Near-Infrared Dye-Conjugated Amphiphilic Hyaluronic Acid

Nov 17, 2014 - Yujie SuYuan LiuXiangting XuJianping ZhouLin XuXiaole XuDun .... Anton Liopo , Richard Su , Dmitri A. Tsyboulski , Alexander A. Oraevsk...
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Near-Infrared Dye-Conjugated Amphiphilic Hyaluronic Acid Derivatives as a Dual Contrast Agent for In Vivo Optical and Photoacoustic Tumor Imaging Koji Miki,*,† Tatsuhiro Inoue,† Yasuhito Kobayashi,† Katsuya Nakano,† Hideki Matsuoka,‡ Fumio Yamauchi,§ Tetsuya Yano,§ and Kouichi Ohe*,† †

Department of Energy and Hydrocarbon Chemistry and ‡Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan § Corporate R&D Headquarters, Canon, Inc., 30-2, Shimomaruko 3-chome, Ohta-ku, Tokyo 146-8501, Japan S Supporting Information *

ABSTRACT: Amphiphilic hyaluronic acid (HA) derivatives bearing hydrophobic indocyanine green dye derivatives and hydrophilic poly(ethylene glycol) were synthesized through the use of condensation and copper-catalyzed click cyclization reactions. The amphiphilic HA derivatives dissolved in water and formed self-assemblies in which the near-infrared dyes were tightly packed and arranged to form dimers or H-aggregates. By irradiating an aqueous solution of HA derivatives with nearinfrared light, photoacoustic signals were detected along with fluorescence emission. Self-assemblies consisting of HA derivatives could smoothly accumulate in tumor tissues by passive tumor targeting. By utilizing HA derivatives as a contrast agent, tumor sites were clearly visualized by optical imaging as well as by photoacoustic tomography.

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INTRODUCTION Photoacoustic tomography (PAT) is a powerful and noninvasive technique with which to visualize organs as well as diseased sites such as tumor tissues, and the approach has recently been investigated with a view to its clinical application.1−3 The ultrasonic wave generated from a PAT probe molecule upon photoirradiation is a more cell-permeable signal compared with fluorescence; therefore, PAT can be used to obtain images of structures deeper beneath the surface of the subject (several centimeters depth) than can be recorded by using optical imaging techniques. Furthermore, the resolution of PAT is scalable with the ultrasonic frequency (20−200 μm range), which is much better than that of optical imaging (1 to 2 mm range). However, PAT has a relatively low sensitivity, and the accumulation of large numbers of probe molecules in the target is usually necessary to detect photoacoustic (PA) signals with adequate intensity. In this context, the efficient accumulation of PAT probe molecules, such as π-conjugated carbon materials,4−6 metal nanoparticles,7−9 and near-infrared dyes and derivatives,10−15 in the target region has been investigated, especially in the field of tumor imaging. Although a PAT agent that consists of tumor-targeting oligopeptides linked to a near-infrared dye has recently been reported,10 an efficient method with which to accumulate large numbers of © 2014 American Chemical Society

PAT probe molecules in a specific site is still required for practical clinical application. During the past two decades, polymeric self-assemblies and their related aggregates have been investigated intensively, and they have been used as tumor-imaging agents or carriers of antitumor drugs.16−21 Such nanoparticles can passively and specifically accumulate in tumor tissues through the phenomenon known as the enhanced permeability and retention (EPR) effect.22 Considering the challenges facing PAT with respect to accumulation of probe molecules at the site of interest, we envisioned that a self-assembly containing large numbers of PAT probe molecules in a hydrophobic core would be a suitable contrast agent with which to visualize tumor sites by PAT. Recently, we reported the synthesis of polysaccharide analogues through ring-opening metathesis polymerization (ROMP) and dihydroxylation and demonstrated the use of block-type amphiphilic copolymers bearing near-infrared fluorescent indocyanine green (ICG) dye moieties in highcontrast optical tumor imaging in vivo.23−27 Most recently, we Received: September 29, 2014 Revised: November 11, 2014 Published: November 17, 2014 219

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Figure 1. (a) Amphiphilic Janus-type polysaccharide analogues for optical tumor imaging and (b) amphiphilic Janus-type HA derivatives for optical tumor imaging and PA tumor tomography. Wako Pure Chemicals (Japan) and used without further purification. 1-Hydroxybenzotriazole (HOBt) and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) were purchased from Watanabe Chemical Industry (Japan). DMSO and DMF were distilled over CaH2 before use. Dialysis membrane, Spectra/Por 6 (molecular weight cutoff (MWCO): 1000, 3500, and 25000), was purchased from Spectrum Laboratories (Rancho Dominguez, CA). 3-Azido-1-propylamine (APA) was prepared from 3-chloro-1-propylamine hydrochloride according to the reported method.43 Poly(ethylene glycol) B bearing a propargyl group was prepared according to our previous report.26 The synthetic procedure of indocyanine green derivative A bearing a propargyl group is shown in the SI. Transmission electron microscopy (TEM, JEM-1400, JEOL, Tokyo, Japan) was used to visualize the morphology of dried self-assemblies. Samples were dropped onto a TEM copper grid covered with a carbon film (200 mesh, Nisshin EM, Japan) and dried for 3 h. The distribution of diameters was determined by using TEM images of at least one hundred of self-assemblies. Dynamic light scattering (DLS) was measured on FPAR-1000 (Otsuka Electronics, Japan) at 20 °C. The sample solutions were diluted in H2O and filtered prior to analysis (syringe filter, 0.45 μm pore size). The concentrations of the analyzed sample solutions were 0.05 g/L. The measurements were performed at scattering angles of 90° at 25 °C. The critical aggregation concentration (cac) values of the dyecontaining polymers were determined using static light scattering (SLS, SLS-6000, Otsuka Electronics, Japan). The measurement was conducted according to the reported method.44 The results are summarized in the SI. Synthesis of 2. HA derivative 2 was prepared by condensation reaction (Scheme 1). HA derivative in which 10% of carboxylates are grafted with APA is abbreviated as 2′. To a solution of 1a (0.25 g, 0.40 mmol of the repeating unit) in anhydrous DMSO (4.0 mL) were added PyBOP (1.25 g, 2.4 mmol), HOBt (0.32 g, 2.4 mmol), and APA (0.32 g, 3.2 mmol) at room temperature. After stirring at room temperature for 20 h, the mixture was diluted with H2O (10 mL). The aqueous solution was washed with CH2Cl2 (10 mL × 2) and dialyzed against H2O for 1 day by using Spectra/Por 6 (MWCO = 1000). HA derivative 2a (81 mg, 0.18 mmol, 44%) was obtained as a white solid after lyophilization. 1H NMR (400 MHz, D2O, 25 °C) δ = 1.67 (s, 2H), 1.87 (s, 3H), 3.05−3.80 (m, 14H), 4.34−4.40 (m, 2H). Tetrabutylammonium salts of HA 2b and 2c were similarly synthesized. HA derivative 2a′ and 2b′ were synthesized by using

reported that Janus-type amphiphilic polysaccharide analogues conjugated with ICG dye moieties could be used for the ultrarapid visualization of tumor tissues by optical imaging in vivo (Figure 1a).28 During the course of our study, we found that by replacing the hydrophobic alkyl chains in Janus-type amphiphilic polymers with hydrophobic ICG dye derivatives, self-assemblies with an ICG dye-condensed hydrophobic core were formed (Figure 1b). With a view to clinical application, the naturally occurring polysaccharide hyaluronic acid (HA) was chosen as the main chain polymer of the amphiphilic Janustype polymers. There are a few examples of HA derivatives conjugated with near-infrared fluorescent dyes for use as tumorspecific contrast agents and carriers. Recently, Kwon, Kim, Park, and coworkers reported the synthesis of cholanic-acidand cyanine-dye-grafted HA derivatives and demonstrated the application of such compounds as tumor-targeting nanocarriers.29−38 Chung and coworkers reported optical imaging of tumor and lymph nodes by using nanogels consisting of ICG-conjugated HA derivatives.39 In these studies, cyanine dyes were used as a fluorescent molecule. To our knowledge, there are few examples of the use of tumor-targeting HA derivatives conjugated with large numbers of ICG dye moieties as a PAT contrast agent. During the preparation of this manuscript, the application of polymeric micelles including cyanine dyes to PA imaging was reported.40 Here we report on amphiphilic Janus-type HA derivatives bearing both hydrophobic ICG dye moieties and poly(ethylene glycol) (PEG) moieties, and we demonstrate the application of the selfassemblies in optical tumor imaging as well as in PA tumor tomography in vivo.

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MATERIALS AND METHODS

Materials. Sodium salts of HA with various molecular weights, 5, 8, and 25 K, were prepared from commercially available sodium salt of HA. Tetrabutylammonium salts of HA 1 were synthesized from sodium salts of HA by using cation exchange resin.41,42 Experimental details to prepare 1 are summarized in the Supporting Information (SI). 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF) were purchased from nacalai tesque (Japan). Copper(I) iodide (CuI) was purchased from 220

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against H2O for 1 day by using Spectra/Por 6 (MWCO = 3500). ICGcontaining HA derivative 3a (Mn = 5.9K) (20 mg, 3.4 μmol, 86%) was obtained as a greenish white solid after filtration by syringe filter (0.45 μm pore size, PVDF filter membrane, Whatman, Florham Park, NJ) and lyophilization. 1H NMR (400 MHz, D2O, 25 °C) δ = 1.60−1.70 (m, 0.23H), 1.89 (br s, 3H), 3.20−3.85 (m, 11H), 4.30−4.50 (m, 2H). The grafting efficiency of ICG moieties into 3 was determined by comparing UV−vis absorbance of 3 with that of A as a standard compound at 790 nm in DMF. Synthesis of HA Derivatives 4. ICG- and PEG-grafted HA derivatives 4 were synthesized by click reaction (Table 1). To a solution of 2b (Mn = 9.2K) (23 mg, 2.5 μmol of polymer involving 50 μmol of the repeating unit) in dry DMF (2.0 mL) were added A (8.8 mg, 13 μmol), B (0.18 g, 88 μmol), CuI (2.9 mg, 15 μmol), and DBU (2.3 mg, 15 μmol) at room temperature. After stirring at 60 °C overnight, the reaction mixture was diluted with H2O (2.0 mL). The aqueous solution was dialyzed against H2O for 1 day by using Spectra/ Por 6 (MWCO = 25 000). ICG-containing HA derivative 4d (Mn 45K) (65 mg, 1.5 μmol, 60%) was obtained as a dark green solid after filtration by syringe filter (0.45 μm pore size) and lyophilization. 1H NMR (400 MHz, D2O, 25 °C) δ = 1.80−2.15 (m, 3H), 3.10−3.90 (m, 183H), 4.30−4.50 (m, 2H), 7.93 (br s, 1H). The 1H NMR spectrum shows that azido groups were completely converted to triazole. The grafting efficiency of ICG moieties into 4 was determined by comparing UV−vis absorbance of 4 with that of A as a standard compound at 790 nm. Preparation of Self-Assemblies. Amphiphilic HA derivative 3 or 4 was dissolved in H2O (5 mg/mL) and stored at room temperature for 30 min in dark. The resulting solution was filtered by syringe filter (0.45 μm pore size). Measurement of Fluorescence Intensity of HA Derivatives. Solutions of HA derivatives ([ICG] = 1.0 × 10−4 to 1.0 × 10−5 M, 5 μL) in H2O were diluted with 45 μL of DMF/H2O (including 0.5 wt % Triton X-100, v/v 1:1) or phosphate-buffered saline (PBS, pH 7.4). Fluorescence intensities of HA derivatives were measured by an IVIS Imaging System 200 Series (Xenogen, excitation wavelength: λexc = 745 nm, detected wavelength: λem = 820 nm). Images were analyzed using Living Image 2.50-Igor Pro 4.09 software (Xenogen) to quantify the fluorescence intensity, according to the manufacturer’s instructions. Measurement of PA Signal. By irradiating an aqueous solution of HA derivatives with pulse laser light, the ultrasonic wave generated from the sample was detected with a piezoelectric element. The intensity of PA signal was determined by amplifying the detected signal with a high-speed preamplifier, followed by acquiring the amplified signal with a digital oscilloscope. Titanium sapphire laser (Lotis TII, Belarus) was used as a light source. Laser wavelengths were set to 720 and 790 nm for samples and 710 and 780 nm for the aqueous solution of commercially available indocyanine green dye for comparison. The conditions of laser irradiation are as follows: energy density, 12 mJ/cm2; pulse width, 20 ns; pulse repetition, 10 Hz. An ultrasonic transducer (Panametrics-NDT V303, Olympus, Japan) was used. The conditions of transducer are as follows: central band, 1 MHz; element size, ϕ0.5; measurement distance, 25 mm (nonfocus); amplification, +30 dB (Ultrasonic Preamplifier Model 5682, Olympus, Japan); optical path length, 0.1 cm. PA signals of aqueous solution (200 μL) ([ICG] = 1.0 × 10−4 to 1.0 × 10−5 M) in a polystyrene cuvette were measured by digital phosphor oscilloscopes (DPO4104, Tektronix, USA). The measurement conditions are as follows: trigger, detection of PA signal with a photodiode; data acquisition, 128 times (128 pulses). The PA signal (V) was given by calculating the difference between the values of maximum and minimum intensities of signals. The normalized PA signal (V/J·abs) was given by dividing PA signal (V) by absorbance of samples and the laser power (J) used. Optical Imaging Experiments in Vivo. All animal experiments were approved by the Animal Research Committee of Kyoto University and carried out in accordance with its guidelines. For the current study, the suspension of colon 26 mouse colon cancer cells (1 × 106, RIKEN) was subcutaneously inoculated into the femurs of 6-

Scheme 1. Synthesis of HA Derivatives Having Azido Groups

small amounts of condensation reagents and APA. To a solution of 1a (0.12 g, 0.20 mmol of the repeating unit) in anhydrous DMSO (2.0 mL) were added PyBOP (26 mg, 0.050 mmol), HOBt (6.8 mg, 0.050 mmol), and APA (7.6 mg, 0.075 mmol) at room temperature. After stirring at room temperature for 20 h, the mixture was diluted with H2O (5 mL). The aqueous solution was washed with CH2Cl2 (5 mL × 2) and dialyzed against H2O for 1 day by using Spectra/Por 6 (MWCO = 1000). Tetrabutylammonium salt of HA 2a′ (61 mg, 0.15 mmol, 74%) was obtained as a white solid after lyophilization. The grafting efficiency of APA was determined by 1H NMR. (See the SI.) Synthesis of HA Derivatives 3. ICG-grafted HA derivatives 3 were synthesized by click reaction (Scheme 2). To a solution of 2a′ (Mn = 5.1K) (20 mg, 3.9 μmol of polymer involving 5.0 μmol of azido groups) in dry DMF (2.0 mL) were added A (7.7 mg, 11 μmol), CuI (1.0 mg, 5.0 μmol), and DBU (0.8 mg, 5.0 μmol) at room temperature. After stirring at 60 °C overnight, the reaction mixture was diluted with H2O (2.0 mL). The aqueous solution was dialyzed

Scheme 2. Synthesis of HA Derivatives 3 Having ICG Moieties

221

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Table 1. Synthesis and Properties of HA derivatives 4 bearing ICG and PEG moieties

4

2

c/d

yield (%)a

s/tb

4a 4b 4c 4d 4e 4f 4g

2a 2a 2a 2b 2b 2b 2c

0.00/2.00 0.25/1.75 0.50/1.50 0.25/1.75 1.00/1.00 1.20/0.80 0.75/1.25

93 68 81 60 81 79 34

0:100 12:88 34:66 15:85 66:34 78:22 16:84

a

DDLS (nm)c

cac (g/L)d e

275 209 157 160

27 ± 8

± ± ± ±

e

73 56 38 76

2.3 3.0 7.9 9.2

e

10−4 10−4 10−4 10−4

e

1.8 × 10−3

111 ± 45 b

× × × ×

1

Calculated from a molecular weight based on the grafting efficiencies of ICG and PEG moieties. Determined by H NMR and UV−vis spectroscopy. cMeasured by dynamic light scattering. dMeasured by static light scattering. eNot determined.

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week-old nude mice (BALB/c Slc-nu/nu mice, Japan SLC). Solutions of HA derivatives in PBS were prepared, filtered (0.45 μm pore size), and kept in dark at room temperature before injection. Each solution of HA derivatives (100 μL in PBS, [ICG] = 5.0 × 10−4 M) was intravenously injected in each experiment. Optical imaging experiments to detect the distribution of the fluorescensce probes in tumorbearing mice were carried out with an IVIS Imaging System 200 Series (λexc = 745 nm, λem = 820 nm). In all imaging experiments, the contrast agent in the same batch was injected to at least three mice to confirm reproducibility, and representative images are shown. To emphasize the areas where imaging probes specifically accumulated, we set a threshold and represent intense fluorescence above it. Blood Concentration of Self-Assemblies [%ID(blood)]. Solutions of HA derivatives in H2O were injected to the tail vein of a female outbred BALB/c Slc-nu/nu mouse (7- to 9-week old, Japan SLC). Blood (2 μL) was harvested from the tail vein of the mouse after 24 h from the injection and was mixed with 1% Triton X-100 aqueous solution (9 μL) and DMF (9 μL). By measuring the fluorescence intensity of the mixture with an IVIS Imaging System 200 Series (λexc = 745 nm, λem = 820 nm), we determined a series of % ID(blood) with respect to an injected dose. Accumulated Ratio of HA Derivatives in a Tumor Site [%ID/ g(tumor)]. After in vivo tumor imaging experiments, mice were euthanized under CO2, followed by the surgical resection of tumor tissues. DMF (20.25 times as large as the weight of tumor tissues) was added to a mixture of tumor tissues in 1% Triton X-100 aqueous solution (1.25 times as large as the weight of tumor tissues). By measuring the fluorescence intensity of the mixture with an IVIS Imaging System 200 Series (λexc = 745 nm, λem = 820 nm), a series of %ID(tumor) with respect to an injected dose was determined. PA Imaging and Tomography in Vivo. The preparation of tumor-bearing mice was same as that shown in the Accumulated Ratio of HA Derivatives in a Tumor Site [%ID/g(tumor)] section. HA derivatives (100 μL in PBS, [ICG] = 5.0 × 10−4 M) were intravenously injected into the tail vein of tumor-bearing mice. PA signals from the tumor site were measured before injection of HA derivatives and after 24 h from injection with Nexus 128 (Endra, wavelength of laser: 790 nm). From the data of PA signals in a region of interest (ROI, 2 cm × 2 cm × 2 cm), 3D reconstruction data including tomographic images were acquired by utilizing a software (GEHC MicroView, GE Healthcare).

RESULTS AND DISCUSSION Synthesis of Amphiphilic HA Derivatives Bearing ICG Moieties. At first, the condensation reaction of tetrabutylammonium salt of HA 1a, prepared from sodium salt of HA having molecular weight of 5K, with 3-azido-1-propylamine (APA) was carried out (Scheme 1). Among the condensation reagents examined, we found that the combination of PyBOP and HOBt was the most effective, affording 2a with 100% APAgrafting efficiency.45 By changing the amounts of PyBOP, HOBt, and APA used, 2a′ with 10% APA-grafting efficiency was obtained in good yield. The use of 1b and 1c, prepared from sodium salts of HA having molecular weights of 8K and 25K, afforded the HA derivatives 2b (l/m 100:0, from 1b), 2b′ (l/m 10:90, from 1b), and 2c (l/m 100:0, from 1c) in moderate to high yields in a similar manner. (See the SI.) We next examined the conjugation of ICG moieties with the azido groups of 2 under copper catalysis.46−48 When the [3 + 2] cyclization reaction of 2a′ and A was carried out with a catalytic amount of CuSO4 and sodium ascorbate in water, no reaction took place; this was probably because of the low solubility of A in water. Upon examining alternative reaction conditions, we found that the CuI-catalyzed cyclization reaction of 2a′ in DMF succeeded, affording the corresponding ICGconjugated HA derivative 3a (Scheme 2). The reaction of 2b′ proceeded in a similar fashion to afford 3b. However, both reactions were difficult to reproduce reliably. We were pleased to find that CuI-catalyzed cyclization reaction of A and B, both bearing a propargyl group, with 2 proceeded smoothly to afford amphiphilic HA derivatives 4 (Table 1). We confirmed that the click reaction proceeded to completion by measuring the integration of the proton signal arising from the triazole ring. The UV−vis spectra of HA derivatives in DMF were similar to the spectrum of A in DMF, and no signals arising from aggregated ICG moieties were observed. Accordingly, the grafting efficiency of ICG to PEG moieties was easily determined by comparing the absorbance (λmax = 790 nm) of the ICG moieties in the HA derivatives in DMF with that of A as a standard compound. The grafting efficiency could be controlled by changing the amounts of A and B used in the reaction. In most cases, A was more reactive 222

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HA derivatives. The PEG content of HA derivatives also affects the cac values. HA derivatives with higher PEG content formed more stable self-assemblies. This might be caused by the entanglement of PEG moieties within the hydrophilic shell. We previously reported that the cac values of self-assemblies consisting of amphiphilic Janus-type polysaccharide analogues were 0.22 to 0.73 mg/L (Figure 1a).28 On the basis of these values, we assumed that the disassembly of self-assemblies of HA derivatives would be suppressed during in vivo experiments. We also checked the stability of self-assemblies under physiological conditions and found that the mean diameters of aggregates of 4b and 4d did not change after several days in phosphate-buffered saline (PBS, pH 7.4) or after 18 h in fetal bovine serum (10% in PBS). (See the SI.) UV−vis Absorbance, Fluorescence, and PA Signals of Self-Assemblies of Amphiphilic HA Derivatives. Kamat and coworkers reported that the absorbance maxima of monomeric ICG molecules, H-aggregates of ICG molecules including dimers, and their J-aggregates were observed at approximately 780, 700, and 900 nm, respectively.50 In DMF/ H2O (1:1 v/v) solution, the absorbance maximum of HA derivative 4c was detected at 790 nm along with a shoulder around 720 nm (Figure 3). This indicates that almost no self-

than B, presumably because of the difference in their molecular sizes. PEG-grafted HA derivative 4a without ICG moieties was also synthesized from 2a and B according to a similar procedure, but we were unable to generate self-assemblies in water. Considering that 2a consists of approximately 12 repeating units, it is apparent that one or two azido units in HA derivative 4b conjugates with an ICG moiety and the rest conjugate with PEG moieties. HA derivatives 4d and 4g, with similar ICG content to 4b, could be prepared from 2b and 2c, respectively. HA derivatives 4c and 4e, with 1:2 and 2:1 ratios of ICG to PEG, were synthesized by the click reaction with a large amount of A; however, these were slightly insoluble in water. HA derivative 4f, in which 78% of the carboxylate units were conjugated with ICG moieties, was synthesized, but it was almost completely insoluble in water. Aqueous solutions of 4c and 4e were obtained by slowly adding solutions of the compounds dissolved in DMF to water (DMF was removed by dialysis); however, the similar treatment of 4f afforded an insoluble solid. Formation of Self-Assemblies of Amphiphilic HA Derivatives. Amphiphilic HA derivatives 3, 4b−e, and 4g dissolved in water and formed self-assemblies. The representative TEM images of dried spherical assemblies are shown in Figure 2. The diameters of the self-assemblies were 30−270

Figure 2. TEM images of self-assemblies of HA derivative (a) 4b and (b) 4d. Figure 3. UV−vis spectra of 4c in DMF/H2O (v/v 1:1, dashed line), 4c in H2O (solid line), and 4b in H2O (dashed-dotted line). Concentration: ca. 0.05 g/L. All spectra were normalized by the absorbance at 790 nm.

nm, as determined by DLS. In the case of 4a, no scattering intensity was detected by DLS because of the lack of hydrophobic groups. HA derivatives 3, for which only one or two carboxylate groups were grafted with ICG moieties, formed self-assemblies in water despite no PEG grafting. HA derivatives 4b, 4d, and 4g, containing small numbers of hydrophobic ICG moieties, as well as 3, may form nanogel-like aggregates in water. In contrast, HA derivatives 4c and 4e, containing more ICG moieties in one polymer chain, can potentially form micellar or related aggregates such as multimicellar aggregates or multilayer vesicles in water. The cac of these self-assemblies was determined to be 0.23 to 1.8 mg/L. By comparing the cac values of HA derivatives 4b, 4d, and 4g (having similar ICG content), we could establish that HA derivatives with lower molecular weights formed more stable self-assemblies under dilute conditions. This might be caused by the size of segments of the self-assemblies.49 In the case of amphiphilic HA derivatives with lower molecular weights, the self-assembly consists of many smaller segments. Therefore, the dissociation of several smaller segments from a self-assembly would not significantly destabilize the resulting self-assembly even under dilute conditions. In contrast, the dissociation of larger segments composed of HA derivatives with larger molecular weights leads to the formation of distorted assemblies, thus destabilizing the self-assembly of

assembled forms of HA derivative 4c with an ICG-condensed hydrophobic core were generated in the solution. In the absorbance spectra of HA derivative 4c in water, the absorbance maximum of dimers or H-aggregates of ICG moieties was observed at ∼720 nm, together with that of the monomeric ICG moiety. The absorption intensity at 720 nm of 4c with higher ICG content was stronger than that at 790 nm. This indicates that some of the ICG moieties in the selfassemblies interact tightly with each other to form aggregates in a hydrophobic core. In contrast with 4c, in the case of HA derivative 4b with lower ICG content, the absorption intensity at 720 nm was weaker than that detected at 790 nm. Although the structures of self-assemblies of 4b and 4c differ, as previously mentioned, this result implies that hydrophobic ICG moieties efficiently form aggregates in the hydrophobic core of self-assemblies of HA derivatives with a high ICG content. The fluorescence of a solution of HA derivatives 4b−e in PBS (pH 7.4) and in DMF/H2O (including 0.5 wt % Triton X100, 1:1 v/v) was measured with an optical imaging device (Table 2). Whereas HA derivatives 4b−e in DMF/H2O 223

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Table 2. Fluorescence Intensities and Relative Intensities of PA Signals of HA Derivatives HA derivative 4b 4c 4d 4e

FLDMF/H2O (cps·M−1) 1.2 5.4 2.3 1.9

× × × ×

1013 1012 1012 1012

FLPBS (cps·M−1)

FLDMF/H2O/FLPBS

× × × ×

8 19 40 50

1.5 2.9 5.8 1.6

1012 1011 1010 1010

Table 3. Relative Intensities of PA Signals of HA Derivatives Compared with Commercially Available ICG Dyea

a Sample solutions were prepared by diluting 5 μL of aqueous solution of HA derivatives ([ICG] = 1.0 × 10−4 to 1.0 × 10−5 M) with 45 μL of DMF/H2O (including 0.5 wt % Triton X-100, v/v 1:1) or PBS (pH 7.4). Fluorescence intensities of HA derivatives (FLDMF/H2O and FLPBS) at 820 nm were measured (excitation wavelength: 745 nm). FLDMF/H2O and FLPBS are normalized by the absorbance at 790 nm.

HA derivative

PA (V/J·abs) at 720 nm

PA/ PAICG

PA (V/J·abs) at 790 nm

PA′/ PA′ICG

4b 4c 4d 4e

284 265 269 251

1.27 1.18 1.20 1.12

383 309 380 391

1.25 1.02 1.25 1.29

a

Photoacoustic (PA and PA′) signals of HA derivatives in H2O ([ICG] = 1.0 × 10−4 to 1.0 × 10−5 M) were measured by irradiating a sample solution with pulse laser light at 720 and 790 nm, respectively. PA and PA′ were normalized by the power of pulse laser and the absorbance of HA derivatives. The PA signals of commercially available indocyanine green dye (PAICG and PA′ICG) were measured by irradiating a sample solution with pulse laser light at two absorbance maxima (710 and 780 nm), respectively. PAICG and PA′ICG normalized by the power of pulse laser and the absorbance of indocyanine green dye were 224 and 303 V/J·abs, respectively.

emitted strong fluorescence at 820 nm (λexc = 745 nm), weak fluorescence was detected from the HA derivatives in PBS. The latter result was probably because of aggregation-induced fluorescence quenching. Upon irradiating aqueous solutions of HA derivatives with pulsed laser light at 720 and 790 nm, ultrasonic waves from the HA derivatives were detected by using a piezoelectric element. Representative PA signals generated upon irradiation are shown in Figure 4. The relative intensities of PA signals of HA derivatives compared with those obtained by using commercially available ICG dye are summarized in Table 3. To demonstrate the conversion efficiency of absorbed photoenergy to PA signal clearly, we normalized PA signals to the laser power as well as to the absorbance of HA derivatives. Although there was no clear relationship between the PA signal intensities at 720 and 790 nm, the normalized PA signals of HA derivatives were somewhat stronger than that of commercially available ICG dye. Because commercially available ICG dye also forms aggregates in water,50 it is reasonable to assume that there is no significant difference between “normalized” PA signals of commercially available ICG dye and ICG derivatives in polymeric self-assemblies. Commercially available ICG dye itself does not accumulate in tumor tissues specifically,27 whereas self-assemblies with an ICG-condensed core can carry large numbers of hydrophobic ICG molecules to a tumor site through the EPR effect. In this context, we next examined the use of HA derivatives that were developed in this study, which generate PA signals as well as near-infrared fluorescence, in tumor imaging in vivo.

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IN VIVO TUMOR IMAGING To evaluate HA derivatives as tumor-imaging agents, we first carried out optical tumor imaging in vivo. The fluorescence images of mice 24 h after injection are shown in Figure 5a−c. HA derivatives 4c−e were efficiently accumulated in tumor tissues (colon 26 cells) that were subcutaneously inoculated into femurs. The signal-to-noise (S/N) ratios of HA derivatives 4c−e estimated by comparing the fluorescence intensity detected at a tumor site with those detected at a health site (a leg) were 2.2 ± 0.4, 2.1 ± 0.4, and 2.2 ± 0.4, respectively (Table 4). These results indicate that HA derivatives could be used to visualize the tumor site with high contrast in fluorescence tumor imaging. The accumulated ratios [%ID/g(tumor)] of HA derivatives in the tumor site with respect to the total injected dose (ID) were determined by measuring the amounts of HA derivatives extracted into DMF from the harvested tumor tissues. Among the HA derivatives 4c−e examined, 4d accumulated in the tumor site most efficiently. The blood concentration [% ID(blood)] of HA derivatives was determined by measuring the

Figure 4. PA signals of 4d measured by irradiating pulsed laser at (a) 720 and (b) 790 nm. 224

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differences between the values for each compound were similar. These results are consistent with the observed S/N ratios for optical tumor imaging. PA imaging of tumor-bearing mice was carried out before and after intravenous injection of HA derivatives (Figures 5d− i). By irradiation with pulsed laser light at 790 nm, the blood vessels around the tumor site were visualized because hemoglobin generates a PA signal (Figures 5d−f). Although background signals from hemoglobin were also detected, strong PA signals generated from the tumor site were detected (Figures 5g−i). It is noted that no PA signal enhancement was observed by using saline as a control contrast agent.(See the SI.) The PA signals (PA24) detected 24 h after injection of HA derivatives 4c−e were 3.8−7.4 times stronger than those detected before injection (PA0). There was no significant difference between PA24/PA0 ratios of HA derivatives tested. However, the tumor site was clearly visualized in all cases. By utilizing the same PA imaging device, PAT of subcutaneously inoculated tumor tissues could be obtained (Figure 6 and see

Figure 5. Near-infrared fluorescence images (cps) of tumor-bearing mice 24 h after injection of HA derivatives (a) 4c, (b) 4d, and (c) 4e (100 μL of a PBS solution, [ICG] = 5.0 × 10−4 M). PA images of tumor-bearing mice before injection: (d) for 4c, (e) for 4d, and (f) for 4e; 24 h after injection of (g) 4c, (h) 4d, and (i) 4e (100 μL, [ICG] = 5.0 × 10−4 M). Arrows point out tumor sites. Color code: low intensity black, high intensity yellow. Thresholds were appropriately established. Colon 26 mouse colon cancer cells (1 × 106, RIKEN) were subcutaneously inoculated into the femurs of nude mice 1 week before the imaging experiment.

Figure 6. Images of PAT (a) before and (b) 24 h after injection of 4c. PAT data (movies) are provided in the Supporting Information.

also the Supplementary Movies in the SI). The PA signals from the tumor site as well as from blood vessels were enhanced. Additionally, the PA signals around tumor tissues (background signals) were also enhanced because of the permeation of HA derivative 4c from the tumor vasculature by the EPR effect. On the basis of these results, the use of HA derivative 4c as a contrast agent was successfully applied in the tomographic visualization of a tumor site. In summary, we achieved the facile synthesis of amphiphilic HA derivatives bearing hydrophobic ICG dye derivatives through the use of condensation and cyclization reactions. Amphiphilic HA derivatives formed spherical self-assemblies with a dye-condensed hydrophobic core in water. Because ICG moieties formed dimers or H-aggregates in the dye-condensed hydrophobic core, enhanced PA signals as well as fluorescence were detected upon irradiating an aqueous solution of HA derivatives with near-infrared light. Furthermore, the HA derivatives, which could efficiently accumulate in tumor tissues by passive tumor targeting, could be applied as a dual contrast agent for high-contrast optical tumor imaging and PA tumor tomography. Considering the enhancement of PA signals as well as the efficient accumulation in tumor tissues, we believe that polymeric self-assemblies with a near-infrared dyecondensed hydrophobic core represent one of the most promising high-performance photoacoustic tumor-imaging agents available.

Table 4. Blood Concentration of Self-Assemblies and Their Amounts Accumulated in Tumor Tissues HA derivative

S/N in optical imaginga

4c 4d 4e

2.2 ± 0.4 2.1 ± 0.4 2.2 ± 0.4

%ID/g(tumor)b %ID(blood)c 6.9 ± 2.7 11.8 ± 2.7 3.0 ± 0.7

5.0 ± 2.0 15.0 ± 4.0 0.8 ± 0.8

PA24/PA0d 5.7 ± 1.9 3.8 ± 0.4 7.4 ± 3.2

a

Signal-to-noise ratio (S/N) was determined by comparing fluorescence intensity in tumor site with that at the femurs. b Accumulated ratio (%ID/g) of HA derivatives in a tumor site with respect to the total injected dose per 1 g of the tumor measured after 24 h. cValue corresponds to residual ratio (%ID) of HA derivatives in blood vessel with respect to the total injected dose measured after 24 h, which means blood circulation. dRatio of PA signal intensities around a tumor site detected before injection of HA derivatives (PA0) and those detected 24 h after injection (PA24).

fluorescence intensities of HA derivatives in blood harvested 24 h after injection. Among the HA derivatives 4c−e examined, 4d circulated in blood vessels for the longest time, probably because of the stealth effect of PEG brushes. These data indicate that HA derivatives that can remain in blood vessels for a longer time could accumulate through the EPR effect in tumor tissues more efficiently. Although the values of %ID/ g(tumor) and %ID(blood) determined for HA derivatives 4c−e differed significantly depending on the compound, the 225

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis of 2 and A, details of tumor imaging, PAT data, NMR spectra, and other characterization details. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +81 75 383 2497. *E-mail: [email protected]. Phone: +81 75 383 2495. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by Modality Xn program of Kyoto University/Canon joint research project−Innovative TechnoHub for Integrated Medical Bio-Imaging from MEXT Budget for the Promotion of Science and Technology, a Grant-in-Aid for Young Scientists (A), and a Grant-in-Aid for Scientific Research on Innovative Areas “New Polymeric Materials Based on Element-Blocks (No. 2401)” of The Ministry of Education, Culture, Sports, Science, and Technology, Japan. We acknowledge Professor Teruyuki Kondo and Dr. Yu Kimura in Kyoto University for TEM measurements.

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