Synthesis and characterization of ICG-Fe NPs
The preparation process of ICG-Fe NPs is shown in Fig. 1. First, free ICG was reacted with Fe3+ ions in water to form a water-insoluble, hydrophobic ICG-Fe complex, implying that cationic Fe3+ ions and anionic ICG molecules were associated via the electrostatic interaction with their electric charges cancelled. As can be seen in Fig. 2a, the color of the ICG solution changed from green to brownish-black with sedimentation upon increasing the Fe3+ ion concentration, indicating the occurrence of complexation. The formation of the ICG-Fe complex was confirmed by absorption and fluorescence emission spectra (Fig. 2b, c). The absorption band at 793 nm and the fluorescence of ICG gradually decreased with increasing the molar ratio of Fe3+ from 1:0 to 1:10 (ICG:Fe3+). Upon increasing the Fe3+ ion concentration, the absorption band of free ICG shifted hypsochromically by ~8 nm, from 793 to 785 nm. The complex formed between the Fe3+ and ICG molecules was found to quench the fluorescence of ICG, likely owing to the paramagnetic property of Fe3+ [21,22,23,24]. This fluorescence quenching was further proved by the fluorescence lifetime as shown in Additional file 1: Figure S2. The fluorescence lifetime of ICG-Fe NPs was 0.94 ns, while free ICG exhibited 1.61 ns of fluorescence lifetime. The shortened fluorescence lifetime of ICG-Fe NPs supports that complexation between Fe3+ and ICG induced fluorescence quenching [25]. As the complex exhibited a higher quenching efficiency at the molar ratio of 1:10, we chose this condition for the metal-complexed ICG nanoformulation study. To characterize the composition of the ICG-Fe complex, FT-IR spectra of free ICG and the ICG-Fe complex were obtained (Additional file 1: Figure S1), where the ICG-Fe complex retained the characteristic peaks of free ICG near 1000, 1500, and 1400 cm−1, corresponding to the vinyl, C=C and S=O stretches, respectively [26]. Furthermore, the ICP-OES results showed that the parent counterion of free ICG (Na+) was almost replaced by Fe3+ with an ICG:Fe molar ratio of 3:1 (Additional file 1: Table S1).
Next, ICG-Fe NPs were synthesized by encapsulating a hydrophobic ICG-Fe complex into a water-dispersed Pluronic F127 polymeric micelle. The transmission electron microscope (TEM) image indicated that the obtained ICG-Fe NPs were spherical in shape with an average diameter of 17.1 \(\pm\) 2.6 nm (Fig. 3a). The nanoparticle loading of the ICG-Fe complex was also confirmed by UV/Vis absorption and fluorescence spectra (Fig. 3b, c). The absorption spectrum of ICG-Fe NPs was slightly red-shifted with broadening and an increased shoulder peak due to the formation of ICG dye aggregates in the nanoparticles [27,28,29]. Additionally, ICG-Fe NPs exhibited quenched fluorescence, which is similar to that of the ICG-Fe complex. These results indicate that the optical properties of the ICG-Fe complex were stably maintained after water-dispersed nanoformulation.
The photothermal effect of ICG-Fe NPs was investigated using a 785 nm laser with an energy density of 1 W (Fig. 3d). The heating curves showed that ICG-Fe NPs readily reached over 60 °C within 10 min of laser irradiation. In contrast, free ICG increased the temperature up to 48.2 °C within 4 min, followed by a gradual decrease. ICG-Fe NPs showed a better temperature elevation efficiency than free ICG, despite reduced light absorption (Fig. 3b). To compare the photothermal heating performances between free ICG and ICG-Fe NPs, their optical properties were evaluated under laser irradiation. As shown in Fig. 4a–c, there was observed temporal declines in the UV/Vis absorbance and fluorescence emission in the case of free ICG, indicating significant photobleaching under the irradiation condition, whereas the photostability of ICG-Fe NPs was shown substantially enhanced. The enhanced photostability of ICG-Fe NPs is attributable to (1) the fluorescence quenching by paramagnetic Fe3+ to shorten the excited-state lifetime of ICG, and (2) nanoparticle encapsulation of ICG to disturb its contact to oxygen molecules, both of which might reduce the chance of photobleaching by photo-oxidation (reaction with oxygen molecules in the excited state). Along with photostability, we measured the stability of ICG-Fe NPs in the physiologically relevant FBS-containing medium. The emission intensities of free ICG and ICG-Fe NPs were evaluated depending on the concentration of FBS (Fig. 4d). The fluorescence intensity of free ICG significantly increased in proportion to FBS concentration as ICG was adsorbed to serum proteins to form more fluorescent ICG-protein complexes that are known to be promptly excreted from systemic circulation, being unfavorable for tumor-specific accumulation [12, 30, 31]. In sharp contrast, ICG-Fe NPs retained fluorescence quenching of the ICG-Fe complex regardless of FBS concentration, indicating the excellent protection of ICG from release and contact with the biological environment. In addition, the constant quenched state of ICG-Fe NPs in the biological environment can promote the non-radiative thermal decay, which can result in an increase in the PTT efficiency. Based on these results, we confirmed that ICG-Fe NPs confer considerable physiological/optical stability, being favorable for the improved tumor accumulation and PTT ability in vivo.
Next, we investigated the PA signaling properties of ICG-Fe NPs. As depicted in Fig. 4e, f, where PA signals were produced under laser irradiation and increased in proportion to their concentration from 0.023 to 0.375 mg/mL based on the ICG content. Although ICG-Fe NPs presented only a similar level of PA signals to those of free ICG under the given condition, it is anticipated that their enhanced photo and physiological stability over time would be beneficial for tumor-specific PA imaging in vivo.
In vitro phototoxic property
The intrinsic cytotoxicity of ICG-Fe NPs was examined using an MTT assay in HT-29 cells. As shown in Fig. 5a, no obvious cytotoxicity was observed after the incubation of the cells with ICG-Fe NPs at different concentrations for 24 h, indicating that ICG-Fe NPs are biocompatible. In addition, the photothermally induced cytotoxicity by ICG-Fe NPs was evaluated after irradiation with the 785 nm laser at a power density of 1 W for 5 min (Fig. 5b). The laser-alone treatment exhibited no cytotoxicity; however, free ICG and ICG-Fe NPs under the given irradiation condition induced considerable cell death of over 50%. No significant difference was found between free ICG and ICG-Fe NPs, likely because in both cases, the temperature elevations were shown to be similar during the initial 5 min of laser irradiation (Fig. 3d). These results conclude that ICG-Fe NPs are safe in the absence of light but able to be cytotoxic under light due to the efficient photothermal heating property, being useful for PTT.
PA and NIRF dual-modal tumor imaging in vivo
The feasibility of ICG-Fe NPs for in vivo PA and NIRF dual-modal imaging was evaluated in an HT-29 tumor-bearing mouse model. When the size of the tumor reached approximately 100 mm3, the mice were intravenously injected with ICG-Fe NPs, which were imaged using MSOT and IVIS systems. As shown in Fig. 6a, a low PA signal was detected before the injection of ICG-Fe NPs, whereas after the administration of ICG-Fe NPs, the tumor manifested an obvious PA signal that was the strongest 24 h post-injection. Meanwhile, the NIRF signal was also accumulated at the tumor with time after the injection of ICG-Fe NPs (Fig. 6b). In the case of free ICG-treated mice, however, the fluorescence signal was initially distributed throughout the body with the highest accumulation at the liver rather than the tumor, and as expected, cleared from the body much faster than ICG-Fe NPs. Since ICG-Fe NPs showed relatively low fluorescence signals compared with free ICG owing to the fluorescence quenching, we also conducted the in vivo tumor imaging with the injection of Cy5.5-labeled ICG-Fe NPs (Additional file 1: Figure S3), which confirmed similar tumor accumulation and in vivo distribution with cy5.5 fluorescence 2 h post-injection. For further validation, the organs and tumors were harvested 24 h post-injection. As presented in Fig. 6c, the majority of free ICG was present in the liver, whereas ICG-Fe NPs exhibited substantial accumulation in the tumor, liver, and kidney. These results clearly suggest that ICG-Fe NPs can significantly improve the tumor targetability via the enhanced permeability and retention (EPR) effect with reduced body clearance compared with free ICG, to potentiate dual-modal tumor imaging via the systemic targeting of ICG-Fe NPs.
In vivo PTT of cancer
The PTT efficacy of ICG-Fe NPs was investigated in mouse models whose tumor region was exposed to irradiation for 10 min after 24 h of ICG-Fe NP injection. When monitored with an infrared thermal camera, the ICG-Fe NP-treated tumor gradually increased the temperature up to 56.4 °C within 4 min after laser irradiation, exceeding the destructive threshold for irreversible tumor ablation, while the laser-alone treatment did not show any temperature increase under the given irradiation condition (Fig. 7a). After PTT, a therapeutic response was observed. As shown in Fig. 7b, mice treated with PBS alone showed sustained tumor growth. In stark contrast, tumor growth was considerably suppressed with obvious signs of necrosis, such as redness and scabbing on the tumor surface in the ICG-Fe NP-treated group under laser irradiation, indicating the outcome of efficient PTT. For a more rigorous study on tumor size reduction, we conducted MRI analysis of the ICG-Fe NP-treated mice after PTT. MRI images and estimated tumor volume showed that the tumor appeared to be destructive over time; in particular, there was a statistically significant shrinkage in the tumor volume from 4 days to 14 days after treatment, suggesting that systemically targeted ICG-Fe NPs exert a remarkable therapeutic impact for PTT of cancer (Fig. 7c, d).
Histological examination of the major organs was conducted to evaluate the in vivo toxicity of ICG-Fe NPs. H&E staining images of liver and kidney collected from ICG-Fe NP-treated mice revealed no obvious tissue damage compared with those of the control group (Additional file 1: Figure S4). Furthermore, liver function parameters of the ICG-Fe NP-treated group, including aspartate aminotransferase and alanine aminotransferase, were measured within the normal range (Additional file 1: Table S2). In addition, the body weight did not obviously change during cancer PTT after intravenous injection of ICG-Fe NPs (Additional file 1: Figure S5). This demonstrates that ICG-Fe NPs hold potential as a biocompatible theranostic agent with minimal in vivo toxicity.