Self-Assembly Behavior of Emissive Urea Benzene Derivatives

Mar 7, 2017 - In this study we describe the construction of a system composed of thermally responsive molecules that can be induced to accumulate in t...
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Self-Assembly Behavior of Emissive Urea Benzene Derivatives Enables Heat-Induced Accumulation in Tumor Tissue Takeru Araki,† Shuhei Murayama,‡ Kazuteru Usui,† Takashi Shimada,† Ichio Aoki,*,‡ and Satoru Karasawa*,†,§ †

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Department of Molecular Imaging and Theranostics, National Institute of Radiological Sciences (NIRS), QST, Anagawa 4-9-1, Inage, Chiba 263-8555, Japan § PRESTO, Japan Science and Technology Agency, Kawaguchi, 332-0012, Japan ‡

S Supporting Information *

ABSTRACT: In this study we describe the construction of a system composed of thermally responsive molecules that can be induced to accumulate in tumor tissues by heating. EgX molecules consisting of an urea−benzene framework and oligoethylene glycol (OEG) functional groups with an emissive aminoquinoline formed nanoparticles (NPs) ∼10 nm in size at 23 °C with a fluorescence quantum yield of 7− 10%. At higher temperatures, additional self-assembly occurred as a result of OEG dehydration, and the NPs grew to over 1000 nm in size; this was accompanied by low critical solution temperature behavior. EgXs accumulated in tumor tissues of mice at a body temperature of around 33−35 °C, an effect that was accelerated by external heating around the tumor to approximately 40 °C as a result of increased particle size and enhanced retention in tissue. These EgX NPs can serve as a tool for in vivo monitoring of tumor progression and response to treatment. KEYWORDS: Self-assembly, nanoparticles, LCST, tumor imaging, fluorescence

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enhanced permeability and retention (EPR) effect refers to drug delivery systems that take advantage of these features,6−8 although vessel permeability and retention by tumor tissue are highly dependent on particle size. We have investigated the thermal self-assembly behavior of urea−benzene derivatives (UBDs) with amphiphilic side chains consisting of oligoethylene glycol (OEG) and alkyl chains; those with side chain at the 1, 3, and 5 positions of the benzene ring (tri-UBD-Cn) showed thermally induced size changes in aqueous solution accompanied by low critical solution temperature (LCST) (Scheme 1)3,9,10 that was dependent on the length of the alkyl chain. Additionally, UBD conjugated with 2,2,6,6-tetramethyl-1-piperidinyloxl (TEMPO) and proton-responsive tertiary amines showed pH dependence of molecular motion and water proton relaxivity, suggesting that it can serve as a metal-free functional magnetic resonance imaging (MRI) contrast agent (Scheme 1).9 Exploiting the differences in heat production between tumor and normal tissue (Figure 1), we constructed thermally responsive molecules (EgX, where X = OEGs of different lengths) comprising a fluorophore and UBD framework (Scheme 1). The fluorophore of the aminoquinoline derivative

umor tissue shows a higher temperature than normal tissue due to higher activity and energy requirements.1−3 Heat production rates per cell differ between patients with high- and low-grade lymphoma (3.9 and 2.8 pW, respectively; Figure 1).1 Fluorescence imaging using polymers to introduce thermally sensitive fluorophores into cells allow inter- and intracellular temperature monitoring.2,4 Heated tumor tissue radiates less heat than normal tissue, resulting in tumor cell death by hyperthermia.5 In addition, vascular endothelial cells in tumor tissue are surrounded by void spaces (Figure 1).6 The

Figure 1. Schematic illustration of differences in morphology and temperature between normal and tumor tissues. Tumor tissue exhibits void spaces corresponding to the EPR effect. © XXXX American Chemical Society

Received: December 27, 2016 Revised: February 1, 2017

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DOI: 10.1021/acs.nanolett.6b05371 Nano Lett. XXXX, XXX, XXX−XXX

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were observed at 6.0−4.0 and 4.0−1.5 ppm, respectively. When the concentration was increased to 0.1 mM, no spectral changes were observed. In contrast, at concentrations above 0.25 mM, individual signals shifted, suggesting self-assembly; for instance, in the aromatic region, signals corresponding to Ph and Q shifted to lower and higher fields, respectively, while those associated with OEGs and alkyl chains shifted to higher fields. The resultant shielding of H atoms is typical self-assembly behavior,14 whereas the deshielding shifts detected only for Ph was likely due to the formation of hydrogen bonds15 between oxygen and hydrogen atoms in urea groups and Ph, respectively; this was supported by molecular orbital calculations. Deshielding of protons often gives rise to the UBD analogues Tri-UBD and TEMPO-UBD.9,10 Plots of chemical shifts as a function of concentration revealed a noncontinuous point corresponding to the critical aggregation concentration (CAC) (Figure 2b). In addition, the sigmoidal curve showing the noncontinuous point was indicative of cooperative selfassembly.16 Association constants (K) estimated from Benesi− Hildebrand plots17 were 9.8 × 103, 7.6 × 103, and 4.4 × 103 M−1 for Eg3, Eg4, and Eg6, respectively (Figures 2c and S19− S21). The rank order of K values was Eg3 > Eg4 > Eg6, indicating that the driving force of EgX self-assembly at 23 °C was hydrophobicity (i.e., enthalpy-driven aggregation). An enlargement of the 1H NMR spectrum around the aromatic region showing the concentration dependence of Eg6 and of HA chemical shifts in EgXs is shown in Figure 2, whereas full 1 H NMR spectra as well as the concentration dependence of EgXs (X = 3, 4, and 6) and Benesi−Hildebrand plots of HA−HI of EgXs are shown Figures S12−S21. K and CAC values along with the photophysical properties of EgXs in H2O are summarized in Table 1. To clarify the molecular structure of the self-assembled NPs, the two-dimensional (2D) 1H NMR nuclear Overhauser effect (NOE) spectrum of Eg4 was examined in 0.25 mM D2O, which was higher than the CAC. A strong correlation between HA and HD in Q and HE in Ph was observed, although there was no correlation between H atoms connected in neighboring C atoms (Figure 3a), suggesting through-space interactions between the Q and Ph. In addition, Eg4 had a bent form above the CAC before self-assembly (Figure 3c). Molecular mechanics calculations under aqueous conditions (MMFFaq)18 were performed at the single-molecule level for Eg4 using Spartan ‘08 software (Waveform, Irvine, CA, USA). Monte Carlo searching18 of the conformer distributions at ground state with MMFFaq19 suggested that, in the lowest-energy structures, there was close contact between Ph and Q, yielding the bent form. In the aromatic region, the closest distances between HA and HD in Q and HE in Ph were 4.85 and 3.25 Å, respectively, which were in accordance with experimental results of NOE.

Scheme 1. Molecular Structures of Tri-UBD-Cn, TEMPOUBD, and EgXs

2,4-bis(trifluoromethyl)quinolin-7-amine (TFMAQ)11−13 and the UBD core with amphiphilic side chains were connected via a linker. To evaluate thermal responsiveness, tri-, tetra-, and hexaethylene glycol chains were incorporated into a secondary nitrogen atom of TFMAQ to obtain Eg3, Eg4, and Eg6, respectively, as functional molecules (Scheme 1). EgXs selfassembled in water (saline), forming nanoparticles (NPs) that were 7−9 or 5−7 nm in size. Upon heating, the NPs showed different LCST values depending on the extent of dehydration of the OEG chain. Above the LCST value, micrometer-sized particles were observed in cloudy solution. The NPs showed thermally induced accumulation in mouse tumor tissues, which was monitored by fluorescence imaging. Results and Discussion. We prepared three fluorophore unitsi.e., TFMAQ−EgX−EgBr (X = 3, 4, and 6)with OEG as a controlling hydrophobic unit. Separately, we also prepared a UBD core unit (UBD−OH) with a hydroxyl group and two amphiphilic side chains incorporated into the benzene ring. A CH3CN solution of TFMAQ−EgX−EgBr and UBD−OH was stirred for 24 h at room temperature to yield Eg3, Eg4, and Eg6, which showed fluorescence and thermal responsiveness. The steps in EgX synthesis are summarized in Scheme 2 and shown in detail in Scheme S1. 1 H nuclear magnetic resonance (NMR) spectra of EgXs at various concentrations (0.05−10 mM in heavy water; deuterium oxide, D2O) were obtained at 23 °C (Figure 2a). For 0.05 mM Eg6, aromatic signals attributable to quinoline (Q) and benzene (Ph) rings were detected in the range of 8.0− 6.5 ppm, while signals arising from the OEGs and alkyl chains Scheme 2. Steps in the Synthesis of EgXs (X = 3, 4, and 6)

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Figure 2. (a) Enlarged 1H NMR spectrum of the aromatic region showing the concentration dependence of Eg6. (b) Concentration dependence of HA chemical shifts in EgXs. (c) Benesi−Hildebrand plot of HA in EgXs.

Table 1. Values of CAC, K, λmaxFl, and θFl in Aqueous Solutiona estimated values related to selfassembly behavior Eg3 Eg4 Eg6 a

fluorescence properties in H2O

CAC

Kb (M−1)

λmaxFl (nm)

θFl

Eg4 > Eg3. These results indicate that LCST is around 35 °C, which is the body temperature of mice. Thermal behaviors in different transmittance solutions were also confirmed visually (Figure 4b). LCST in 0.25 mM saline and the concentration dependence of LCST for EgXs (X = 3, 4, and 6) are shown in Figures 4a, S24, and S25. Changes in size in response to heat were assessed by dynamic light scattering (DLS) measurements below and above the LCST (Figure 5 and Table 2). In the case of Eg6 in 0.25 mM saline, the hydrodynamic diameters (DH) were 7 nm at 25 °C− 40 °C and of 290−4000 nm at 40 °C−45 °C, indicating the formation of micelle-like NPs and large globular microparticles below and above the LCST, respectively. Eg3 and Eg4 showed

(X = 3, 4, and 6) in various solvents are shown in Figure S22, and photophysical values of EgXs in H2O and in various solvents are summarized in Tables 1 and S1. The thermal response of the EgXs was investigated based on changes in their transmittance (800 nm); that is, we monitored self-assembly in transparent and opaque solutions (Figure 4). In 0.25 mM Eg6, there was no change in transmittance until 39 °C; an abrupt decrease due to LCST was observed at 40 °C, indicating that OEG dehydration occurred and resulted in the formation of self-assembled globular molecules.20−22 By further increasing the temperature above 50 °C, there was a subtle increase in transmittance, suggesting precipitation followed by a return to a transparent solution. Although these behaviors are characteristic of water-soluble polymers during the coil−globule transition, they were exhibited by UBD even as supramolecules. LCST values showed a gradual decrease until 2 mM to reach saturation at 39 °C, demonstrating the concentration dependence of Eg6 (Figure S24c). In contrast, an increase in LCST was observed below 0.05 mM. Similar thermal behaviors were observed for 0.25 mM Eg3 and Eg4 at 35 and 37 °C, respectively (Figure S24a,b). The differences in LCST values among EgXs can be explained by differences in hydrophilicity, which increased as a function of OEG length in

Table 2. LCST Values and EGX Sizes in 0.25 mM Salinea size (nm) Eg3 Eg4 Eg6 a

D

LCST (°C)

below LCST

above LCST

35 (38) 37 (39) 40 (43)

8 (7) 8 (7) 7 (5)

740−2900 (620−740) 850−3300 (310−520) 290−4000 (∼1000)

Numbers in parentheses indicate values obtained in aqueous solution. DOI: 10.1021/acs.nanolett.6b05371 Nano Lett. XXXX, XXX, XXX−XXX

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would therefore not exhibit an EPR effect (Figure 1). On the other hand, when the tumor temperature is above LCST, EgX NPs were expected to form micrometer-sized globules that would accumulate in the void spaces of tumor tissue (Figure 6c) and would be detectable by fluorescence imaging. BALB/c nude mice bearing colon-26 tumors on the lower back were used to test our hypothesis. The rectal temperature of mice was maintained under the LCST of each EgX (Eg3, 4, and 6). EgXs (2 mM, 200 μL) were administered via the mouse coccygeal (tail) vein. The tumor tissue of the locally heated group was maintained at around 45 °C using flowing hot water for 1.5 to 4 h after EgX administration. After euthanization, tumor tissue and organs (liver, kidney, heart and lungs, spleen, and intestine) were extirpated and imaged with excitation and emission wavelengths of 455 and 500−720 nm, respectively (Figures 7 and S39−S42).

similar size changes in response to heat; NPs of identical size were detected below and above the LCST. The particle sizes below the LCST were consistent with the radius of Eg4 (2.0 nm) obtained by MMFFaq calculation (Figure 3b). Particle sizes were smaller in aqueous solution than in saline due to the inhibition of dehydration in the absence of metal ions (Table 2). The concentration dependence of DH values in saline is shown in Figures S26−S37. The morphology of self-assembled molecules was confirmed by transmission electron microscopy (TEM) (Figure 5c,d). Images were acquired in water and not saline to eliminate the presence of salts. Spherical NPs were mostly 5−20 nm in diameter, although a small fraction was >50 nm. The identical sizes observed by TEM as compared to those obtained by DLS may be due to formation of the micelles that have no void space into core. A transmission electron micrograph of Eg6 in 0.25 mM aqueous solution below LCST and a histogram of particle size distribution are shown in Figure 5c and d. Images of Eg3 and 4 are shown in Figure S38, and EgX sizes and LCST are summarized in Table 2. LCST values were plotted as a function of concentration along with morphological changes (Figure 6a). Below the CAC,

Figure 7. Fluorescence ex vivo imaging following EgX injection. (a−f) Eg3, 4, and 6 without (a−c) and with (d−f) heating of local tumor tissue. Enlarged images of tumors are shown in the center. Arrows indicate tumors (white solid lines), liver (yellow solid lines), kidney (red solid lines), heart and lungs (sky blue solid lines), spleen (orange solid lines), and intestines (red dotted lines).

Upon local heating, all of the EgXs exhibited fluorescence in tumor tissue, with Eg3 and 4 showing a higher signal in the area surrounding the tumor. This suggested that EgXs selfassembled as a result of local heating and were retained in the tissue. In contrast, without heating there was almost no fluorescence signal enhancement in the tumor for Eg6, although weak signals were observed for Eg3 and 4. It is possible that Eg3 passively accumulated in the tumor as a result of a stronger hydrophobic effect caused by the shorter Eg chain. Thus, local heating enhanced the accumulation of EgX in the tumor and surrounding tissue. Conclusions. In conclusion, we prepared thermally responsive aminoquinoline derivatives with LCSTs that were dependent on the length of the OEG chain. That is, responsiveness to temperatures close to body temperature was achieved by varying the OEG length. EgXs formed NPs ∼10 nm in size and microparticles of ∼1000 nm in size below and above the LCST, respectively, in response to heating. In a mouse tumor model, local heat application enhanced the accumulation of injected EgX in tumor tissues, which can be explained as a thermally triggered EPR effect (Figure 1). Future studies will focus on further developing this system to incorporate emissive molecules with a longer wavelength that can pass through biological membranes, as well as magnetic moleculs carrying radical and Gd complexes that can be used as MRI contrast agents.10

Figure 6. (a) Relationship between the LCST, concentration, and Eg6 size along with morphological changes. Colored bars indicate the body temperature of mice (35 °C ± 2 °C). (b, c) Illustration of plausible behaviors of EgXs forming NPs around 10 nm in size (b) or microparticles around 1000 nm in size (c) in tumor tissue.

EgX monomers were in equilibrium between bent and straight forms, whereas above the CAC, EgXs formed micelle-like NPs with a bent form and a size of ∼10 nm. Moreover, above the LCST, the driving force of NP self-assembly was OEG dehydration, which yielded globular micrometer-sized particles. We speculated based on the abruptness and sensitivity of this thermally responsive size change that the particles would accumulate in tumor tissues. When tumor tissue temperature is below LCST (i.e., the same as normal tissue; Figure 6b), EgXs would pass through the tissue owing to their small size and E

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Nano Letters Experimental Section. Materials. Unless otherwise stated, solvents and reagents were used without additional purification. Details on the preparation of TFMAQ−EgX−EgBr (X = 3, 4, and 6) and UBD−OH are provided in the Supporting Information. For the synthesis of 1,1′-(3-(10-(2,4-trifluoromethyl-7-aminoquinoline-N-diyl)-1,4,7,13,16,19-hexaoxaicosane-1-yl)benzene-1,3-triyl)bis(3-(2,5,8,11,14,17,20heptaoxahexacosan-26-yl)urea) (Eg3), a mixed solution of UBD−OH (788 mg, 814 μmol), TFMAQ−Eg3−EgBr (506 mg, 814 μmol), and potassium carbonate (563 mg, 4.07 mmol) in 5 mL acetonitrile was refluxed for 47 h. After salt removal, the filtrate was evaporated and separated three times by chromatography on a silica gel using CH2Cl2/MeOH (100:1− 20:1) as an eluent to obtain Eg3 (385 mg, 256 μmol), which was a fluorescent yellowish oil with a yield of 31%. Peaks in the infrared (IR) spectrum (neat on NaCl) were observed at 3368, 2926, 2868, 1692, 1619, 1555, 1349, 1277, and 1134 cm−1. The 1 H NMR (CDCl3, 500 MHz) peaks were as follows: δ 7.97 (dd, J = 9.6, 1.9 Hz, 1H), 7.61 (s, 1H), 7.44 (dd, J = 9.6, 2.7 Hz, 1H), 7.29 (s, 2H), 7.25 (d, J = 2.7 Hz, 1H), 6.91 (d, J = 1.7 Hz, 2H), 6.74 (s, 1H), 5.43 (t, J = 5.5 Hz, 2H), 4.12−4.10 (m, 2H), 3.80−3.78 (m, 2H), 3.74−3.71 (m, 6H), 3.68−3.60 (m, 52H) 3.57−3.52 (m, 10H), 3.45 (t, 6.4 Hz, 4H), 3.36 (s, 6H), 3.35 (s, 3H), 3.20 (q, J = 6.7 Hz, 4H), 1.60−1.54 (m, 4H), 1.53−1.48 (m,4H), and 1.37−1.35 (m, 8H). The 13C NMR (CDCl3, 126 MHz) peaks were as follows: δ 159.9, 156.0, 150.5, 1501, 147.6 (q, J = 35.0 Hz), 141.2, 135.6 (q, J = 32.2 Hz), 124.6, 123.2 (q, J = 275.3 Hz), 121.3 (q, J = 275.7 Hz), 120.0, 116.0, 109.0, 106.4, 101.9, 99.5, 71.9, 71.2, 70.8, 70.7, 70.7, 70.6, 70.6, 70.5, 70.5, 70.0, 69.8, 68.5, 68.4, 67.3, 60.0, 58.9, 51.0, 50.1, 39.8, 29.9, 29.3, 26.5, and 25.7. The calculated and actual m/z values for C70H116F6N6Na2O22 [M + 2Na]2+ determined by highresolution mass spectrometry (HRMS) electrospray ionization (ESI) were 776.3916 and 776.3891, respectively. The synthesis of 1,1′-(3-(10-(2,4-trifluoromethyl-7-aminoquinoline-N-diyl)-1,4,7,13,16,19,22-octaoxatriicosane-1-yl)benzene-1,3-triyl)bis(3-(2,5,8,11,14,17,20-heptaoxahexacosan26-yl)urea) (Eg4) was carried out in a manner similar to Eg3 but using TFMAQ−Eg4−EgBr. The reaction yield was 37%. Peaks in the IR spectrum (neat on NaCl) were observed at 3360, 2867, 1693, 1619, 1552, 1462, 1349, 1278, and 1134 cm−1. The 1H NMR (CDCl3, 500 MHz) peaks were as follows: δ 7.97 (dd, J = 9.5 and 1.8 Hz, 1H), 7.61 (s, 1H), 7.44 (dd, J = 9.5, 2.5, 1H), 7.27 (s,2H), 7.25 (d, J = 2.7 Hz, 1H), 6.90 (d, J = 1.8 Hz, 2H), 6.77 (s, 1H), 5.41 (t, J = 5.5 Hz, 2H), 4.12−4.10 (m, 2H), 3.80−3.78 (m, 2H), 3.73−3.70 (m, 6H), 3.68−3.59 (m, 54H) 3.57−3.52 (m, 12H), 3.45 (t, J = 6.4 Hz, 4H), 3.36 (s, 6H), 3.35 (s, 3H), 3.21 (q, J = 6.7 Hz, 4H), 1.60−1.55 (m, 4H), 1.54−1.49 (m, 4H), and 1.37−1.36 (m, 8H). The 13C NMR (CDCl3, 126 MHz) peaks were as follows: δ 159.9, 156.0, 150.5, 150.1, 147.6 (q, J = 35.2 Hz), 141.2, 135.6 (q, J = 32.1 Hz), 124.6, 123.1 (q, J = 275.4 Hz), 121.3 (q, J = 275.7 Hz), 120.0, 116.0, 109.0, 106.4, 101.8, 99.5, 71.9, 71.8, 71.2, 70.8, 70.7, 70.6, 70.6, 70.6, 70.5, 70.5, 70.0, 69.8, 68.4, 68.4, 67.4, 59.0, 58.9, 51.1, 51.0, 39.8, 29.9, 29.3, 26.5, and 25.8. The calculated and actual m/z values for C72H120F6N6Na2O23 [M + 2Na]2+ determined by HRMS (ESI) were 798.4047 and 798.4035, respectively. The synthesis of 1,1′-(3-(10-(2,4-trifluoromethyl-7-aminoquinoline-N-diyl)-1,4,7,13,16,19,22,25,28-nonaoxanonaicosane1-yl)benzene-1,3-triyl)bis(3-(2,5,8,11,14,17,20-heptaoxahexacosan-26-yl)urea) (Eg6) was carried out in a manner similar to Eg3 but using TFMAQ−Eg6−EgBr. The reaction yield was

63%. Peaks in the IR spectrum (neat on NaCl) were observed as 3368, 2927, 2868, 1694, 1617, 1559, 1464, 1349, 1278, and 1134 cm−1. The 1H NMR (CDCl3, 500 MHz) peaks were as follows: δ 7.97 (dd, J = 9.7, 1.9 Hz, 1H), 7.61 (s, 1H), 7.44 (dd, J = 9.6, 2.8 Hz, 1H), 7.27 (s,1H), 7.24 (d, J = 2.7 Hz, 1H), 6.92 (d, J = 1.6 Hz, 2H), 6.75 (s, 1H), 5.39 (t, J = 5.3 Hz, 2H), 4.13−4.11 (m, 2H), 3.80−3.79 (m, 2H), 3.72−3.71 (m, 6H), 3.68−3.59 (m, 62H) 3.58−3.52 (m, 12H), 3.45 (t, J = 6.4 Hz, 4H), 3.36 (s, 6H), 3.35 (s, 3H), 3.21 (q, J = 6.7 Hz, 4H), 3.04 (m, 4H), 1.62−1.56 (m, 4H), 1.55−1.50 (m, 4H), and 1.37− 1.36 (m, 8H). The 13C NMR (CDCl3, 126 MHz) peaks were as follows: δ 159.6, 155.9, 150.5, 150.1, 147.6 (q, J = 34.9 Hz), 141.2, 135.6 (q, J = 32.4 Hz), 124.6, 123.2 (q, J = 275.5 Hz), 121.3 (q, J = 275.6 Hz), 120.0, 116.0, 108.9, 106.4, 101.8, 99.5, 71.9, 71.2, 70.8, 70.7, 70.7 70.6, 70.6, 70.5, 70.5, 70.0, 69.8, 68.4, 68.4, 67.4, 59.0, 51.0, 39.8, 30.0, 29.3, 26.5, and 25.8. The calculated and actual m/z values for C76H128F6N6Na3O25 [M + 3Na]3+ determined by HRMS (ESI) were 569.2837 and 569.2827, respectively. LCST Behavior. Transmittance at 800 nm was monitored using normal saline and water samples containing 0.05−2 mM EgX, which included concentrations below and above the CAC determined by 1H NMR spectroscopy. The temperature of the sample in a cuvette was set at 25 °C and was increased by increments of 1 °C up to 70 °C. Each measurement was obtained after maintaining the temperature for 120 s. Temperature Control in Mice with and without Local Heating around the Tumor Tissue. After anesthetization, rectal temperatures of individual mice were maintained at 33 °C for Eg3 and at 35 °C for Eg4 and 6which were below the corresponding LCST valuesusing a heating lamp. For local heating around the tumor tissue, the temperature of the surrounding tissue was maintained above 40 °C using a heating pad with circulating hot water (i.e., tumors contacting the pad were maintained at 45 °C). A schematic illustration of the procedure used for temperature control is shown in Figure S38.

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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.nanolett.6b05371. Experimental details, copies of 1H and 13C NMR spectra for all new materials, concentration dependence of 1H NMR spectra; LCST behavior and DLS measurements of EgXs, transmission electron micrographs of Eg3 and 4 schematic illustration of temperature control for mouse bioimaging, and fluorescence imaging using EgXs (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail address: [email protected] (S.K.). *E-mail address: [email protected] (I.A. for in vivo study). ORCID

Satoru Karasawa: 0000-0002-3107-442X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Noboru Koga for many helpful discussions and Ms. Sayaka Shibata for assistance with animal F

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Nano Letters experiments. This work was partially supported by the PRESTO Program on Molecular Technology from the Japan Science Technology Agency (JST). Optical imaging and in vivo studies were also financially supported by the Center of Innovation Program (COI) stream (Vision 1, 3) of the JST and by a Jisedai Grant/Innovative Cancer Grants (No. 16771085) from the Japan Agency for Medical Research and Development (AMED).

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