)

To optimize the amount of Gd³⁺ complexes on the surface of FLAMEs, three types of FSGs with different concentrations of Gd³⁺ were prepared (Scheme 7.2). The synthetic intermediate FLAME-Py was prepared by the reaction of FLAME with different amounts of 2-((3-(trimethoxysilyl)propyl)dithio)pyridine (1 eq. for FSG1, 10 eq. for FSG2, and 100 eq. for FSG3). Then, 1 eq., 10 eq., or 100 eq. of Gd³⁺ complexes were conjugated to the FLAMEs via a thiol-disulfide exchange reaction to afford FSG1–3, respectively.

Scheme 7.2

Preparation of FLAME-SS-Gd³⁺ (FSG). (a) 2-((3-(trimethoxysilyl)propyl) dithio)pyridine, isopropanol; (b) Gd-DOTA-SH, MeOH

Next, the number of fluorine atoms and Gd³⁺ ions per nanoparticle were calculated as n _19F and n _Gd, respectively (Table 7.3). The quantity of attached Gd³⁺ ions was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES), and the amount of the fluorine atoms was quantified by ¹⁹F NMR in comparison with that of an internal standard, sodium trifluoroacetate. The average diameter of FLAME was 53.4 nm with a 5 nm-thick silica shell, as measured by transmission electron microscopy. If FLAME has a single size of 53.4 nm, the mole of PFCE per one nanoparticle (m _PFCE) could be calculated as follows:

$$ {m}_{\mathrm{PFCE}}=\frac{w_{\mathrm{PFCE}}}{M{W}_{\mathrm{PFCE}}}=\frac{d_{\mathrm{PFCE}}\times {V}_{\mathrm{core}}}{M{W}_{\mathrm{PFCE}}}=\frac{d_{\mathrm{PFCE}}\times \frac{4}{3}\pi {r}_{\mathrm{core}}^3}{M{W}_{\mathrm{PFCE}}}\approx 1.4\times {10}^{-19}\left(\mathrm{mol}/\mathrm{particle}\right) $$

where w _PFCE is the weight of PFCE in FLAME, MW_PFCE is the molecular weight of PFCE, d _PFCE is the density of PFCE (1.86 g/cm³), V _core is the volume of PFCE in FLAME, and r _core is the radius of the FLAME core (21.7 nm). Thus, the number of fluorine atoms per one nanoparticle (n _19F) was calculated as:

$$ {n}_{{}{}^{19}\mathrm{F}}={m}_{\mathrm{PFCE}}\times 20\times {N}_{\mathrm{A}}\approx 1.7\times {10}^6\left({}{}^{19}\mathrm{F}\;\mathrm{atom}/\mathrm{particle}\right) $$

where N _A is Avogadro’s constant. Since the amount of the Gd³⁺ ions was measured by ICP-AES, the molar ratio of the Gd³⁺ ions to PFCE for FSG1, FSG2, and FSG3 was calculated to be 0.011, 0.026, and 0.038, respectively. Therefore, the number of Gd³⁺ ions per nanoparticle (n _Gd) was calculated as:

$$ {\displaystyle \begin{array}{l}\mathrm{FSG}1:{m}_{{\mathrm{Gd}}^{3+}}/{m}_{\mathrm{PFCE}}=0.011\\ {}{n}_{Gd}={m}_{{\mathrm{Gd}}^{3+}}\times {N}_{\mathrm{A}}=0.011\times {m}_{\mathrm{PFCE}}\times {N}_{\mathrm{A}}\approx 9.1\times {10}^2\left({\mathrm{particle}}^{-1}\right)\\ {}\mathrm{FSG}2:{m}_{{\mathrm{Gd}}^{3+}}/{m}_{\mathrm{PFCE}}=0.026\\ {}{n}_{Gd}={m}_{{\mathrm{Gd}}^{3+}}\times {N}_{\mathrm{A}}=0.026\times {m}_{\mathrm{PFCE}}\times {N}_{\mathrm{A}}\approx 2.1\times {10}^3\left({\mathrm{particle}}^{-1}\right)\\ {}\mathrm{FSG}3:{m}_{{\mathrm{Gd}}^{3+}}/{m}_{\mathrm{PFCE}}=0.038\\ {}{n}_{\mathrm{Gd}}={m}_{{\mathrm{Gd}}^{3+}}\times {N}_{\mathrm{A}}=0.038\times {m}_{\mathrm{PFCE}}\times {N}_{\mathrm{A}}\approx 3.1\times {10}^3\left({\mathrm{particle}}^{-1}\right)\end{array}} $$

Table 7.3 Physical properties of FLAME and FSGs

The ς-potentials of FSGs gradually shifted towards the positive direction with increasing amounts of surface Gd³⁺ ions (Table 7.3). This was because the slightly electronegative silanol groups on the FLAME surface were decreased owing to the coupling with 2-((3-(trimethoxysilyl)propyl)dithio)pyridine. The n _Gd and ς-potential data indicated that different concentrations of Gd³⁺ complexes were successfully introduced on the FLAME surface.

The ¹⁹F NMR spectrum of FLAME without paramagnetic ions exhibited a sharp peak. In contrast, the ¹⁹F NMR peaks of FSGs were decreased and more broad according to the concentration of surface Gd³⁺ on account of the PRE effect (Fig. 7.7a). Although the ¹⁹F NMR of FSG1 exhibited a sharp peak, the T ₂ of FSG1 (120 ms) was shorter than that of FLAME (420 ms) (Table 7.3). The T ₂ of FSG2 and FSG3 was 66 ms, 27 ms, respectively. As such, the PRE effect was observed in all FSGs.

Fig. 7.7

(a) ¹⁹F NMR spectra of FSGs incubated with or without TCEP. C_PFCE: 0.6 mM, C _TCEP: 1.0 mM, incubation time: 4 h, accumulation time: 10 min 55 s. (b) ¹⁹F NMR signal to noise ratio of FSGs in the presence of TCEP (Blue: FSG1, Red: FSG2, Green: FSG3). C_PFCE: 0.15 mM

¹⁹F NMR spectra and T ₂ of FSGs were measured after treatment with a reducing agent, tris(2-carboxyethyl)phosphine (TCEP) (Fig. 7.7). Addition of TCEP made the ¹⁹F NMR peaks of all FSGs sharper and taller as compared to those before the addition. The T ₂ values of FSG1–3 were significantly increased upon addition of TCEP within 2 h, and were comparable to that of FLAME. All Gd³⁺ complexes were cleaved upon addition of more than 2 mM TCEP (Fig. 7.7b). The highest ¹⁹F NMR SNR of FSG1–3 was obtained at 2 mM TCEP, and the values were 16.2 for FSG1, 19.5 for FSG2, and 17.9 for FSG3. The signal enhancement factors in response to the reductant were 3.1, 9.7, and 12.7 for FSG1–3, respectively. Thus, FSG3 was the most sensitive ¹⁹F NMR probe in the detection of the reducing environment.

The ¹⁹F NMR signals of the FSGs increased upon addition of other reducing agents such as glutathione, cysteine, and dithiothreitol (Fig. 7.8). In particular, addition of glutathione induced the greatest ¹⁹F NMR signal enhancement. Although there are some concerns about the stability of reduction-triggered nanoparticles in normal tissues, rational optimization of the disulfide linkage will lead to practical in vivo applications.

Fig. 7.8

¹⁹F NMR spectra of FSG2 (C_PFCE = 0.15 mM) incubated with several thiol-based reducing agents (3 mM). Left to right, control (without reductant), glutathione (GSH), cysteine (Cys), 1,4-dithiothreitol (DTT). The accumulation time was 1 min 22 s. Incubation time was 4 h

Finally, ¹⁹F MR phantom images of FSGs solutions with or without TCEP were obtained by varying T _E,eff. In general, the MRI signal of the long T ₂ component is well observed at both short and long T _E,eff. In contrast, the MRI signal of samples with moderately short T ₂ is only visible at short T _E,eff, and that of the extremely short T ₂ component is not observed even at short T _E,eff. As expected from the ¹⁹F NMR results, almost no ¹⁹F MRI signals of FSG2 and FSG3 were detected without TCEP at any T _E,eff due to the strong PRE effect (Fig. 7.9a, b). In contrast, the ¹⁹F MRI signals of FSG1 were observed at T _E,eff ≤ 84 ms because of the moderately short T ₂. However, the measurement of FSG1 without TCEP at T _E,eff ≥ 108 ms extinguished the undesired ¹⁹F MRI signals. Reductive reactions induced a noticeable ¹⁹F MRI signal enhancement in FSG1–3 at any T _E,eff (filled circles). At T _E,eff = 12 ms, approximately 60- and 40-fold increases were observed in FSG2 and FSG3, respectively. Although the signal the enhancement of FSG1 was only two-fold at T _E,eff = 12 ms, a 50-fold increase was observed at T _E,eff = 108 ms. These results indicated that FSG2 was the most effective probe for detecting reducing environments. One of the advantages of FSGs is the high sensitivity, because the ¹⁹F NMR/MRI signals of 1.7 × 10⁶ fluorine atoms in the core were decreased by ca. 1.0 × 10³ Gd³⁺ complexes on the FLAME surface. The ratios of fluorine atoms to Gd³⁺ complexes (Table 7.1) are the highest among known PRE-based probes, of which the ratios were single digits. This high ratio led to the high signal amplification.

Fig. 7.9

¹⁹F MRI signal enhancement of FSGs by TCEP. (a) ¹⁹F MRI phantom images of FSG1–3 with or without TCEP. (b) Plot of ¹⁹F MRI signal intensity of FSG1–3 at different T _E,eff with (filled circles) or without (open circles) TCEP. ¹⁹F MRI RARE method: the matrix size was 128 × 64 and the slice thickness was 30 mm. T _R was 3000 ms. The NEX was 64. The acquisition time was 25 min 36 s

1 Supplementary Electronic Material (S)

(MP4 508249 kb)