Indocyanine Green-Labeled Polysarcosine for in Vivo Photoacoustic

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Indocyanine green-labeled polysarcosine for in vivo photoacoustic tumor imaging Kohei Sano, Manami Ohashi, Kengo Kanazaki, Akira Makino, Ning Ding, Jun Deguchi, Yuko Kanada, Masahiro Ono, and Hideo Saji Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00715 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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Bioconjugate Chemistry

Pre-injection

24 h post-injection high

tumor

ICG-labeled polysarcosine

blood vessel

ACS Paragon Plus Environment

low


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Bioconjugate Chem Indocyanine green-labeled polysarcosine for in vivo photoacoustic tumor imaging

The names, affiliations, and addresses of the authors Kohei Sanoa,b,¶, Manami Ohashia,¶, Kengo Kanazakia,c, Akira Makinoa,d, Ning Dinga, Jun Deguchia, Yuko Kanadaa, Masahiro Onoa, Hideo Sajia,*

a

Department of Patho-Functional Bioanalysis Graduate School of Pharmaceutical

Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto, Japan, 606-8501 b

Kyoto University Hospital, 54 Kawaharacho, Shogoin, Sakyo-ku, Kyoto, Japan,

606-8507 c

Medical Imaging Project, Corporate R&D Headquarters, Canon Inc., 3-30-2

Shimomaruko, 10 Ohta-ku, Tokyo, Japan, 146-8501 d

Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki,

Eiheiji-cho, Yoshida-gun, Fukui, Japan, 910-1193

* Corresponding author: Professor Hideo Saji, PhD

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Department of Patho-Functional Bioanalysis Graduate School of Pharmaceutical Sciences, Kyoto University; 46-29 Yoshida Shimoadachimachi, Sakyo-ku, Kyoto 606-8501, Japan Tel: +81-75-753-4556 Fax: +81-75-753-4568 E-mail: [email protected]

¶ These authors contributed equally to this work.

Table of Contents/Abstract Graphic

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Abstract Photoacoustic (PA) imaging has been considered as an attractive imaging modality for sensitive and in depth imaging of biomolecules with a high resolution in vivo. PA imaging probes utilizing fluorescence dyes, including indocyanine green (ICG), have been proposed to enhance PA signal intensity. On the other hand, nanomicelles modified with polysarcosine (PSar), a biocompatible hydrophilic polymer, on their surface have previously achieved rapid tumor uptake, suggesting active transport of PSar into tumor tissues. Thus, we hypothesized that PSar-based materials might be utilized as diagnostic probes for targeting tumors and therefore evaluated the potential of PSar labeled with an ICG derivative, ICG-PSar, as a PA imaging probe for targeting cancer. In this study, ICG-PSars with differing molecular weights (10, 20, and 30 kDa) were synthesized. In

vitro cellular uptake studies using ICG-PSar demonstrated rapid uptake in colon26 tumor cells partially via macropinocytosis-mediated endocytosis. In vivo fluorescence imaging and biodistribution study indicated that ICG-PSar30k exhibited high accumulation in the tumor (8.4% dose/g), with high tumor-to-blood ratios reaching 4.6 at 24 h post injection of the probe. Finally, in vivo PA imaging studies showed that PA signal increased in tumors (251%) but not in blood vessels, achieving high contrast tumor imaging at 24 h after ICG-PSar30k probe injection. These results suggest that

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ICG-PSar has potential as a tumor-targeting PA imaging probe.

Keywords: photoacoustic imaging, polysarcosine, indocyanine green, tumor imaging

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Introduction Photoacoustic (PA) imaging detects ultrasonic waves that are thermoelastically induced by optical absorbers irradiated with a pulsed laser.1, 2 Since ultrasonic waves have much lower tissue scattering leading to deeper penetration (multiple centimeters) and high spatial resolution (sub-millimeter), PA imaging has the potential for broader clinical translation compared to other optical imaging modalities. For PA imaging, a near-infrared (NIR) light ranging from 650-900 nm is used since this wavelength range can relatively penetrate through tissues, enabling visualization of deeper tissues from the surface.3 Metal nanoparticles (i.e., gold nanorods and iron oxide nanoparticles)4, 5 and fluorescence dyes6 have been proposed as exogenous PA signal emitters. Among these, indocyanine green (ICG), an FDA-approved NIR fluorescence dye, is expected to achieve sensitive PA signals as well as fluorescence signals,2 and protein-based probes (monoclonal antibody and human serum albumin) labeled with ICG have been developed for tumor imaging.7, 8 On the other hand, over the last decades, nanomedicines have shown appeal in preclinical and clinical studies. Some water-soluble polymers such as polyethylene glycol (PEG) have been clinically used to improve bioavailability and pharmacokinetics of the attached bioactive medicines. The most prominent effect is an increased half-life

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in circulation due to size-related evasion from hepatic uptake by reticuloendothelial system (RES) and slower renal clearance, which could result in passive accumulation in well-vascularized tumors via enhanced permeability and retention (EPR) effect.9 In a recent study, ICG-labeled PEG used as a tumor-targeted PA imaging probe demonstrated time-dependent accumulation in the tumor via the EPR effect.10 However, due to slow tumor uptake (highest uptake was achieved at 24 or 48 h post-injection) and prolonged half-life in circulation, the tumor-to-background ratio was not high enough to obtain high contrast images. On the other hand, polysarcosine (PSar) is categorized as a water-soluble polypeptide, and is expected to suppress capture by RES due to the formation of a hydration layer on its surface as well as PEG.11 The high biocompatibility, including low immunogenicity12 and low-nonspecific protein binding,13, 14 properties are well-characterized because PSar consists of N-methylated glycine, a natural amino acid. PSar

could

be

synthesized

by

ring-opening

polymerization

of

sarcosine

N-carboxyanhydride.11 Furthermore, nanomicelles modified with PSar on their surface have been previously reported to achieve rapid tumor uptake and high contrast tumor imaging.15 Therefore, these findings motivated us to investigate the possibility of using PSar itself as a diagnostic drug carrier for targeting tumors. Therefore, in this study, ICG-labeled PSar was synthesized and the possibility of using

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it as a tumor-targeted PA probe was evaluated by assessing in vitro cellular uptake, in

vivo biodistribution, and in vivo PA imaging, and its utility was compared against that of ICG-PEG.

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Results Synthesis ICG-PSar conjugates were synthesized according to Scheme 1. PSars with molecular weights of 8030, 17540, and 31500 were synthesized, and each PSar has hereinafter been referred to as PSar10k, PSar20k, or PSar30k according to molecular weight. The conjugation ratios of ICG to PSar were approximately equal to 1 for each ICG-PSar conjugate (1.0, 1.1, and 1.0 for ICG-PSar10, ICG-PSar20k, and ICG-PSar30k, respectively). As defined by Native-PAGE in order to determine the chemical purity of probes, the fractions of covalently bound ICG to PSar were more than 95% for ICG-PSars (Fig. 1).

PSar ICG

ICG-PSar

Scheme 1. Synthesis scheme of ICG-labeled polysarcosine (PSar).

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Figure 1. Fluorescence image of ICG-PSars after electrophoresis. Lane

1,

ICG-PSar10k;

2,

ICG-PSar20k; 3, ICG-PSar30k; 4, ICG-Sulfo-OSu.

Cellular uptake study The cellular uptake of ICG-PSar30k or ICG-PEG30k was evaluated by in vitro fluorescence imaging (Fig. 2). At 1 and 6 h after incubation, the fluorescence signals of ICG-PSar30k in colon26 cells were significantly higher than ICG-PEG30k (P < 0.01) (Fig. 2A). Fluorescence signals of ICG-PSar30k were significantly (94%) reduced by incubation at 4°C than that at 37°C (Fig. 2A). In order to determine the cellular uptake mechanism, inhibition experiments using various types of endocytosis inhibitors (Fig. 2B) was performed. The uptake of ICG-PSar30k was significantly (25%) inhibited with amiloride (macropinocytosis inhibitor), while ICG-PEG uptake did not change.

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A

Figure 2. Cellular uptake of ICG-PSar30k and ICG-PEG30k by

Relative cellular uptake

1.2 #

1

37˚C_1 h

colon26 tumor cells. (A) Cellular uptake at 1 and 6 h after incubation

37˚C_6 h

0.8

4˚C_1 h 0.6 0.4 0.2

4˚C_6 h

at 37°C or 4°C. Data are shown as the ratios to fluorescence signal intensities at 6 h after incubation

* ‡

0 ICG-PSar30k

B Relative cellular uptake

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.4

control (6 h) chlorpromazine genistein

ICG-PEG30k

with ICG-PSar30k. *P < 0.01 vs. ICG-PEG30k at 1 h, #P < 0.01 vs.

amiloride cytochalasin D

ICG-PEG30k at 6 h, ‡P < 0.01 vs. ICG-PSar30k at 6 h, (B) Evaluation of cellular uptake mechanisms

1.2 1 0.8

**

using endocytosis inhibitors. Data are shown as ratios to fluorescence signal intensities of the control. **P < 0.01 vs. control.

0.6 0.4 0.2 0 ICG-PSar30k

ICG-PEG30k

In vivo fluorescence imaging study To confirm the tumor accumulation of ICG-PSars, each preparation was intravenously injected into colon26 tumor-bearing mice, and whole-body fluorescence images were obtained at 1, 6, 24, and 48 h post-injection (Fig. 3A). Each ICG-PSar showed maximum fluorescent signals in the tumor at 6 h post-injection, and these signals slightly decreased with time, which was determined by analysis of fluorescent signal intensity (Fig. 3B). Higher fluorescent signal intensity was observed in tumor regions as the molecular weight of PSar was increased (Fig. 3A and 3B). In the ex vivo fluorescence imaging study, as the molecular weight of PSar increased, the tendency for

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ICG uptake in the liver was relatively increased coincidentally with a decreased uptake in the kidney (Fig. 4A-C). On the other hand, ICG-PEG30k was gradually accumulated in the tumor, and higher uptake level was exhibited, however, relatively high background signals were observed (Fig. 3 and Fig. 4D).

Figure 3. In vivo fluorescence imaging of colon26 tumor-bearing mice injected with ICG-PSar10k, ICG-PSar20k, ICG-PSar30k, and ICG-PEG30k. (A) In vivo fluorescence images of mice injected with ICG-labeled probes. (B) Fluorescence signal intensity in the tumor analyzed from fluorescence images in (A). Data are represented as means ± s.d. (n = 3). *P < 0.01 vs ICG-PSar10k, ICG-PSar20k, and ICG-PSar30k. #P < 0.05 vs ICG-PEG30k and ICG-PSar10k. **P < 0.01 vs ICG-PSar10k.

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Figure 4. Ex vivo fluorescence signal intensity of ICG-PSar10k (A), ICG-PSar20k (B), ICG-PSar30k (C), and ICG-PEG30k (D). At 1, 6, 24, and 48 h post-injection, whole-organ specimens were harvested and images using fluorescent imager. 12



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In vivo biodistribution study For quantitative evaluation of probe biodistribution, the accumulation of ICG-PSar and ICG-PEG30k in the tumor and blood was determined based on the fluorescence intensity of ICG extracted from the tissues (Fig. 5). Among the ICG-PSar series examined, ICG-PSar30k showed the highest accumulation in the tumor (8.4% dose/g at 24 h post-injection) (Fig. 5A), which was consistent with in vivo and ex vivo fluorescence imaging data (Fig. 3, 4). The retention of ICG-PSar in circulation tended to be prolonged as the molecular weight of PSar was increased (Fig. 5B). The tumor-to-blood (T/B) ratios of ICG-PSar increased with time, and at 24 h after injection, the T/B ratios were 14.1±4.0, 17.3±2.7, and 4.6±1.3 for ICG-PSar10k, ICG-PSar20k, and ICG-PSar30k, respectively (Fig. 5C). On the other hand, ICG-PEG30k was gradually taken up by the tumor and the tumor uptake was significantly higher than ICG-PSar series, however, the prolonged retention of ICG-PEG30k in the blood resulted in significantly lower T/B ratios (0.6±0.0 at 24 h post-injection) (Fig. 5C). Due to the highest tumor uptake and sufficient T/B ratios to achieve high contrast images, ICG-PSar30k was investigated for further in vivo imaging studies. As defined by SDS-PAGE (Fig. 6), there was no metabolite in the urine for up to 24 h after injection, indicating that the ICG-PSars were excreted without any metabolism.

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20

ICG-PSar20k

%dose/g tissue

16

B

*

ICG-PSar10k

‡

ICG-PSar10k #, †

‡

ICG-PSar30k ICG-PEG30k

12

60

%dose/g tissue

A

** 8

ICG-PSar20k

*


40 #

*

20

*

4 0

0 0

12 24 36 Time after Injection (h)

C 50 Tumor-to-blood ratios

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ICG-PSar10k 40


‡‡

0 0

††

*


48

§

20 10

0

¶

ICG-PSar20k

30

48

# #


48

Figure 5. Accumulation of ICG-PSar (10k, 20k, and 30k) and ICG-PEG30k in the tumor (A) and blood (B). The accumulation of ICG (%dose/g) was determined by measuring ICG content in tumor homogenate and blood sample by fluorescence imaging. (C) Time-dependent change of tumor-to-blood ratios. *P < 0.01 vs ICG-PSar10k, ICG-PSar20k, and ICG-PSar30k. #P < 0.01 vs ICG-PSar10k and ICG-PSar20k. †P < 0.05 vs ICG-PSar30k. **P < 0.05 vs ICG-PSar10k. ‡P < 0.01 vs ICG-PSar20k and ICG-PSar30k. ¶P < 0.01 vs ICG-PEG30k and ICG-PSar20k. §P < 0.05 vs ICG-PEG30k and ICG-PSar20k. ††P < 0.01 PSar20k vs ICG-PSar10k and ICG-PSar30k. ‡‡P < 0.05 ICG-PSar10k vs ICG-PSar30k.

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Figure 6. Fluorescence image of urine from mice injected with ICG-PSars after electrophoresis. Lane 1, ICG-PSar10k (in urine); 2, ICG-PSar10k (in PBS); 3, ICG-PSar20k (in urine); 4, ICG-PSar30k (in urine); 5, ICG-PSar20k (in PBS); 6, ICG-PSar30k (in PBS).

Protein binding assay To understand the rapid clearance of ICG-PSar from circulation compared with ICG-PEG, binding of ICG-PSar30k and ICG-PEG30k to serum albumin was investigated. Both PSar30k and PEG30k did not exhibit any interaction with BSA. Both ICG-PSar30k and ICG-PEG30k showed weak binding contrast (Kb (105 M-1): 0.40±0.15 and 0.44±0.25 for ICG-PSar30k and ICG-PEG30k, respectively), but, showed no differences in binding affinity.

In vivo PA imaging study PA images of colon26 tumor bearing mice that received intravenous injection of ICG-PSar30k and ICG-PEG30k (40 nmol ICG/PBS 150 µL) were acquired at 24 h post-injection (Fig. 7). In our previous study, fluorescence imaging has higher

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sensitivity as compared to PA imaging.7 Therefore, compared to fluorescence imaging, high-dose of ICG-PSar30k or ICG-PEG30k was injected into mice in order to complement detection sensitivity. Even if the injection dose of ICG-PSar to the mouse was increased, tumor uptake did not change (10.6 %ID/g and 11.2%ID/g for 5 nmol and 40 nmol ICG injected, respectively, at 6 h post-injection). Both probes successfully detected the tumor tissues (251±25% and 382±81% increase of PA signals in the tumor for ICG-PSar30k and ICG-PEG30k). Although ICG-PEG30k yielded intense PA signals compared to ICG-PSar30k, ICG-PSar30k achieved high contrast PA images due to low PA signals from the blood. The T/B ratios analyzed from PA images were 1.10±0.05 and 1.86±0.39 for ICG-PEG30k and ICG-PSar30k, respectively (P < 0.05).

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Figure 7. In vivo photoacoustic images of colon26 tumor-bearing mice injected with ICG-PSar30k (A) and ICG-PEG30k (B). Dotted circles and white arrowheads indicate tumor regions and blood vessels, respectively.

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Discussion In this study, the usefulness of ICG-labeled PSar as a PA imaging probe targeted for cancer was elucidated by evaluating its cellular uptake mechanism in vitro, in vivo biodistribution, and its potential for in vivo PA tumor imaging. Recently, an amphiphilic micelle with PSar on the surface, called as a lactosome, was reported as a tumor-targeted probe aiming at diagnosis and therapy. A NIR fluorescence dye-labeled lactosome clearly visualized the tumor regions with high background contrast at early time points (3-24 h) after probe administration,15 which could help deduce the active delivery mechanism of PSar. Therefore, in vitro cellular uptake of ICG-PSar was examined and compared with PEG, another water-soluble polymer. As expected, ICG-PSar30k was rapidly and markedly taken up by the tumor cells compared to ICG-PEG30k, suggesting the presence of a PSar-specific uptake pathway. In the inhibition study, amiloride significantly suppressed internalization of ICG-PSar30k into tumor cells. Although ICG-PSar could be mainly taken up by the tumor via a passive EPR effect, these results indicate the cellular uptake of ICG-PSar partially via macropinocytosis, which could facilitate in vivo tumor accumulation of ICG-PSar early (6 h) after injection. In this study, we revealed the involvement of active transport mechanism of ICG-PSar, however, target molecule of PSar was not yet identified,

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therefore, it was difficult to select cell line suitable for this study. In the future, we have a plan to seek the target molecule of PSar and to conduct PA imaging study using mice inoculated with suitable tumor cell line. On the other hand, ICG-PSar showed rapid clearance from blood compared to ICG-PEG, resulting in high T/B ratios (4.6 and 0.6 for ICG-PSar30k and ICG-PEG30k, respectively at 24 h post-injection). There was no difference in binding affinity to albumin between ICG-PSar and ICG-PEG, which suggested little involvement of protein binding. The molecular weights of the repeating unit are 71 and 44 for PSar and PEG, respectively, therefore, the structure of PSar might be tightly packed. Since the smaller PSar would be cleared rapidly through glomerular filtration in the kidney and partly taken up via macropinocytosis-mediated endocytosis, high T/B ratios sufficient to achieve high contrast PA imaging were obtained. In fact, in vivo PA imaging study, while ICG-PEG30k increased PA signal not only in tumor but also blood vessels, ICG-PSar30k clearly visualized tumor regions without increasing PA signal in blood vessels, resulting in high contrast images (T/B ratios in PA images: 1.10 and 1.86 for ICG-PEG30k and ICG-PSar30k). In this study, we compared ICG-PSar30k with ICG-PEG30k with same molecular weight. Our previous study using ICG-PEG series demonstrated that ICG-PEG20k could be a candidate for PA tumor imaging, however,

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ICG-PSar30k was also superior to ICG-PEG20k in high contrast tumor imaging. In metabolite analysis, no metabolite of ICG-PSar was confirmed in the urine for up to 24 h post-injection. In addition to rapid clearance of ICG-PSar from the body, sarcosine is a natural occurring amino acid, which can be degraded endogenously by sarcosine dehydrogenase. Because ICG was FDA-approved, each component would have a favorable toxicity profile, although formal toxicity studies of ICG-PSar would be required in the future. A potential of PA imaging for intraoperative diagnosis was recently demonstrated.16, 17 Since ICG-PSar30k achieved high contrast tumor imaging, ICG-PSar30k could be used as a powerful tool for accurate intraoperative diagnosis resulting in accurate resection of tumors.

Conclusions ICG conjugated polysarcosine was designed and synthesized as a tumor-targeted PA imaging probe. ICG-PSar30k showed active cellular uptake via macropinocytosis in vitro and high tumor accumulation and high T/B ratios. Furthermore, the PA imaging study using ICG-PSar30k achieved high contrast tumor imaging without increasing signal in blood vessels, indicating the usefulness of ICG-PSar30k as a tumor-targeted

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PA imaging probe.

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Experimental Section General 1

H NMR spectra were recorded on a JEOL JNM-LM400 with TMS as an internal

standard. Coupling constants are reported in hertz. Multiplicity was defined by s (singlet), d (doublet), and br (broad). Gel permeation chromatography (GPC) was performed with a Shimadzu system (a LC-20AD pump with a SPD-20A UV detector,

λ = 254 nm) using GPC column KD 803 (Showa Denko K.K., Tokyo, Japan) and N,N-dimethylformamide (DMF) as the mobile phase at a flow rate of 1.0 mL/min. Polyethylene glycol was used as standards to calibrate the GPC.

Materials 2-[7-[1,3-Dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benzo[e]indol-2-ylidene]-1,3,5-he ptatrienyl]-1,1-dimehyl-3-[5-(3-sulfosuccinimidyl)oxycarbonylpentyl]-1H-benzo[e]indo lium, inner salt, sodium salt (ICG-Sulfo-OSu) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Z-Sar-OH was obtained from Kokusan Chemical Co., Ltd. (Tokyo, Japan). Neopentylamine was purchased from Tokyo Chemistry Industry Co., Ltd. (Tokyo, Japan). Amiloride was obtained from Carbiochem (Darmstadt, Germany). Genistein and cytochalasin D were purchased from Wako Pure

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Chemical Industries, Ltd. (Osaka, Japan). Chlorpromazine was obtained from Nacalai Tesque (Kyoto, Japan). Other reagents were of reagent grade and were used without further purification unless otherwise indicated.

Synthesis of Sarcosine N-Carboxyanhydride Thionyl chloride (5 mL) was added to Z-Sar-OH (5.0 g, 22.4 mmol) and the mixture was stirred at 55°C. After stirring for 10 min, petroleum ether (100 mL) was added to the resulting yellow reaction mixture. The precipitate was filtered and dried in vacuo for 30 min. After dissolving in ethyl acetate at 40°C, the filtrate was collected. Petroleum ether was added to the filtrate, and the precipitate was filtered and dried in vacuo for 30 min. The same purification step was repeated thrice, and sarcosine N-carboxyanhydride (Sar-NCA) was obtained as a white powder. 1H NMR (400 MHz, CDCl3) δ [ppm] = 4.14 (s, 2H), 3.06 (s, 3H)

Synthesis of Polysarcosine (PSar) Neopentylamine (6.3–11.7 mg) was dissolved in dry dimethyl formamide (2 mL), and dried over anhydrous magnesium sulfate. Neopentylamine was added to Sar-NCA at the Sar-NCA:neopentylamine molar ratio of 140:1, 280:1, or 420:1, and stirred for 110 h

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under an argon atmosphere at room temperature. Diethyl ether was added to the reaction mixture, centrifuged at 2000 rpm for 15 min at 4°C, and the supernatant was collected. The same purification step was repeated thrice, and the precipitate was dried in vacuo. The molecular weight of each PSar was measured by GPC. Each PSar has hereinafter been referred to as PSar10k, PSar20k, or PSar30k according to molecular weight. 1H NMR (400 MHz, CDCl3) δ [ppm] = 4.6-3.8 (br, H (2n)), 3.1-2.6(br, H, (3n)), 0.82(d, 9H)

Synthesis of ICG-PSar PSar (9.2 mg, 12.7 mg, and 18.4 mg for PSar10k, PSar20k, and PSar30k, respectively) was mixed with ICG-Sulfo-OSu in 100 µL dimethyl sulfoxide (DMSO)) at the molar ratio of PSar:ICG-Sulfo-OSu = 1:1 in 0.1 M borate buffer (pH 8.6) at room temperature for 24 h with light shielding. The solvent was dialyzed against methanol with a pre-treated regenerated cellulose membrane Spectra/Por® 7 dialysis tubing (MWCO: 3.5 kDa) (Spectrum Laboratories, Inc., Rancho Dominguez, CA, USA) in order to remove the unconjugated ICG derivatives. The concentration of ICG was determined by measuring absorption at 794 nm with a UV-Vis NIR system (UV-1800, Shimadzu Co., Kyoto, Japan). To determine the chemical purity, the purified ICG-PSar (100 pmol ICG)

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was separated by Native-PAGE. The gel was imaged with IVIS Imaging System 200 (ex/em 745/820 nm, PerkinElmer Inc., Waltham, MA, USA).

Synthesis of ICG-PEG ICG-PEG30k was prepared according to a previous report.10 Briefly, mono-amino PEG30k (0.54 µmol in 1 mL chloroform, SUNBRIGHT ME-EA series, NOF Co., Tokyo, Japan) was mixed with ICG-Sulfo-OSu (1.08 µmol in 100 µL DMSO) at a molar ratio of PEG:ICG = 1:2, followed by incubation at room temperature for 24 h with light shielding. After solvent evaporation, the resulting mixture was dissolved in methanol (2 mL) and dialyzed against methanol with a pre-treated regenerated cellulose membrane to remove the unconjugated ICG.

Cell culture Colon26, a mouse cell line derived from rectal cancer, was provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. The cells were grown in DMEM (Life Technologies Co., Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin in 5% CO2 at 37°C.

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Cellular uptake study To evaluate the cellular uptake of ICG-PSar30k and ICG-PEG30k, in vitro fluorescence imaging was performed. Colon26 cells (8×105) were plated in a 6-well plate and allowed to grow for 2 days at 37°C. ICG-PSar30k or ICG-PEG30k (500 µM ICG) was added to the medium. After incubation for 1 or 6 h at 37°C, the cells were washed with PBS and collected in tubes by centrifugation. Two time points (1 and 6 h after incubation) were selected in order to investigate the cellular uptake mechanism at the early time post-incubation. The fluorescence images of cell pellets (1.2–2.4×106 cells) were acquired by the IVIS imaging System 200 (ex/em 745/820 nm). The fluorescence signal intensities were corrected against cell numbers. Cellular uptake was also investigated at 4°C after 1 or 6 h-incubation to evaluate the involvement of active transport mechanisms. For these hypothermal experiments, colon26 cells were incubated in DMEM containing 10% FBS and 25 mM HEPES.

Evaluation of cellular uptake mechanism Colon26 cells (8×105) were plated in a 6-well plate and allowed to grow for 2 days at 37°C. In order to determine the cellular uptake mechanism of ICG-PSar30k and ICG-PEG30k, endocytosis inhibitors (amiloride (inhibitor of macropinocytosis, final

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concentration: 1 mM), genistein (inhibitor of caveola-mediated endocytosis, 200 µM), chlorpromazine (inhibitor of clathrin-mediated endocytosis, 7 µM), or cytochalasin D (inhibitor of phagocytosis, 5 µM) were added to the medium 1 h before addition of ICG-PSar30k or ICG-PEG30k (500 µM ICG). Inhibitors were dissolved in dimethyl sulfoxide (3 µL), and then diluted with DMEM (997 µL) in order to adjust concentration. At 6 h after incubation at 37°C with ICG-PSar30k or ICG-PEG30k, the cells were washed with PBS and collected in tubes. The fluorescence images of cell pellets (0.6– 2.4×106 cells) were acquired by the IVIS imaging System 200 in the same conditions as mentioned above.

Tumor model Animal studies were conducted in accordance with the institutional guidelines of Kyoto University, and the experimental procedures were approved by the Kyoto University Animal Care Committee. Five-week-old female BALB/c-nu/nu nude mice were purchased from Japan SLC Inc. (Hamamatsu, Japan). The animals were housed in air-conditioned rooms under a 12-h light/dark cycle and allowed free access to food (D10001, Japan SLC, Inc.) and water. Colon26 cells (1×106) suspended in 50 µL PBS

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were injected subcutaneously into the right flank of mice, and the experiments were conducted when the tumors grew to about 7-8 mm at 7 days after inoculation. During the procedure, mice were anesthetized with isoflurane.

In vivo fluorescence imaging study ICG-PSar or ICG-PEG30k (5 nmol ICG/150 µL PBS) was intravenously administered into colon26 tumor bearing mice and the fluorescence images were acquired by the IVIS imaging System 200 (ex/em 745/820 nm) at 1, 6, 24, and 48 h post-injection (n = 3 at each time point). We selected four time points in order to investigate the appropriate time point to achieve high background ratios.

In vivo biodistribution study In order to determine the optimal molecular weight of PSar, accumulation of ICG-PSar in the tumor and blood was evaluated. At 1, 6, 24, and 48 h after each ICG-PSar (5 nmol) was intravenously injected in the colon26 tumor bearing mice, tumors and blood were harvested. The tumors were homogenized after adding 1% Triton-X aqueous solution (1.25× µL 1% Triton-X for × mg tumor tissue). The concentration of ICG-PSar in the tumor homogenate and in blood was measured according to a protocol reported

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previously.7 In vivo biodistribution of ICG-PEG30k was also evaluated. To analyze metabolites, urine was collected at 24 h after injection of ICG-PSars and SDS-PAGE was performed. The gel was imaged with an IVIS imaging System 200 (ex/em 745/820 nm).

Protein binding assay Binding affinities of each probe to serum albumin were measured as reported previously.10, 18 ICG-PSar30k, ICG-PEG30k, PSar30k (each 0–60 µM), or ICG (0–14

µM) was incubated with bovine serum albumin (BSA) (2 µM) for 30 min to allow equilibration, and the fluorescent intensity of tryptophan in BSA was measured by a spectrofluorophotometer (RF-5300PC, Shimadzu Co., ex/em 279/342 nm). The binding affinity of each compound to BSA was calculated by the Hill equation for a static quenching interaction.

In vivo photoacoustic imaging study Colon26 tumor bearing mice received an intravenous injection of ICG-PSar30k or ICG-PEG30k (40 nmol ICG/150 µL PBS). Before and at 24 h after administration, mice were imaged with a Nexus 128 instrument (797 nm, 120 angles, 100 pulses per angle)

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(Endra Inc., Ann Arbor, MI, USA) under isoflurane anesthesia (n = 3). PA images were reconstructed by volumetric rendering using Osirix software. PA signal intensity was analyzed by Image J or AMIDE after normalization with irradiated laser intensity. We set the regions of interest in the tumor and blood vessels on PA images and analyzed the PA signal intensity, and T/B ratios were calculated for ICG-PEG30k and ICG-PSar30k.

Statistical analysis Quantitative data have been expressed as mean ± SD. Means were compared using two-way factorial ANOVA followed by the Tukey-Kramer test. P-values

Indocyanine Green-Labeled Polysarcosine for in Vivo Photoacoustic

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