19F MRI Probes with Tunable Chemical Switches

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.

Toward this ends, 19 F 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 19 F MRI contrast

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 19 F 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.

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 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 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 Gd 3+ complexes is occurred through dipole-dipole interaction. The transverse (Γ 2 ) PRE rates of Gd 3+ are described by the Solomon-Bloembergen (SB) equations (Solomon 1955;Bloembergen and Morgan 1961;Lipari and Szabo 1982): 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; 1 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 Γ 2 PRE rates of Curie-spin relaxation are given by (Bertinin et al. 2002): 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 Gd 3+ complexes, SPIOs have higher T 2 relaxivity than Gd 3+ complexes (Table 7.2). Thus, SPIO is efficient for decreasing the 19 F NMR/MRI signals of PFCE near the FLAME core compared with Gd 3+ complexes.

Gadolinium Based-19 F 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 19 F MRI small molecule probes for detection of enzyme activity (Mizukami et al. 2008). The probes consist of fluorine compound, enzyme substrate, and Gd 3+ complex. Gd 3+ complex was conjugated with fluorine compounds through enzyme substrate. The distance between fluorine compound and Gd 3+ complex was approximately 2.2 nm, determined by molecular mechanic method. Since PRE effect is effective at such close distance, 19 F NMR/MRI signal of the probes were decreased. Upon addition of enzyme, Gd 3+ complexes were away from fluorine compounds, which results in high 19 F NMR/MRI signal enhancements.
In the case of FLAME, most of PFCE compounds are more than 50 Å away from the surface-modified Gd 3+ complexes due to the thickness of the silica shell. Thus, it was assumed that the PRE effect might not sufficiently attenuate the 19 F NMR/ MRI signals of FLAME.
The authors first confirmed whether the PRE of the Gd 3+ complexes on the FLAME surface was effective. Different concentration of Gd 3+ diethylenetriaminepentaacetate (DTPA) complexes were attached to FLAME to yield FLAME- DTPA-Gd1-2 (Scheme 7.1). The 19 F NMR spectrum of FLAME-DTPA without Gd 3+ exhibited a sharp, single peak (T 2 = 420 ms). Meanwhile, that of FLAME-DTPA-Gd became a broader peak as Gd 3+ concentration increased ( Fig. 7.4a). The T 2 of FLAME-DTPA-Gds decreased in Gd 3+ concentration dependent manner (T 2 = 68, 40 ms for FLAME-DTPA-Gd1, 2 respectively). Although the 19 F MRI signal of FLAME-DTPA were observed due to the long T 2 , that of FLAME-DTPA-Gd was decreased with Gd 3+ concentration increasing ( Fig. 7.4b). These results indicated that the 19 F NMR/MRI signals of PFCE in FLAME were affected by the PRE from the surface-modified Gd 3+ complexes. Therefore, the author expected that activatable 19 F MRI probes with high 19 F MRI signal enhancement would be achieved by introducing a cleavable linker between FLAME and the surface-modified Gd 3+ complexes. 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 Gd 3+ complexes (where PRE is not efficient), FLAME-DTPA FLAME-DTPA-Gd2 FLAME-DTPA-Gd1 the fluorine compounds can access the inner shell of FLAME on the measurement time scale. Near the inner shell, although the contribution of one Gd 3+ complex to the PRE effect is small, the PRE effect from multiple surface Gd 3+ 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 Gd 3+ -modified nanoemulsions, where the distance between the Gd 3+ 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. Next, the authors designed activatable FLAMEs, FLAME-SS-Gd 3+ (FSG), to image reducing environments. Gd 3+ 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 19 F NMR/MRI signals (Fig. 7.6). When the disulfide of FSG was reduced, the Gd 3+ complexes were cleaved from the FLAME surface. Then, the T 2 of the encapsulated PFCE would be elongated and the 19 F NMR/MRI signal intensity would increase.
To optimize the amount of Gd 3+ complexes on the surface of FLAMEs, three types of FSGs with different concentrations of Gd 3+ 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 Gd 3+ complexes were conjugated to the FLAMEs via a thiol-disulfide exchange reaction to afford FSG1-3, respectively. Next, the number of fluorine atoms and Gd 3+ ions per nanoparticle were calculated as n 19F and n Gd , respectively (Table 7.3). The quantity of attached Gd 3+ ions was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES), and the amount of the fluorine atoms was quantified by 19 F 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: 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/cm 3 ), 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:  Table 7.3 Physical properties of FLAME and FSGs ς-potential/mV n 19F a n Gd a n 19F /n Gd a T 2, TCEP− /ms T 2, TCEP+ /ms FLAME −24.8 ± 1.7 1.7 × 10 6 0 − 420 − b FSG1 −12.6 ± 2.4 1.7 × 10 6 9.1 × 10 2 1.8 × 10 3 120 383 FSG2 3.9 ± 1.4 1.7 × 10 6 2.1 × 10 3 7.7 × 10 2 66 365 FSG3 5.7 ± 1.5 1.7 × 10 6 3.1 × 10 3 5.3 × 10 2 27 371 where N A is Avogadro's constant. Since the amount of the Gd 3+ ions was measured by ICP-AES, the molar ratio of the Gd 3+ ions to PFCE for FSG1, FSG2, and FSG3 was calculated to be 0.011, 0.026, and 0.038, respectively. Therefore, the number of Gd 3+ ions per nanoparticle (n Gd ) was calculated as: The ς-potentials of FSGs gradually shifted towards the positive direction with increasing amounts of surface Gd 3+ 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 Gd 3+ complexes were successfully introduced on the FLAME surface.
The 19 F NMR spectrum of FLAME without paramagnetic ions exhibited a sharp peak. In contrast, the 19 F NMR peaks of FSGs were decreased and more broad according to the concentration of surface Gd 3+ on account of the PRE effect ( Fig. 7.7a). Although the 19 F 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.
19 F 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 19 F 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 Gd 3+ complexes were cleaved upon addition of more than 2 mM TCEP (Fig. 7.7b). The highest 19 F 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 19 F NMR probe in the detection of the reducing environment.
The 19 F 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 19 F 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.   Finally, 19 F 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 19 F NMR results, almost no 19 F 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 19 F 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 19 F MRI signals. Reductive reactions induced a noticeable 19 F 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 19 F NMR/MRI signals of 1.7 × 10 6 fluorine atoms in the core were decreased by ca. 1.0 × 10 3 Gd 3+ complexes on the  (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. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence and indicate if changes were made. The images or other third party material in this chapter are included in the chapter's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.