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pH-Sensitive Radical-Containing-Nanoparticle (RNP) for the L-Band-EPR Imaging of Low pH Circumstances Toru Yoshitomi,† Rie Suzuki,† Takashi Mamiya,|,# Hirofumi Matsui,§,|,# Aki Hirayama,§,∇ and Yukio Nagasaki*,†,‡,§,|,⊥ Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Satellite Laboratory, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), Division of Gastroenterology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba, 305-8573, Ibaraki, Center for Integrative Medicine, Tsukuba Universiy of Technology, Kasuga 4-12-7, Tsukuba, 305-8521, Ibaraki, Japan. Received May 15, 2009; Revised Manuscript Received July 24, 2009
For the imaging of low pH circumstances in ViVo, a pH-sensitive radical-containing-nanoparticle (RNP), which has an intense electron paramagnetic resonance (EPR) signal, was designed and developed using a self-assembling amphiphilic block copolymer (PEG-b-PCTEMPO) composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(chloromethylstyrene) (PCMS) segment in which the chloromethyl groups were converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of PEG-b-PCMS block copolymer with 4-aminoTEMPO. This RNP formed core-shell-type micelles in the physiological environment, and the cumulant average diameter of the RNP was about 50 nm. The cytotoxicity and acute toxicity studies for the RNP revealed that the median inhibitory concentration (IC50) of TEMPO radicals in RNP core and median lethal dose (LD50) of RNP were >8 mmol N(TEMPO)/L and >600 mg/kg (>960 µmol N(TEMPO)/kg), respectively, indicating fairly low toxicity. The blood circulation of the RNP was evaluated using ICR mice. Contrary to the rapid clearance of low-molecular-weight TEMPO derivatives such as 4-hydroxy-TEMPO (TEMPOL) from the bloodstream, the EPR signal of the RNP remained for a fairly long period of time. Actually, the signal was observed in the blood for more than 2 h, as monitored by EPR spectroscopy. The compartmentalization of the TEMPO radicals in the RNP core improved the stability in the bloodstream. Since an amino group was introduced in each repeating unit of the PCTEMPO segment, the disintegration of the RNP was caused by the protonation of the amino groups in response to the acidic pH environment (pH < 6.0), as confirmed by the dynamic light scattering (DLS) measurements. In addition, a drastic change in the EPR spectra from broad to sharp triplet was observed, accompanying the disintegration. This change was based upon the mobility of the TEMPO moieties covalently conjugated in the hydrophobic segment, which was confirmed by the rotational correlation time of the TEMPO moieties on the PCTEMPO segment. Note that the peak intensity of the EPR signal increased at around the phase transition point (ca. pH ) 6.0). When pH-sensitive RNP solutions at pH values 5.6 and 7.4 were visualized using an L-band EPR imaging system, the phantom images showed a remarkable on-off regulation in response to the acidic pH environment. These results demonstrate that pH-sensitive RNPs are expected to serve as nanoprobes for the in ViVo EPR imaging of low pH circumstances.
Recently, the non-invasive in ViVo imaging of low pH circumstances has attracted attention as a new means of detecting disease and response to clinical treatment. Low pH circumstances, which are an aberration of normal pH homeostasis, are caused by ischemia (1, 2), infection (3) or inflammation (4), extracellular acidosis in tumors (5, 6), wound healing (7), and so forth. However, there is currently no clinical tool available to capture an image of low pH circumstances in ViVo. In order
to image acidic pH regions in ViVo, various molecular probes have been developed so far for magnetic resonance imaging (MRI) (8-10), positron emission tomography (PET) (11), and optical imaging (12). Under in ViVo conditions, however, the diagnostic efficiency of such probes is still controversial. A more desirable design criterion for the imaging of acidic pH regions in ViVo is believed to be the development of nanoprobes capable of the on-off regulation of the signal in response to the acidic pH environment (8, 12).
*
[email protected]. † Graduate School of Pure and Applied Sciences, University of Tsukuba. ‡ TIMS, University of Tsukuba. § TARA, University of Tsukuba. | Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba. ⊥ MANA, NIMS. # Division of Gastroenterology, Graduate School of Comprehensive Human Sciences, University of Tsukuba. ∇ Center for Integrative Medicine, Tsukuba Universiy of Technology.
Nitroxyl radicals, such as 2,2,6,6-tetramethylpiperidinyloxy (TEMPO), are organic molecules that have a stable radical and are therefore susceptible to electron paramagnetic resonance (EPR). Recently, the research on in ViVo EPR imaging has been stepped up by several groups (13, 14). Some groups have reported on pH-sensitive nitroxyl radicals of the imidazolidine type or the imidazoline type for the noninvasive measurement of pH (15, 16) Under in ViVo conditions, however, such lowmolecular-weight nitroxyl radicals pose several problems, such as nonspecific accumulation in normal tissues, preferential renal
INTRODUCTION
10.1021/bc900214f CCC: $40.75 2009 American Chemical Society Published on Web 08/17/2009
pH-Sensitive RNP for L-Band-EPR Imaging
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Figure 1. Schematic illustration of the pH-sensitive radical-containing-nanoparticle (RNP) prepared from PEG-b-PCTEMPO for the in ViVo EPR imaging of low pH circumstances.
clearance, and rapid reduction of the nitroxyl radical form to the corresponding hydroxylamine form (17, 18). To solve these difficulties, we have designed and developed a radical-containing-nanoparticle (RNP) using a self-assembling amphiphilic block copolymer (PEG-b-PCTEMPO) composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(chloromethylstyrene) (PCMS) segment in which the chloromethyl groups were converted to TEMPOs via the amination of PEG-b-PCMS block copolymer with 4-aminoTEMPO (Figure 1) (19). This RNP formed core-shell-type micelles in the physiological environment; the cumulant average diameter of the RNP was about 50 nm, and the RNP emitted an intense EPR signal. We have previously reported that the TEMPO radicals in the core of the RNP showed fairly high resistance against reducing reactions compared with low-molecular-weight 4-hydroxyl-TEMPO (TEMPOL) even in the presence of 3.5 mM ascorbic acid (19). Thus, this RNP is anticipated to serve as a high-performance bionanoparticle which can be used in ViVo. In this study, we evaluated the toxicity and the blood circulation of the RNP in view of its use in ViVo. Since an amino group was introduced into each repeating unit on the PCTEMPO segment, the RNP emitted pH-sensitive EPR signals in response to the acidic pH environment due to the protonation of the amino groups. We demonstrated the drastic change in the EPR signal of the RNP as a function of pH. A pH-dependent phantom study of the RNP for the in ViVo EPR imaging of low pH circumstances using L-band EPR spectroscopy is also described herein.
EXPERIMENTAL PROCEDURES Synthesis of MeO-PEG-b-PCTEMPO. MeO-PEG-b-PCMS was synthesized by the radical telomerization of chloromethylstyrene (CMS) using MeO-PEG-SH (Mn ) 5000) as a telogen, according to procedure described in the previous report (19) (see Supporting Information Scheme S1 and Figure S1). After the synthesis of PEG-b-PCMS, the chloromethyl groups on the PCMS segment of the block copolymer MeO-PEG-b-PCMS were converted to stable radicals via the amination of MeOPEG-b-PCMS with 4-amino-TEMPO in DMSO (see Supporting Information Scheme S1 and Figure S2). Preparation of the RNP. The RNP was prepared from MeOPEG-b-PCTEMPO by the dialysis method described previously (19). Briefly, 400 mg of MeO-PEG-b-PCTEMPO was dissolved in 100 mL of DMF, and the polymer solution was transferred
into a membrane tube (Spectra/Por, molecular-weight cutoff size 3500; Spectrum, USA) and then dialyzed for 24 h against 2 L of water, which was changed after 2, 5, 8, and 20 h. Dynamic light scattering (DLS) measurement was carried out to determine the diameter of the obtained RNP after dialysis. In order to adjust the concentration of the RNP solution, the obtained RNP solution was concentrated by a centrifugal evaporator (∼60 mg/ mL), followed by DLS measurement of the concentrated RNP to confirm the size of the RNP after the concentration. Cytotoxicity Studies. Cell viability was determined by a quantitative colorimetric assay using WST-8 (DOJINDO Laboratories, Kumamoto, Japan). Briefly, 1 × 104 cells were seeded per well on a 96-well flat-bottom plate. At 24 h after treatment, 10 µL of WST-8 reagent was added to each well containing 100 µL of medium, according to the manufacturer’s instructions. The plate was incubated for 1 h at 37 °C and read at an absorbance of 450 nm by a plate reader. Median Lethal Dose (LD50) Studies. Male ICR mice (5 weeks old, ca. 25 g) were purchased from Charles River Japan, Inc. The LD50 value of the RNP was determined by injecting the RNP saline solution with increasing doses into the ICR mice via the tail vein. There were five mice in each dose group. The doses of intravenous administration were elevated as follows: 75, 150, 300, and 600 mg/kg mouse body weight. The mice in all groups were observed for 2 weeks, and the number of surviving mice was recorded. Blood Circulation of the RNP. The blood circulation of the RNP was examined as follows: after anesthesia with pentobarbital sodium (40 mg/kg), 200 µL of RNP saline solution (12 mg/mL) were injected into the tail vein of male ICR mice (ca. 25 g), and then blood samples were collected at 2 min, 10 min, 30 min, 1 h, and 3 h. Plasma samples were obtained by centrifugation (6200 rpm, 2000 × g, 10 min) of the blood. It was confirmed that no EPR signal was observed in the blood cell precipitates after centrifugation. The EPR signal intensities in these plasma samples were measured by an X-band EPR spectrometer (JES-TE25X, JEOL, Tokyo, Japan) at room temperature. In the case of the EPR intensities at 0 min, 50 µL of the RNP solution (12 mg/mL) was added to the blood (500 µL), followed by centrifugation (6200 rpm, 2000 × g, 10 min) of the blood and EPR measurement, assuming that the total blood volume in mice is 80 mL/kg. A free-TEMPOL saline solution with the same spin concentration as the RNP was used as a control. The EPR measurements were carried out under
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the following conditions: frequency, 9.41 GHz; power, 8.00 mW; field, 333.8 ( 5 mT; sweep time, 1.0 min; modulation, 0.1 mT; time constant, 0.1 s. All procedures and animal care were approved by the Committee on the Ethics of Animal Experimentation of the University of Tsukuba, and were conducted according to the Guidelines for Animal Experimentation of the University of Tsukuba. Potentiometric Titrations. An aqueous solution of RNP (1.0 mL: 20 mg/mL) was added to a solution of 0.01 M HCl (4.0 mL), followed by the adjustment of the NaCl concentration to 0.15 M (40 mL). The resulting RNP solution (40 mL) was titrated with 0.01 M NaOH containing 0.15 M NaCl at 25 °C. An automatic titrator (DL-25, Mettler) was used for the titration. In this case, the titrant was added in quantities of 0.05 mL after the pH values were stabilized (minimal interval: 30 s), and the titration curves in the pH range from about 3 to about 10 were recorded. Dynamic Light Scattering (DLS) Experiments and X-Band Electron Paramagnetic Resonance (EPR) Experiments as a Function of pH. The light scattering intensities of the pHsensitive RNP were measured as a function of pH using a light scattering spectrometer (Nano ZS, ZEN3600, Malvern Instruments, Ltd., U.K.) equipped with a He-Ne laser that produces vertically polarized incident beams at a detection angle of 173° at 25 °C. Briefly, 3.5 mg/mL of the RNP solution was prepared as stock solution A after the formation of the RNPs by the dialysis method. Britton-Robinson buffers (100 µL each) with various pH values, which were prepared from a stock solution containing 1 M phosphoric acid, 1 M boric acid, and 1 M acetic acid and by adjusting the pH value with NaOH, were added to stock solution A (400 µL). The mixtures with various pH values were transferred to the cells immediately, followed by DLS measurement. A nanoparticle (CMS-NP: CNP) prepared from PEG-b-PCMS was used as a control. After DLS measurement, the samples were transferred to a capillary tube, followed by EPR measurement. To measure the EPR spectra of the pH-sensitive RNP as a function of pH, the EPR spectra were recorded at room temperature on a JEOL JESTE25X spectrometer. The EPR measurements were carried out under the following conditions: frequency, 9.4190 GHz; power, 8.00 mW; field, 333.8 ( 50 mT; sweep time, 2.0 min; modulation, 0.1 mT; time constant, 0.1 s. Phantom EPR Imaging. Phantom images were obtained using a capillary glass tube (1.2 mm i.d.) containing 80 mg/mL of RNP solution at pH 5.6 or 7.4. 2D spatial images perpendicular to the axes of the tubes using an L-band EPR spectrometer manufactured by JEOL (Tokyo, Japan), consisting of a 1 GHz microwave unit and a bridged four-gap loop-gap resonator (38 mm in diameter and 28 mm in length), were obtained in this study. The EPR conditions for these phantom studies were as follows: magnetic field, 370.0 ( 5.0 mT; modulation width, 2.0 mT; time constant, 0.03 s; microwave power, 0.25 mW; scanning time, 30 s. EPR images were constructed on 256 points × 256 points.
RESULTS AND DISCUSSION Synthesis of the Polymer and Preparation of the RNP. As described in the previous report (19), MeO-PEG-b-PCMS was synthesized by the radical telomerization of CMS using MeO-PEG-SH as a telogen, as shown in Supporting Information Scheme S1. The molecular weight distribution of MeO-PEGb-PCMS was evaluated by size exclusion chromatography (SEC), as shown in Supporting Information Figure S1a. The SEC profile of MeO-PEG-b-PCMS and the molecular weight of the PCMS segment after polymerization were almost the same as those of acetal-PEG-b-PCMS reported previously (19). The polymer backbone of PEG-b-PCMS consisted of PEG with a
Yoshitomi et al.
Figure 2. Cell viability against RNP (closed square), TEMPO (open triangle), and 4-amino-TEMPO (open circle) in colon cells (WST assay). The plotted data represent the average ( SEM of three experiments.
molecular weight of 5000 g/mol for the hydrophilic segment and 20 repeating units of PCMS for the hydrophobic segment (MW ) 3300), as determined by the 1H NMR data based on the Mn of PEG (see Supporting Information Figure S1b). MeOPEG-b-PCTEMPO was synthesized by the substitution of the chloromethyl groups of the PCMS segment of MeO-PEG-bPCMS with amino-TEMPO via amination in DMSO. After the purification of the obtained PEG-b-PCTEMPO, the substitution ratio of the modified TEMPO moieties per repeating unit of PCMS was 80%, as determined by EPR spectroscopy using the standard curve of free-amino-TEMPO in chloroform. The thusobtained PEG-b-PCTEMPO was dialyzed against water to obtain the RNP. As shown in Supporting InformationFigure S3, the RNP after dialysis showed a unimodal distribution in the histogram analysis. The average diameter and polydispersity factor (µ2/Γ2), as determined by the cumulant method, were about 50 nm and 0.133, respectively. Toxicity Study of the RNP. TEMPO derivatives are known as biologically active substances (20, 21). Suy et al. have reported that the promotion of apoptosis by TEMPO is one of its physiological activities in cancer cells (22). Since each RNP carries several thousand TEMPO moieties (stable radicals) in its core, it is interesting to investigate the behavior of RNP in ViVo. The cytotoxicity of the RNP was investigated by subjecting colon cells to the WST assay. As shown in Figure 2, the dosedependent cell viabilities were observed in colon cells treated with TEMPO, 4-amino-TEMPO, and the RNP. The number on the horizontal axis of the figure indicates the concentration of TEMPO radical, as determined by EPR spectroscopy. The colon cell viabilities were progressively reduced after 24 h with increasing concentrations of low-molecular-weight TEMPO and amino-TEMPO. The median inhibitory concentrations (IC50) of TEMPO and 4-amino-TEMPO were 8.3 mM and 4.8 mM, respectively. Because amino groups generally show high toxicity, the cytotoxicity of 4-amino-TEMPO might be higher than that of TEMPO. In contrast, the RNP induced no change in cell viability regardless of the concentration of RNP up to the concentration of 8 mmol N(TEMPO)/L. The extremely low toxicity of RNP is probably because the outer PEG layer constitutes an excellent stealth shield around the amino-TEMPO moieties in the RNP core. The median lethal dose (LD50) of the RNP in ICR mice was then evaluated. For the evaluation of the LD50 value, RNP solution at a concentration of 60 mg/ mL was employed, because the size of the RNP did not change up to this concentration (data not shown). 250 µL of RNP saline solution (0, 7.5, 15.0, 30.0, 60.0 mg/mL, which correspond to 0, 75, 150, 300, 600 mg/kg-dose, respectively) were intravenously administered to ICR mice (ca. 25 g).
pH-Sensitive RNP for L-Band-EPR Imaging
Figure 3. Survival rate of ICR mice injected with RNP. The RNP doses of the intravenous injections were gradually increased as follows: 0, 75, 150, 300, 600 mg/kg mouse body weight.
Figure 4. Blood circulation of the RNP (closed circle) and TEMPOL (open circle). The EPR intensity changes in the blood were measured at 2, 5, 30, 60, and 180 min after injecting RNP saline solution into the mice.
As shown in Figure 3, no dead mice were observed for 2 weeks after the administration of the RNP (0-300 mg/kg). Even at 600 mg/kg (concentration of amino-TEMPO moieties: 960 µmol N(TEMPO)/kg), 60% of the mice lived. Stefano et al. have reported that the LD50 of poly(L-lysine) with a molecular weight of 28 000-42 000 (polymerization degree ) 135-203) was between 15 and 30 mg/kg (concentration of amino groups: 72.5-145 µmol N/kg) (23). The extremely low toxicity of RNP, viz., IC50 > 8 mmol N(TEMPO)/L and LD50 > 600 mg/kg (>960 µmol N(TEMPO)/kg), is considered to be due to the confinement of the polyamine segment of the block copolymer in the RNP core. The antioxidant character of the RNP might also have contributed to the reduced toxicity. Blood Circulation of Nitroxyl Radicals. In order to obtain information on the circulation of nitroxyl radicals in the bloodstream, an EPR study was carried out. Figure 4 shows the time course of the spin concentration of the RNP in the blood after intravenous injection. When low-molecular-weight TEMPOL was administered, almost no EPR signals was observed even after 2 min in the bloodstream. The half-life of TEMPOL in blood is reported to be about 15 s (24). It is interesting to note, in contrast, that the EPR signal in the bloodstream was observed even 2 h after tail-vein injection of the RNP. The half-life of the RNP was 60 times longer (15 min) than that of low-molecular-weight TEMPOL. The rapid clearance of low-molecular-weight TEMPOL from the bloodstream was probably due to preferential renal clearance and the rapid reduction of the TEMPO radical form to the corresponding hydroxylamine form in the blood. We have previously reported that the TEMPO radicals in the RNP core showed high reduction resistance in the presence of 3.5 mM ascorbic acid (19). The compartmentalization of the TEMPO radicals in the RNP core resulted in the reduced reactivity of the TEMPO radicals toward ascorbic acid. The increased reduction resistance of the RNP
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Figure 5. (a) Experimental titration curve obtained for aqueous solutions of CNP prepared from PEG-b-PCMS (open circle) and RNP prepared from PEG-b-PCTEMPO (closed circle) using 0.01 M NaOH. (b) The degree of protonation (R) as a function of solution pH for the pHsensitive RNP prepared from PEG-b-PCTEMPO.
must be one of the reasons for the prolonged blood circulation time. Since the observed EPR signals were broad singlet in blood (data not shown), it is considered that the TEMPO moieties are still located in the solid core of the RNP. Thus, slow dissociation is one of the important factors to improve blood circulation of nitroxyl radicals in bloodstream. pH Response of the RNP. Potentiometric Titration and Dynamic Light Scattering (DLS) Measurements. Polyamine with the appropriate hydrophobicity is known to show phase transition as a function of pH (25, 26). For example, the poly[2(N,N-diethylamino)ethyl methacrylate] (PEAMA) homopolymer exhibits precipitation above pH 7.5 owing to the deprotonation of the amino groups of the PEAMA (27). It is reported that the self-assembly structure of block copolymer micelles can be controlled by the environmental pH when PEAMA is used as one of the segments in the block copolymer (28, 29). Because the PCTEMPO segment possesses an amino group in each repeating unit, a similar phenomenon is anticipated in response to acidic pH environments due to the protonation of the amino groups. In order to confirm the pH-response of the RNP, potentiometric measurement of the RNP was performed. As shown in Figure 5a, the CNP, prepared as a control from PEGb-PCMS (no amino groups), did not show buffer action at all, while the titration curve of the RNP showed a clear inflection point at pH 6-8, which was regarded as a buffer action. From the obtained titration curves, the pKa of RNP was confirmed to be 6.5. The R-pH curves calculated using the HendersonHasselbach equation indicated that the amino groups on the PCTEMPO segment were almost completely protonated at pH values below 5.0, as shown in Figure 5b. In order to investigate the effect of the protonation of the PCTEMPO segment on the self-assembly structure of the RNP, the light scattering intensities of RNP solutions were measured as a function of pH. As shown in Figure 6, the light scattering intensity of CNP prepared from PEG-b-PCMS (no amino groups) did not change at all, whereas it drastically decreased at pH values below 6.0 in the case of RNP prepared from the PEG-b-PCTEMPO block copolymer. According to the Rayleigh approximation (I ∝ d6, where I is the scattering intensity and d is the particle size) (30), it is clear that the RNP disintegrated at pH values below 6.0. These results suggest that the PCTEMPO segment cannot form the hydrophobic core of the RNP due to the increased solubility of the PCTEMPO segment, which may be due to the protonation of the amino groups. On the basis of the obtained data, viz., titration and DLS measurements, it is concluded that the disintegration of the RNP at around pH ) 6.0 was due to the protonation of the amino groups on the PCTEMPO segment and the molecular dissolution of the PEG-b-PCTEMPO copolymer in the acidic solution. Electron Paramagnetic Resonance (EPR) Measurement. EPR spectra are known to give information on the local environment
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Figure 8. (a) Rotational correlation times (τc) of the TEMPO moieties on the PCTEMPO segment of RNP as a function of pH, which were calculated using eq 1 (see Supporting Information Figure S4).(b) EPR signal height of RNP as a function of pH. Figure 6. Effect of pH on the light scattering intensity of RNP prepared from PEG-b-PCTEMPO (closed circle) and CNP prepared from PEGb-PCMS (open triangle). The normalized scattering intensity (%) is expressed as the value relative to that at pH 8.2.
Figure 9. Phantom images of the RNP obtained by L-band EPR spectroscopy at pH 5.6 and 7.4. EPR spectrum of the RNP at pH 5.6 (a) and pH 7.4 (b). (c) Phantom images of the RNP at pH 5.6 (left) and pH 7.4 (right).
Figure 7. X-band EPR spectra of the RNP as a function of pH, at pH 4.0, 5.5, 5.6, 5.9, 6.0, 7.4, and 8.2, in Britton-Robinson buffer prepared from a stock solution containing 1 M phosphoric acid, 1 M boric acid, and 1 M acetic acid, and by adjusting the pH value with NaOH.
around the spin probe (31). The EPR measurement of the polymers carrying nitroxyl radicals thus gives us the dynamics of the polymer chain (32, 33). Since the assembly of PEG-bPCTEMPO depends on the protonation of the amino groups, as described above, the EPR spectra must be influenced by the environmental pH. In order to obtain detailed information on the EPR signals, the EPR measurement of the RNP under various pH conditions was carried out using an X-band EPR spectrometer. Figure 7 shows the EPR spectra of the RNP under various pH conditions, from pH ) 4.0 to 8.2. Contrary to the clear triplet signals of low-molecular-weight TEMPO derivatives resulting from the interaction between the 14N nuclei and the unpaired electron in the dilute solution (19), broad signals of the RNP were observed at pH values above 6.0, in same manner as the EPR signal after the dialysis. The EPR spectra of the RNP at pH values above 6.0 are due to the confinement of the TEMPO radicals in the solid core of the RNP. With decreasing pH, the EPR signals gradually changed, and typical triplet signals were observed under acidic pH (pH < 6.0) conditions, which is consistent with the disintegration region of the RNP. Since the spin probe is covalently conjugated in the PCTEMPO segment of the PEG-b-PCTEMPO block copolymer, the relative anisotropy observed in the EPR spectrum is directly related to the rotational mobility of the probe. In order to confirm the
change in the rotational mobility factor, the rotational correlation time, τc (see Supporting Information Figure S4), was determined as a function of the environmental pH (Figure 8a). Under neutral conditions, the τc was about 8.0 × 10-10 s. Its abrupt decrease was observed again at around the phase transition region (ca. pH ) 6.0). Actually, the τc was about (3-4) × 10-10 s at pH values below 6.0. The lower τc indicates the higher mobility of the TEMPO moieties, which is certainly correlated with the disintegration of the RNP. Crucially, the mobility of the TEMPO moieties as side chains of the PCTEMPO segments increased along with the disintegration of the RNP core, and the distance between the TEMPO radicals increased due to the electrostatic repulsion between the protonated amino groups. In conjunction with the phase transition, a change in the EPR signal height was observed, as shown in Figure 8b. Indeed, a significant increase in signal height was observed at pH values below 6.0, which confirms that the RNP shows a phase transition at a specific pH (around pH ) 6.0). If this phenomenon is also observed in the L-band EPR spectra, low pH circumstances might be visualized by in ViVo EPR imaging. L-band EPR imaging systems operating in the lower-frequency microwave bands (less than 1.2 GHz), where the dielectric losses are lower, have been developed and utilized for the in ViVo imaging of animals using a spin probe (34). After two different RNP samples (80 mg/mL) were transferred to a capillary glass tube at pH values 5.6 and 7.4, phantom images were obtained using the L-band EPR imaging system. Figure 9a,b shows the EPR spectra of the RNP at pH values 5.6 and 7.4, as measured by L-band EPR spectroscopy. The EPR spectrum of the RNP at pH 5.6 showed a clear triplet signal due to the disintegration of the RNP, in the same manner as the X-band EPR spectroscopy (Figure 9a). On the other hand, almost no narrow-line-width EPR signals were observed at pH 7.4 (Figure 9b). The signalto-noise (S/N) ratios of the spectra at pH values 5.6 and 7.4 were about 12.5 and 7.8, respectively. The improvement of the S/N ratios in response to the acidic pH environment is remarkable. Figure 9c shows phantom images of the RNPs at pH values 5.6 and 7.4. The appropriate threshold (cutoff: 40%)
pH-Sensitive RNP for L-Band-EPR Imaging
was used for de-noising. The phantom images clearly show a remarkable on-off regulation in the capillary glass tube. If the RNP is able to accumulate in low pH circumstances in ViVo, then the RNP might disintegrate and emit strong EPR signals. This is our strategy for imaging of low pH circumustances using pH-sensitive RNP. From these results, the RNP might be used as a nanoprobe for the in ViVo EPR imaging of low pH circumstances.
CONCLUSIONS We have developed an EPR-imaging nanoprobe based on a pH-sensitive radical-containing-nanoparticle (RNP), which consists of PEG-b-PCTEMPO block copolymer. The RNP was confirmed to attain long-term spin circulation in the blood compared with low-molecular-weight TEMPOL. The RNP was also confirmed to show extremely low toxicity due to the confinement of the amino-TEMPO moieties in the RNP core. It is interesting to note that the disintegration and drastic change in the EPR signal of the RNP were observed at pH values below 6.0; this change was caused by the protonation of the amino groups on the PCTEMPO segments in response to the acidic pH environment. The phantom images indicate that the RNP can implement a remarkable on-off regulation of the EPR signal in response to acidic pH environments (pH < 6.0). These results demonstrate that pH-sensitive RNPs are expected to serve as an EPR imaging nanoprobes, which can image low pH circumstances in ViVo.
ACKNOWLEDGMENT We appreciate Dr. Kazuko Toh of the University of Tsukuba for her helpful assistance in this study. This work was partly supported by a Grant-in-Aid for Scientific Research A (21240050) and the World Premier International Research Center Initiative (WPI Initiative) on Materials Nanoarchitronics of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Supporting Information Available: The SEC diagram and H NMR spectrum of MeO-PEG-b-PCMS and MeO-PEG-bPCTEMPO and the size distribution of the RNP are provided. Also, the materials and calculation method for the rotational correlation time (τc) are described. This material is available free of charge via the Internet at http://pubs.acs.org. 1
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