Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive 19F MRI Nanoprobes Kazuki Akazawa,#,† Fuminori Sugihara,#,‡ Tatsuya Nakamura,† Shin Mizukami,§ and Kazuya Kikuchi*,†,‡ †
Division of Advanced Science and Biotechnology, Graduate School of Engineering and ‡WPI-Immunology Frontier Research Center, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan § Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan S Supporting Information *
ABSTRACT: Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. 19F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsive 19 F MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON 19F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd3+ complex-conjugated peptide, DEVD X (X = 1, 2), which is a substrate peptide sequence tandemly repeated X times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high 19F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged by 19F MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo.
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molecule and achieved 19F MRI detection of caspase-3/7 activity in zebrafish.15 However, 19F MRI detection of enzymatic activities in living mice is more challenging because it requires the large quantity and the excellent biodistribution of the probes. Moreover, a large-scale measurement system is required for experiments using mice. In the past decade, nanoemulsions encapsulating perfluorocarbons (PFCs) have emerged as promising 19F MRI contrast agents for monitoring the dynamics and localization of cells in vivo because of their outstanding sensitivity.16,17 We developed a highly sensitive 19F MRI nanoparticle, which we termed FLAME (FLuorine Accumulated silica nanoparticle for MRI contrast Enhancement), containing a liquid PFC core and a robust silica shell.18 FLAME exhibits several remarkable advantageous properties for 19F MRI, including high sensitivity, in vivo stability, and versatile surface modifiability through organic synthesis. Although PFC-encapsulated nanoprobes have been utilized in various biomedical applications, including cancer imaging,18 cell tracking,16,17 and drug release,19 no studies have succeeded in
INTRODUCTION Enzymes are involved in various human diseases, such as cancer and autoimmune diseases, and are often targeted for drug development and diagnosis.1,2 Therefore, sensitive detection and imaging of enzymatic activities in living mammals is one of the ultimate goals of bioimaging. However, current in vivo optical imaging technologies, such as near-infrared fluorescence imaging3 and bioluminescence imaging,4 cannot yield highresolution images of enzymatic activities in the deep tissues of living animals. Magnetic resonance imaging (MRI) is a noninvasive imaging technique that offers high spatial resolution and unlimited tissue penetration for signal detection.5−9 Especially, 19F MRI has attracted the interest of researchers because of the high NMR sensitivity of 19F (83% relative to 1H) and negligible endogenous background signals,10 which render it superior for monitoring specific biological events in living animals. Although some enzyme-responsive 19F NMR/MRI probes based on small-molecule designs have been reported,11−14 it is difficult to apply such small-molecule 19F MRI probes to in vivo experiments because of their low sensitivity. In addition, few 19F MRI probes can detect enzymatic activity in vivo. Liang et al. designed a 19F MRI probe based on the self-assembly and disassembly of a small © XXXX American Chemical Society
Received: March 6, 2018 Revised: April 23, 2018
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DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 1. Schematic of FLAME-DEVD X (X = 1, 2), enzyme-responsive 19F MRI nanoprobes for detecting caspase-3/7 activity.
imaging enzymatic activities using a highly sensitive 19F MRI nanoprobe. In this study, we developed highly organized 19F MRI nanoprobes for detecting caspase-3/7 activity in vivo. Caspase3 and -7 are hallmarks of apoptosis,20−22 and they play important roles in the regulation of cell number and maintenance of tissue homeostasis. Inhibition of caspase-3/7 activity is a common strategy for drug development,23 and the activity is also used as a biomarker for evaluating anticancer therapies that induce apoptosis in cancer cells.24 Therefore, 19F MRI detection of caspase-3/7 activity in living animals will provide invaluable information on the efficacy of caspase-3/7targeted drugs and anticancer therapies. To successfully detect in vivo caspase-3/7 activity, as a novel methodology, we incorporated a tandemly repeated substrate peptide sequence into 19F MRI signal activation strategy, anticipating that this novel method will improve the cleavage efficiency of peptides by caspase-3/7 and promote the kinetic response of the nanoprobe to caspase-3/7 activity. Here, we describe, for the first time, 19F MRI of caspase-3/7 activity in living mice using a PFC-encapsulated 19F MRI nanoprobe with OFF/ON-switching ability.
substrate peptide (DEVD), and a lysine residue (Figure 2a). The lysine is attached to the FLAME surface via an amide
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RESULTS Rationale of the Probe Design. Recently, we developed an activatable 19F MRI nanoparticle probe, FLAME-SS-Gd3+, for visualization of reducing environments.25 The OFF/ONswitching mechanism of the probe was based on the paramagnetic relaxation enhancement (PRE) effect. We found that the transverse relaxation time (T2) values of the PFCs in FLAME-SS-Gd3+ were efficiently decreased and the 19F NMR/ MRI signals were attenuated due to the PRE from the multiple Gd3+ complexes attached to the FLAME surface. After treatment with a reducing agent, tris(2-carboxyethyl)-phosphine (TCEP), the Gd3+ complexes were eliminated and diffused away from the FLAME surface, thereby increasing the 19 F NMR/MRI signals. To advance this 19F NMR/MRI signal activation strategy for enzyme activity imaging, we designed a caspase-3-responsive 19 F MRI nanoprobe, FLAME-DEVD X (X = 1, 2) (Figure 1). FLAME-DEVD X consists of a surface-carboxylated FLAME and Gd3+ complex-conjugated peptides. The peptide region comprises a gadolinium(III) 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetate (DOTA) monoamide complex, enzyme
Figure 2. (a) Chemical structure of the Gd3+ complex-conjugated peptides, DEVD X (X = 1, 2). (b) HPLC analysis of DEVD X after 0, 1, 3, 6, 12, and 24 h of incubation with caspase-3.
bond. The substrate peptide sequence, DEVD, is cleaved at the C-terminus by caspase-3/7.26 The Gd3+-DOTA monoamide complex is stable under both neutral (physiological) and acidic (lysosomal) conditions.27 In the probe, the T2 is very short, and the 19F MRI signal of FLAME-DEVD X decreases before the enzyme reaction due to the PRE effect of the surface Gd3+ complexes. When the peptides are cleaved by caspase-3, the Gd3+ complexes diffuse away from the FLAME surface, B
DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Table 1. Properties of FLAME-COOH and FLAME-DEVD X (X = 1, 2)
resulting in the recovery of the 19F MRI signal. However, compared to small-molecule targets such as TCEP, it is difficult for enzymes to access substrates located between the FLAME and Gd3+ complexes due to steric hindrance. Therefore, we needed to reduce the steric hindrance and enhance the cleavage efficiency of peptides. To achieve these, we constructed DEVD X (X = 1, 2), which has a tandemly repeated (X times) substrate peptide sequence (DEVD). We anticipated that DEVD 2 would show faster cleavage kinetics than DEVD 1 because of the following reasons. First, the steric hindrance would be reduced by elongating the distance between FLAME and Gd3+ complexes. Second, the cleavage efficiency would be improved by increasing the number of the substrates, DEVD, per one Gd3+ complex-conjugated peptide. These two factors would result in an improved 19F MRI signal enhancement rate. Synthesis of the DEVD X (X = 1, 2) Peptides and Enzyme Assay. The Gd3+ complex-conjugated peptides, DEVD X, were synthesized by Fmoc-solid-phase synthesis (Scheme S2). After cleavage from the resin, Gd3+ was coordinated by the DOTA monoamide ligand, and then the Gd3+ complex-conjugated peptides were purified by reversedphase high-performance liquid chromatography (HPLC) (Figure S1). Next, the reactivity of DEVD X with recombinant mouse caspase-3 was examined (Figure 2b). DEVD X (1.0 mM) was incubated with caspase-3 at 37 °C, and the enzyme reaction was monitored by HPLC. Both DEVD 1 and DEVD 2 were cleaved by caspase-3 at the C-terminus of DEVD. The DEVD 1 peak remained after 24 h of incubation with caspase-3. In contrast, the DEVD 2 peak disappeared after 6 h of incubation, indicating that the caspase-3 reactivity with DEVD 2 was faster than that with DEVD 1. All the HPLC peaks were identified as products of hydrolysis by caspase-3 with electrospray ionization-mass spectrometry (ESI-MS). To examine the substrate specificity of other proteases, DEVD 2 was incubated with caspase-1, -7, and -9 as well as cathepsin B and legumain (Figure S2). Hydrolyzed products of DEVD 2 were observed following incubation with caspase-7. About 24% and 27% of DEVD 2 was hydrolyzed after incubation with caspase-1 and -9, respectively. In contrast, about 99% and 97% of DEVD 2 remained after incubation with cathepsin B and legumain. Preparation and Characterization of FLAME-DEVD X (X = 1, 2). FLAME-DEVD X was prepared by reacting DEVD X with N-hydroxysuccinimidyl FLAME (FLAME-NHS; Scheme S3). In our previous study, a short T2 value (approximately 100 ms) was required to sufficiently quench the 19F MRI signals of FLAME.25 Thus, we optimized the amount of the Gd3+ complexes conjugated on the FLAME surface by adjusting the concentrations of peptide and triethylamine in the surface modification reaction. The hydrodynamic diameters and particle concentrations were measured using a tunable resistive pulse sensor (Figure S5). The average hydrodynamic diameters of FLAME-COOH, FLAME-DEVD 1, and FLAME-DEVD 2 were 101 ± 17, 115 ± 20, and 102 ± 18 nm, respectively (Table S2). The T2 values of FLAME-DEVD X were measured by 19F NMR (Table 1). The T2 values of PFCE in FLAME-DEVD X were 88 and 97 ms for X = 1 and 2, respectively. These values were sufficiently lower than that of FLAME-COOH (432 ms). The 19F NMR spectrum of FLAME-COOH (CPFCE = 10 mM) showed a sharp single peak, whereas the spectrum of FLAME-DEVD X showed a broad single peak (Figure S6). Next, surface modification of the Gd3+ complexes was validated by inductively coupled
materials
n19Fa
FLAME-COOH FLAME-DEVD 1 FLAME-DEVD 2
1.3 × 10 9.2 × 107 8.0 × 107 8
nGda
n19F/nGda
T2 (ms)
0 1.5 × 104 8.6 × 104
− 6.1 × 103 9.3 × 102
432 88 97
a
Values were calculated from 19F NMR spectroscopy, ICP-AES, and particle concentration. n19F: the number of 19F atoms in one nanoparticle; nGd: the number of Gd3+ atoms in one nanoparticle.
plasma atomic emission spectrometry (ICP-AES). Gd 3+ complex-conjugated peptides were introduced on the nanoparticle surface after purification by HPLC. In the previous study, we confirmed Gd3+-DOTA complexes on FLAME surface are stable under physiological conditions.25 Therefore, it is reasonable to assume that all DOTA chelators form the stable complex with Gd3+ and there is no free chelator on the nanoparticle surface. The numbers of fluorine atoms and Gd3+ ions per nanoparticle, n19F and nGd, respectively, were calculated from the 19F NMR spectra and ICP-AES data with particle concentration data (Table 1; detailed calculations are given in the Supporting Information). The n19F/nGd values were 6.1 × 103 and 9.3 × 102 for FLAME-DEVD 1 and 2, respectively. Attenuation of the 19F NMR signals and T2 values of FLAMEDEVD X was confirmed to be caused by the PRE effect of the surface Gd3+ complexes. The relationship between R2 and nGd/ n19F was investigated in the previous study (Figure S16).25 Enzyme Assay and in Vitro 19F MRI Study of FLAMEDEVD X (X = 1, 2). We carried out an enzymatic reaction using FLAME-DEVD X and caspase-3. Briefly, FLAME-DEVD X was dispersed in reaction buffer (CPFCE = 6.4 mM) and then incubated with caspase-3 at 37 °C for 12 h. After incubation, the mixture was centrifuged, and the supernatant was analyzed by HPLC (Figure S7). The peak of the cleaved peptide, Gd3+DOTA-Deg-DEVD, was detected in the supernatant of reactions containing FLAME-DEVD X and caspase-3. In contrast, no cleaved products were observed in reactions containing FLAME-DEVD X without caspase-3. Thus, at least some of the Gd3+DOTA-Deg-DEVD peptides on the FLAME surface were cleaved by caspase-3. The peak of Gd3+-DOTADeg-DEVD-Deg-DEVD was not detected from the mixture of FLAME-DEVD 2 with caspase-3. Next, 19F MRI phantom images and the T2 of FLAME-DEVD X (CPFCE = 2.0 mM) were measured at the indicated time points (Figure 3a, Figure S8). Almost no 19F MRI signals for FLAME-DEVD X were observed in the absence of caspase-3. In contrast, marked 19F MRI signal enhancement was observed following incubation of FLAME-DEVD 1 or FLAME-DEVD 2 with caspase-3. The normalized 19F MRI signal intensities for FLAME-DEVD 1 and 2 were 0.37 ± 0.07 and 0.62 ± 0.01, respectively, after 12 h of incubation. The T2 value of FLAME-DEVD 1 gradually increased during incubation with caspase-3, and it reached 230 ms after 12 h of incubation (Figure S8b, Table S3). In contrast, the T2 values of FLAME-DEVD 1 without caspase-3 could not be measured due to the very low 19F MRI signal intensity. During incubation with caspase-3, the T2 value of FLAME-DEVD 2 also increased to 276 ms after 12 h of incubation, while the T2 value of FLAME-DEVD 2 without caspase-3 was short and did not change over time. From Table 1, the surface density of Gd3+ complexes on the nanoparticle was higher for FLAME-DEVD 2 than for FLAME-DEVD 1. Therefore, the higher 19F MRI signal and longer T2 relaxation C
DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Figure 4. 19F MRI of caspase-3/7 activity in apoptotic cell lysates. 19F MRI phantom images of FLAME-DEVD 2 (CPFCE = 2.0 mM) after 12 h of incubation under the following conditions. 1, in buffer; 2, in RAW264 cell lysate treated with TNF-α and cycloheximide; 3, RAW264 cell lysate treated with TNF-α and cycloheximide + a caspase-3 inhibitor (Z-DEVD-FMK); 4, RAW264 cell lysate. Figure 3. (a) 19F MRI phantom images of FLAME-DEVD X (CPFCE = 2.0 mM) with and without caspase-3. (b) Time course of the 19F MRI signal for FLAME-DEVD X with (filled) and without (open) caspase3. The 19F MRI signals of FLAME-DEVD X were normalized to those of a reference standard (FLAME-COOH). Data are presented as means ± SD (n = 3).
with apoptotic cell lysate. This result demonstrates that FLAME-DEVD 2 can detect caspase-3/7 activity in an apoptotic cell lysate by 19F MRI. 19 F MRI of Caspase-3/7 Activity in Living Mice. Finally, we evaluated the potential of using FLAME-DEVD 2 for living animal studies. Before applying FLAME-DEVD 2 to 19F MRI of caspase-3/7 activity in mice, we investigated the in vivo distribution of FLAME using FLAME-COOH, an always ONtype nanoprobe. It is well-known that most of the nanomaterials injected into the bloodstream are recognized and engulfed by phagocytic cells, such as Kupffer cells and splenic macrophages, and accumulate in the liver and spleen.28,29 FLAME-COOH (CPFCE = 5.0 mM) was intravenously injected into a mouse, and then 1H/19F MRI measurements were performed (Figure S12a, Before). As expected, 19F MRI signals were observed in the liver and spleen, indicating that FLAME accumulated in these organs. Therefore, we selected the macrophages in the liver and spleen as an apoptosis model, and we used clodronate liposomes to induce apoptosis in these cells. Clodronate liposomes are commonly used to induce apoptosis in macrophages in vivo by inhibiting ADP/ATP translocase,30,31 and these liposomes are predominantly taken up by macrophages in the liver and spleen.32 After intravenous injection of FLAME-COOH, clodronate liposomes (132 nm) were intravenously injected into the mice, and then 1H/19F MRI images were acquired 1 day later (Figure S12a, After). The results showed that the S/N ratios in the liver and spleen exhibited 0.81-fold and 0.79-fold decrease, respectively, after injection of the clodronate liposomes (Figure S12b). Next, FLAME-DEVD 2 was intravenously injected, and 1H/19F MRI measurements were obtained. 19F MRI signals for FLAMEDEVD 2 were observed in the liver, likely due to nonspecific hydrolysis by liver proteases, whereas no signals were detected in the spleen (Figure 5a, Before). After injection of clodronate liposomes, the S/N ratio in the liver showed 0.96-fold (Case 1) and 0.80-fold (Case 2) decrease (Figure 5b, Liver). However, the 19F MRI signals in the spleen were clearly enhanced, and the S/N ratios in the spleen were 6.2-fold (Case 1) and 4.2-fold (Case 2) higher than before clodronate liposome injection (Figure 5b, Spleen). As a control, PBS liposomes were intravenously administered, and 1H/19F MRI measurements were obtained 1 day later. In contrast to the results obtained using clodronate liposomes, after injection of PBS liposomes, no 19F MRI signals were detected in the spleen, and the S/N ratio in the spleen did not increase (Figure 5c,d). To verify FLAME-DEVD 2 accumulation in the spleen, we conducted fluorescence imaging of spleen tissue slices after treatment with clodronate or PBS liposomes. To detect
time of FLAME-DEVD 2 with caspase-3 showed that more peptides were cleaved in FLAME-DEVD 2 surface. 19F MRI signals and T2 of FLAME-DEVD X did not reach those of FLAME-COOH. These data indicate that peptides on nanoparticle surface were not fully cleaved after 12 h of incubation. Although we measured the amount of cleaved Gd3+ complex-conjugated peptides using ICP-AES, the Gd 3+ concentration was not quantified due to the low emission sensitivity of Gd3+. This result suggests that the tandem repeat in the substrate improved the 19F MRI signal enhancement rate of FLAME-DEVD X. Therefore, we performed all subsequent cellular and mouse experiments using FLAME-DEVD 2. Then, FLAME-DEVD 2 was incubated with caspase-1, -7, and -9. In accordance with the results of the substrate specificity assay for DEVD 2 (Figure S2), FLAME-DEVD 2 with caspase-7 showed a higher 19F MRI signal intensity (0.26 ± 0.03) compared to that with caspase-1 (0.01 ± 0.01) and caspase-9 (0.02 ± 0.01) (Figure S9). This result suggests that FLAME-DEVD 2 showed good selectivity for caspase-3/7. 19 F MRI of Caspase-3/7 Activity in Apoptotic Cell Lysate. To verify that FLAME-DEVD 2 can detect enzymatic activities in the apoptotic cell lysate, we used RAW264.7 cells treated with tumor necrosis factor (TNF)-α in the presence of cycloheximide as an apoptotic cell model. After 4 h of incubation at 37 °C, the cells were collected and lysed with lysis buffer. Caspase-3/7 activity in the cell lysate was verified with the fluorogenic substrate Ac-DEVD-MCA (Figure S10). Lysate from TNF-α-treated RAW264.7 cells showed 14-fold higher fluorescence intensity than that in nontreated cells. Next, FLAME-DEVD 2 (CPFCE = 5.3 mM) was incubated with the apoptotic cell lysate at 37 °C for 12 h, and then FLAME-DEVD 2 was diluted with water (CPFCE = 2.0 mM) for MRI measurement. 19F MRI signals for FLAME-DEVD 2 were enhanced from 0.08 ± 0.05 to 0.27 ± 0.10 after incubation with apoptotic cell lysate (Figure 4, Figure S11). The 19F MRI signals for FLAME-DEVD 2 in buffer were due to the background signals which were not fully quenched by Gd3+ complexes. To confirm whether the 19F MRI signal of FLAMEDEVD 2 was enhanced by caspase-3/7 activity in the apoptotic cell lysate, we pretreated the apoptotic cell lysate with a caspase-3/7 inhibitor (Z-DEVD-FMK) before incubation with FLAME-DEVD 2. This inhibitor suppressed the enhancement of the 19F MRI signal observed for FLAME-DEVD 2 incubated D
DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Figure 5. (a) 19F MRI of caspase-3/7 activity in mouse spleen. FLAME-DEVD 2 (CPFCE = 10 mM, 200 μL) was intravenously injected into a mouse, and 1H/19F MR images were acquired (Before). Then, clodronate (a-1,2) or PBS (c-1,2) liposomes (100 μL) were intravenously injected. After 24 h, 1 H/19F MR images were acquired (After). The positions of the liver (L) and spleen (S) are indicated on the 1H MR images. C.L. = clodronate liposome. P.L. = PBS liposome. (b-1,2 and d-1,2) S/N ratio of the liver and spleen before and after injection of clodronate or PBS liposomes.
FLAME as fluorescence signals in the tissue sections, rhodamine B isothiocyanate (RITC) was conjugated to the FLAME surface. RITC fluorescence was detected in tissue sections after injection of both clodronate and PBS liposomes (Figure S13). The results of fluorescence analysis on the tissue sections indicate that FLAME-DEVD 2 was localized in the spleen regardless of apoptosis induction. In addition, caspase-3/ 7 activities in homogenized spleen samples were measured with Ac-DEVD-MCA. The results showed that caspase-3/7 activity was significantly increased in the spleen after injection of clodronate liposomes (Figure S15). These results demonstrate that enhancement of the 19F MRI signal for FLAME-DEVD 2 in the spleen was induced by caspase-3/7 activity.
probe compounds and short acquisition times are preferable. Although increasing the number of 19F atoms per probe is a common solution, large number of 19F atoms decreases the aqueous solubility of the probes, resulting in signal attenuation. Ahrens et al. reported PFC-based nanoemulsions for tracking cell dynamics, demonstrating markedly increased 19F MRI sensitivity.16,17 However, their applications were limited because of the instability of the PFC-based nanoemulsions in organic solvents and Ostwald ripening.33,34 In contrast, our FLAME nanoprobes consist of PFC-encapsulated nanoparticles with high sensitivity, and the silica shell dramatically improves nanoparticle stability as previously demonstrated in both aqueous solutions and the organic solvents used for organic synthesis.18 In our study, the MRI measurement methods for the assays using recombinant enzymes and cell lysates are not comparable to those for in vivo experiments. However, the second difficult problem remained: OFF/ON signal switching of nanomaterial probes. General OFF/ON signal switching strategies for nanomaterial probes are based on
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DISCUSSION Our new probe resolved the difficult problems with enzymeresponsive 19F MRI probes. The first problem is low sensitivity, particularly in the case of small molecule-based probes. In practical applications, less than millimolar concentrations of the E
DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
of FLAME-DEVD 2 and clodronate liposomes. The possibility that other proteases in the spleen were activated after the treatment with clodronate liposomes was not fully excluded. However, it was reported that clodronate liposomes induce apoptosis in macrophages in the spleen.30,31 In addition, in our experiments using the spleen homogenate, the specificity of caspase-3/7 substrate, DEVD, was confirmed by using caspase3/7 inhibitor. In fact, the fluorescence signal enhancement of fluorogenic probe was suppressed in the presence of caspase-3/ 7 inhibitor (Figure S15). Therefore, most of the 19F MRI signal enhancement in the spleen was induced by caspase-3/7 activity. FLAME-DEVD 2 is the first 19F MRI nanoprobe able to detect intrinsic enzymatic activity in deep tissues of living mammals. Therefore, this result potentially has a strong impact on in vivo imaging studies in mammals. We demonstrated that the novel 19 F MRI platform using PFC-based 19F MRI nanoprobes with exceptional sensitivity and OFF/ON-switching is a powerful tool for visualizing in vivo enzymatic activities. This novel technology will open the door to clarifying the biomolecular networks in living animals using the latest molecular imaging techniques.
complicated systems involving multiple steps, such as assembly or disassembly in response to enzymatic activity.13,15 However, self-assembly efficiency depends on probe concentration, and the regulation of probe concentration is difficult in vivo. Thus, intravital probe delivery affects the efficiency of signal production, and unpredictable factors make it difficult to precisely analyze in vivo enzymatic activities with high sensitivity. In this study, we exploited a simple 19F MRI signal enhancement strategy based on the PRE effect, which enabled enzymatic activity detection. Additionally, the tandemly repeated substrate peptide sequence improved the cleavage efficiency of the peptides and the kinetic response of the nanoprobe to enzymatic activity (Figures 2, 3). These fine probe design strategies led to the outstanding sensitivity to visualize endogenous enzymatic activity in vivo with a practical acquisition time for the experiments using anesthetized mice (approximately 30 min) (Figure 5). This novel substrate design concept can be generally used for improving the reactivity of various enzymes. In our in vivo experiments, the distribution analysis using FLAME-COOH indicates that 1 day is sufficient time for the uptake of FLAMEs by macrophages (Figure S12). Moreover, the results of caspase-3/7 activity check using spleen homogenates showed that caspase-3 and -7 were activated 1 day after injection of clodronate liposomes (Figure S15). Therefore, we conducted the MRI measurements using FLAME-DEVD 2 in this time course and methods. In vivo imaging of caspase-3 activity is very important, as recent studies have reported abnormal caspase-3 activity in several diseases, such as Crohn’s,35 Alzheimer’s,36 and Parkinson’s diseases.37 Based on our in vivo results, FLAME-DEVD 2 can be used to analyze the localization and dynamics of caspase-3/7 activity in these diseases and evaluate the in vivo efficacy of new drugs for the treatment of caspase-3/7-associated diseases by 19F MRI. In future studies, we will focus on controlling the nanoparticle size38,39 and improving nanoprobe delivery to diseased sites following intravenous injection. First, it is critical to prolong the blood circulation time of the nanoprobes, and coating the nanoprobe surface with polyethylene glycol40 or a zwitterion41 may extend the circulation lifetime. In addition, targeted tissue delivery will be achieved by functionalization with activetargeting moieties, such as antibodies, aptamers, or peptides.42,43 Through these strategies, caspase-3/7 activity at a targeted diseased site can be imaged by 19F MRI. In addition, this OFF/ON-switching strategy can be broadly applied to detect the activity of various enzymes, such as cathepsins and matrix metalloproteinases, by varying the amino acid sequence of the substrate.
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MATERIALS AND METHODS Materials and Instruments. Rink amide MBHA resin (loading: 0.79 mmol/g) was purchased from Merck Millipore (Kenilworth, NJ, USA). All other reagents were purchased from Tokyo Chemical Industries (Tokyo, Japan), Wako Pure Chemical Industries (Osaka, Japan), Watanabe Chemical Industry (Hiroshima, Japan), Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), and Peptide Institute, Inc. (Osaka, Japan). Reagents were of the highest grade available and used as received without further purification. Polypropylene tubing (Libra tube) was purchased from HiPep Laboratories (Kyoto, Japan). Clodronate and PBS liposomes (hydrodynamic diameter: 132 nm, clodronate concentration: 5.9 mg/mL) were purchased from Katayama Chemical Co. (Osaka, Japan). Recombinant protein caspase-3 was purchased from Abnova (Taipei, Taiwan). Recombinant caspase-1, -7, and -9 proteins were purchased from BioVision (Milpitas, CA, USA). Cathepsin B was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Recombinant legumain protein was purchased from R&D Systems (Minneapolis, MN, USA). The enzyme activities were as follows. Caspase-1: 1 U = 1 nmol h−1 using Ac-WEHD-pNA as a substrate at 37 °C. Caspase-3: 1 U = 1 nmol h−1 using Ac-DEVD-AFC as a substrate at 37 °C. Caspase-7: 1 U = 1 nmol h−1 using Ac-DEVD-pNA as a substrate at 37 °C. Caspase-9: 1 U = 1 nmol h−1 using AcLEHD-pNA as a substrate at 37 °C. NMR spectra were recorded on a JEOL JNM-AL400 instrument (Tokyo, Japan) at 400 MHz for 1H NMR and 100.4 MHz for 13C NMR; on a Bruker Ascend 500 instrument (Billerica, MA, USA) at 500 MHz for 1H NMR and 125 MHz for 13C NMR using tetramethylsilane as an internal standard, and at 376 MHz for 19 F NMR using sodium trifluoroacetate as an internal standard. Mass spectra (EI, CI, FAB) were obtained using a JEOL JMS700. Hydrodynamic diameter and zeta potential were measured with a nano Partica SZ-100 (Horiba, Kyoto, Japan). Magnetic resonance imaging (MRI) was conducted on a Bruker BioSpec 117/11 system equipped with a 35 mm inner diameter volume coil at a frequency of 500 MHz for 1H and 471 MHz for 19F measurements. Image acquisition and processing were carried out using the ParaVision software (Bruker BioSpin) and ImageJ
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CONCLUSION In this study, we rationally designed enzyme-responsive 19F MRI nanoprobes called FLAME-DEVD X (X = 1, 2) by combining an OFF/ON-switching strategy based on the PRE effect with FLAME. Through this approach, FLAME-DEVD X shows high 19F MRI signal amplification in response to caspase3/7 activity. Furthermore, by incorporating a tandemly repeated substrate peptide, the 19F MRI signal enhancement rate of FLAME-DEVD 2 was improved (Figure 3). Based on the results of the HPLC analysis of DEVD 2 incubated with various proteases (Figure S2) and inhibitor assays with cell lysate (Figure 4), FLAME-DEVD 2 showed good selectivity for caspase-3/7. Finally, caspase-3/7 activity was visualized in the spleen of a living mouse following intravenous administration F
DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
with the guidelines of the Animal Care and Use Committee of Osaka University. C57BL/6Jjcl mice were obtained from CLEA Japan (Tokyo, Japan), anesthetized with sevoflurane, and subjected to MRI for data acquisition. 19F MRI images were acquired 24 h after intravenous (iv) injection of FLAME-DEVD 2 (10 mM), and after iv injection of clodronate or PBS liposomes (100 μL; Katayama Chemical Industries Co., Ltd., Osaka, Japan). The same set of images was acquired 1 day later. Acquired images were converted to a DICOM format and rendered in red hot color using ImageJ software (NIH, Bethesda, MD, USA). The area of the liver and spleen was determined by superimposing the 1H MRI images. The 19F MRI signal intensities in the liver and spleen were measured using ImageJ software. 1H MRI RARE method: the image matrix was 256 × 128, field of view was 8 × 4 cm2, and slice thickness was 2.0 mm. TR was 500 ms. TE,eff was 8 ms. The number of averages was 2. The acquisition time was 1 min 4 s. 19 F MRI RARE method: the image matrix was 128 × 64, field of view was 8 × 4 cm2, and slice thickness was 40 mm. TR was 1000 ms. TE,eff was 8 ms. The number of averages was 128. The acquisition time was 34 min 8 s. MRI Measurement Methods of FLAME-COOH in Living Mouse. 1H MRI RARE method: the image matrix was 256 × 128, field of view was 8 × 4 cm2, and slice thickness was 1.0 mm. TR was 1500 ms. TE,eff was 15.4 ms. The number of averages was 2. The acquisition time was 1 min 36 s. 19F MRI RARE method: the image matrix was 64 × 32, field of view was 8 × 4 cm2, and slice thickness was 40 mm. TR was 1500 ms. TE,eff was 12 ms. The number of averages was 64. The acquisition time was 12 min 48 s. (b) S/N ratio of the liver and spleen before and after treatment with clodronate liposomes. The areas of the liver and spleen were determined by superimposing the 1H MRI images.
software (NIH, Bethesda, MD, USA). Transmission electron microscopy (TEM) images were acquired with a HITACHI H7650 (at 120 kV; Tokyo, Japan). Particle size distribution was measured by dynamic light scattering (DLS) on a nano Partica SZ-100 particle analyzer (Horiba), and a tunable resistive pulse sensor on a qNano nanoparticle analyzer (Meiwafosis Co., Ltd., Shinjuku-ku, Japan). Analytical reverse-phase HPLC was performed on an Inertsil ODS-3 column (4.6 × 250 mm; GL Science, Inc., Tokyo, Japan) using an HPLC system composed of a pump (PU-2080; JASCO, Oklahoma City, OK, USA) and a detector (MD-2010; JASCO). Eluents A (0.1% TFA in CH3CN) and B (0.1% TFA in H2O) were used as the mobile phases. Fluorescence intensity was measured using a plate reader (ARVO MX; PerkinElmer Life Sciences, Waltham, MA, USA). Fluorescence images were acquired with a fluorescence microscope (BZ-X700; Keyence). Inductively coupled plasma (ICP) emission spectra were obtained on a Shimadzu ICPS8100 (Kyoto, Japan). Methods. MRI Measurement of FLAME-DEVD X (X = 1, 2) in the Presence of Caspase-3. A caspase-3 enzyme assay was conducted by incubating FLAME-DEVD X (CPFCE = 6.4 mM) with caspase-3 (0.04 U/μL) in 50 mM HEPES (pH 7.4) containing 100 mM NaCl, 10 mM DTT, 1.0 mM EDTA, 0.1% CHAPS, and 10% glycerol. After incubation at 37 °C for the indicated time periods, the mixture was heated at 95 °C for 5 min, centrifuged (14 000 × g, 4 °C, 30 min), and washed once with H2O. 19F MRI RARE method: the image matrix was 128 × 64, field of view was 8 × 4 cm2, and slice thickness was 40 mm. TR was 3500 ms. TE,eff was 144 ms. The number of averages was 256. The acquisition time was 5 h 32 min 48 s. MRI Measurement of FLAME-DEVD 2 in the Presence of Caspase-1, -7, and -9. An enzyme assay with the caspase family was conducted by incubating FLAME-DEVD 2 (CPFCE = 6.4 mM) was incubated with caspase-1, -7, or -9 (0.06 U/μL) for 12 h in reaction buffer (50 mM HEPES, 50 mM NaCl, 5% glycerol, 10 mM DTT, 0.1% CHAPS, and 10 mM EDTA [pH 7.2] for caspase-1 and 50 mM HEPES, 100 mM NaCl, 10% glycerol, 10 mM DTT, 0.1% CHAPS, and 1.0 mM EDTA [pH 7.4] for caspase-7 and -9). Incubation temperature: 37 °C. 19F MRI RARE method: the image matrix was 128 × 64, field of view was 8 × 4 cm2, and slice thickness was 40 mm. TR was 1000 ms. TE,eff was 18 ms. The number of averages was 512. The acquisition time was 2 h 16 min 32 s. MRI Measurement of FLAME-DEVD 2 in Apoptotic Cell Lysates. FLAME-DEVD 2 (CPFCE = 5.3 mM) was incubated with cell lysates (0.47 μg/μL) in 50 mM HEPES (pH 7.4) containing 100 mM NaCl, 10 mM DTT, 1.0 mM EDTA, 0.1% CHAPS, and 10% glycerol at 37 °C. After 24 h of incubation, the mixture was heated at 95 °C for 5 min, and centrifuged (14 000 × g, 4 °C, 30 min). Then, FLAME-DEVD 2 was dispersed in 80 μL of H2O, aliquoted (at CPFCE = 2.0 mM) into 384-well plates, and 1H/19F MRI measurements were performed. 1H MRI RARE method: the image matrix was 256 × 128, field of view was 8 × 4 cm2, and slice thickness was 2.0 mm. TR was 500 ms. TE,eff was 8 ms. The number of averages was 2. The acquisition time was 1 min 4 s. 19F MRI RARE method: the image matrix was 128 × 64, field of view was 8 × 4 cm2, and slice thickness was 40 mm. TR was 1000 ms. TE,eff was 6 ms. The number of averages was 512. The acquisition time was 2 h 16 min 32 s. Animal (in Vivo) Experimental Procedure (FLAME-DEVD 2). All animal handling and experimentation were approved by the local ethics review board and were performed in accordance
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00167. Materials, instrumental analysis, synthesis of compounds, experimental procedures, and supplementary figures and tables (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: kkikuchi@mls.eng.osaka-u.ac.jp. ORCID
Kazuya Kikuchi: 0000-0001-7103-1275 Author Contributions #
Kazuki Akazawa and Fuminori Sugihara contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by MEXT of Japan (Grant numbers 25220207, 16H00768, and 16K13099 to K.K.), CREST of JST, the Asahi Glass Foundation (to K.K.), the Uehara Memorial Foundation (to K.K.), and the Magnetic Health Science Foundation (to S.M.). G
DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
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(20) Shalini, S., Dorstyn, L., Dawar, S., and Kumar, S. (2015) Old, new and emerging functions of caspases. Cell Death Differ. 22, 526− 539. (21) Hengartner, M. O. (2000) The biochemistry of apoptosis. Nature 407, 770−6. (22) Porter, A. G., and Jänicke, R. U. (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6, 99−104. (23) Lavrik, I. N., Golks, A., and Krammer, P. H. (2005) Caspases: pharmacological manipulation of cell death. J. Clin. Invest. 115, 2665− 72. (24) Blankenberg, F. G. (2008) In vivo detection of apoptosis. J. Nucl. Med. 49, 81−95. (25) Nakamura, T., Matsushita, H., Sugihara, F., Yoshioka, Y., Mizukami, S., and Kikuchi, K. (2015) Activatable 19F MRI Nanoparticle Probes for the Detection of Reducing Environments. Angew. Chem., Int. Ed. 54, 1007−1010. (26) Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia-Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., et al. (1997) A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907−11. (27) Kubicek, V., Havlickova, J., Kotek, J., Tircso, G., Hermann, P., Toth, E., and Lukes, I. (2010) Gallium(III) Complexes of DOTA and DOTA-Monoamide: Kinetic and Thermodynamic Studies. Inorg. Chem. 49, 10960−10969. (28) Blanco, E., Shen, H., and Ferrari, M. (2015) Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941−951. (29) Tsoi, K. M., MacParland, S. A., Ma, X. Z., Spetzler, V. N., Echeverri, J., Ouyang, B., Fadel, S. M., Sykes, E. A., Goldaracena, N., Kaths, J. M., et al. (2016) Mechanism of hard-nanomaterial clearance by the liver. Nat. Mater. 15, 1212−1221. (30) Van Rooijen, N., and Sanders, A. (1994) Liposome-mediated depletion of macrophages − mechanism of action, preparation of liposomes and applications. J. Immunol. Methods 174, 83−93. (31) Lehenkari, P. P., Kellinsalmi, M., Napankangas, J. P., Ylitalo, K. V., Monkkonen, J., Rogers, M. J., Azhayev, A., Vaananen, H. K., and Hassinen, I. E. (2002) Further insight into mechanism of action of clodronate: Inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite. Mol. Pharmacol. 61, 1255−1262. (32) Buiting, A. M. J., Zhou, F., Bakker, J. A. J., vanRooijen, N., and Huang, L. (1996) Biodistribution of clodronate and liposomes used in the liposome mediated macrophage ’suicide’ approach. J. Immunol. Methods 192, 55−62. (33) Kabalnov, A. S., and Shchukin, E. D. (1992) Ostwald ripening theory − applications to fluorocarbon emulsion stability. Adv. Colloid Interface Sci. 38, 69−97. (34) Freire, M. G., Dias, A. M. A., Coelho, M. A. Z., Coutinho, J. A. P., and Marrucho, I. M. (2005) Aging mechanisms of perfluorocarbon emulsions using image analysis. J. Colloid Interface Sci. 286, 224−232. (35) Murthy, A., Li, Y., Peng, I., Reichelt, M., Katakam, A. K., Noubade, R., Roose-Girma, M., DeVoss, J., Diehl, L., Graham, R. R., et al. (2014) A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3. Nature 506, 456−62. (36) D’Amelio, M., Cavallucci, V., Middei, S., Marchetti, C., Pacioni, S., Ferri, A., Diamantini, A., De Zio, D., Carrara, P., Battistini, L., et al. (2011) Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer’s disease. Nat. Neurosci. 14, 69−76. (37) Hartmann, A., Hunot, S., Michel, P. P., Muriel, M. P., Vyas, S., Faucheux, B. A., Mouatt-Prigent, A., Turmel, H., Srinivasan, A., Ruberg, M., et al. (2000) Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 97, 2875−2880. (38) De Jong, W. H., Hagens, W. I., Krystek, P., Burger, M. C., Sips, A., and Geertsma, R. E. (2008) Particle size-dependent organ distribution of gold nanoparticles after intravenous administration. Biomaterials 29, 1912−1919.
REFERENCES
(1) Deu, E., Verdoes, M., and Bogyo, M. (2012) New approaches for dissecting protease functions to improve probe development and drug discovery. Nat. Struct. Mol. Biol. 19, 9−16. (2) Turk, B. (2006) Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discovery 5, 785−799. (3) Weissleder, R., Tung, C. H., Mahmood, U., and Bogdanov, A. (1999) In vivo imaging of tumors with protease-activated near-infrared fluorescent probes. Nat. Biotechnol. 17, 375−378. (4) Laxman, B., Hall, D. E., Bhojani, M. S., Hamstra, D. A., Chenevert, T. L., Ross, B. D., and Rehemtulla, A. (2002) Noninvasive real-time imaging of apoptosis. Proc. Natl. Acad. Sci. U. S. A. 99, 16551−16555. (5) Westmeyer, G. G., Emer, Y., Lintelmann, J., and Jasanoff, A. (2014) MRI-based detection of alkaline phosphatase gene reporter activity using a porphyrin solubility switch. Chem. Biol. 21, 422−9. (6) Louie, A. Y., Hüber, M. M., Ahrens, E. T., Rothbächer, U., Moats, R., Jacobs, R. E., Fraser, S. E., and Meade, T. J. (2000) In vivo visualization of gene expression using magnetic resonance imaging. Nat. Biotechnol. 18, 321−5. (7) Yoo, B., and Pagel, M. D. (2006) A PARACEST MRI contrast agent to detect enzyme activity. J. Am. Chem. Soc. 128, 14032−3. (8) Perez, J. M., Josephson, L., O’Loughlin, T., Högemann, D., and Weissleder, R. (2002) Magnetic relaxation switches capable of sensing molecular interactions. Nat. Biotechnol. 20, 816−20. (9) Ye, D., Shuhendler, A. J., Pandit, P., Brewer, K. D., Tee, S. S., Cui, L., Tikhomirov, G., Rutt, B., and Rao, J. (2014) Caspase-responsive smart gadolinium-based contrast agent for magnetic resonance imaging of drug-induced apoptosis. Chem. Sci. 5, 3845−3852. (10) Tirotta, I., Dichiarante, V., Pigliacelli, C., Cavallo, G., Terraneo, G., Bombelli, F. B., Metrangolo, P., and Resnati, G. (2015) 19F Magnetic Resonance Imaging (MRI): From Design of Materials to Clinical Applications. Chem. Rev. 115, 1106−1129. (11) Mizukami, S., Takikawa, R., Sugihara, F., Hori, Y., Tochio, H., Walchli, M., Shirakawa, M., and Kikuchi, K. (2008) Paramagnetic relaxation-based 19F MRI probe to detect protease activity. J. Am. Chem. Soc. 130, 794−795. (12) Mizukami, S., Takikawa, R., Sugihara, F., Shirakawa, M., and Kikuchi, K. (2009) Dual-function probe to detect protease activity for fluorescence measurement and 19F MRI. Angew. Chem., Int. Ed. 48, 3641−3. (13) Matsuo, K., Kamada, R., Mizusawa, K., Imai, H., Takayama, Y., Narazaki, M., Matsuda, T., Takaoka, Y., and Hamachi, I. (2013) Specific Detection and Imaging of Enzyme Activity by SignalAmplifiable Self-Assembling 19F MRI Probes. Chem. - Eur. J. 19, 12875−12883. (14) Yu, J. X., Kodibagkar, V. D., Liu, L., and Mason, R. P. (2008) A 19 F-NMR approach using reporter molecule pairs to assess betagalactosidase in human xenograft tumors in vivo. NMR Biomed. 21, 704−12. (15) Yuan, Y., Sun, H. B., Ge, S. C., Wang, M. J., Zhao, H. X., Wang, L., An, L. N., Zhang, J., Zhang, H. F., Hu, B., et al. (2015) Controlled Intracellular Self-Assembly and Disassembly of 19F Nanoparticles for MR Imaging of Caspase 3/7 in Zebrafish. ACS Nano 9, 761−768. (16) Ahrens, E. T., Flores, R., Xu, H. Y., and Morel, P. A. (2005) In vivo imaging platform for tracking immunotherapeutic cells. Nat. Biotechnol. 23, 983−987. (17) Kislukhin, A. A., Xu, H. Y., Adams, S. R., Narsinh, K. H., Tsien, R. Y., and Ahrens, E. T. (2016) Paramagnetic fluorinated nanoemulsions for sensitive cellular fluorine-19 magnetic resonance imaging. Nat. Mater. 15, 662−8. (18) Matsushita, H., Mizukami, S., Sugihara, F., Nakanishi, Y., Yoshioka, Y., and Kikuchi, K. (2014) Multifunctional Core-Shell Silica Nanoparticles for Highly Sensitive 19F Magnetic Resonance Imaging. Angew. Chem., Int. Ed. 53, 1008−1011. (19) Nakamura, T., Sugihara, F., Matsushita, H., Yoshioka, Y., Mizukami, S., and Kikuchi, K. (2015) Mesoporous silica nanoparticles for 19F magnetic resonance imaging, fluorescence imaging, and drug delivery. Chem. Sci. 6, 1986−1990. H
DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry (39) Piao, Y., Burns, A., Kim, J., Wiesner, U., and Hyeon, T. (2008) Designed Fabrication of Silica-Based Nanostructured Particle Systems for Nanomedicine Applications. Adv. Funct. Mater. 18, 3745−3758. (40) Jokerst, J. V., Lobovkina, T., Zare, R. N., and Gambhir, S. S. (2011) Nanoparticle PEGylation for imaging and therapy. Nanomedicine 6, 715−728. (41) Yuan, Y. Y., Mao, C. Q., Du, X. J., Du, J. Z., Wang, F., and Wang, J. (2012) Surface Charge Switchable Nanoparticles Based on Zwitterionic Polymer for Enhanced Drug Delivery to Tumor. Adv. Mater. 24, 5476−5480. (42) Torchilin, V. P. (2014) Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nat. Rev. Drug Discovery 13, 813−827. (43) Ling, D. S., Hackett, M. J., and Hyeon, T. (2014) Surface ligands in synthesis, modification, assembly and biomedical applications of nanoparticles. Nano Today 9, 457−477.
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DOI: 10.1021/acs.bioconjchem.8b00167 Bioconjugate Chem. XXXX, XXX, XXX−XXX