Highly reliable, targeted photothermal cancer therapy combined with thermal dosimetry using indocyanine green lactosome

Indocyanine green (ICG) is a near-infrared light-absorbing substance. Thus, when a tumor in which ICG has accumulated is irradiated with a near-infrared (NIR) laser, only the tumor can be heated by a photothermal reaction. We developed ICG lactosome, a novel drug delivery system (DDS) composed of polymeric micelles and ICG that shows selective accumulation in tumor based on an enhanced permeability and retention (EPR) effect. We showed that ICG lactosome accumulated in a tumor by using an intradermal tumor mouse model of a murine colon cancer cell line (Colon26) transfected with Nano lantern luminescent protein (NLC26). Two days after the administration of ICG lactosome, the tumor was irradiated with an 808-nm diode-laser while monitoring tumor temperature. The results showed that the treated tumors were cured when the peak of tumor temperature during NIR irradiation reached 43°C or higher. To verify these results, photothermal therapy (PTT) using ICG lactosome was carried out using a newly developed system that can control the temperature at the NIR irradiation site at a constant level. All of the tumors that had been kept at 43°C during irradiation were cured, while 2 of 5 tumors that had been kept at 42°C were not cured, and none of tumors that had been kept at a temperature below 41°C were cured. ICG lactosome-assisted PTT combined with thermal dosimetry is a highly reliable method for cancer treatment and may afford further clinical opportunities for PTT.


Introduction
Indocyanine green (ICG), which was approved by the U.S. Federal Drug Administration (FDA) in 1959 (Zheng et al. , 2014), has been widely used clinically not only for evaluation of liver function and cardiac output but also as a fluorescent biomedical imaging tool for identification of sentinel lymph nodes of breast cancer (Toh et al. , 2015) and gastric cancer (Okubo et al. , 2018) and for real-time evaluation of blood flow in organs , Milstein et al. , 2016. ICG is also known as a near-infrared (NIR) light-absorbing substance, that is a photothermal agent, and thus when a tissue in which ICG exists is irradiated with NIR light, only the tissue irradiated can be heated by a photothermal reaction (Liu et al. , 2002). Studies on anti-cancer treatment using this phenomenon have also been performed (Chen et al. , 1995a, Chen et al. , 1995b, and a growth inhibitory effect was found in tumor tissue in which ICG was locally injected and then the tissue was irradiated with NIR light (Chen et al. , 1996). However, the properties of ICG have some limitations such as poor photostability, non-specific targeting, and short half-life. In order to overcome these problems and to deliver more ICG to the tumor, many researchers have attempted to develop an ICG-based drug-delivery system (DDS) to exert an enhanced permeability 6 and retention (EPR) effect (Matsumura andMaeda, 1986, Wang et al. , 2018). Various ICG nanoparticles including lipid-based, polymer-based, and mesoporous silica ICG nanoparticles have been developed (Wang et al., 2018). Subcutaneous tumors have been successfully eradicated by a photothermal reaction using ICG nanoparticles (Chen et al. , 2016, Zheng et al., 2014. In our previous study, we established ICG lactosome from self-assembly of poly When tumors in which ICG lactosome had accumulated were exposed to NIR light, the proliferation of tumor cells was greatly inhibited mainly due to the photothermal reaction: the therapeutic effectiveness of ICG lactosome has been shown in peritoneal carcinomatosis (Tsujimoto et al., 2014b)  fluence rate were predetermined before NIR irradiation in those studies, and the optimal therapeutic effects have not been determined. Furthermore, the photothermal efficacy of ICG lactosome as a NIR light-absorbing agent has not been demonstrated.
We have conducted animal experiments on phototherapy using ICG lactosome, but the expected therapeutic effect was not obtained for all. Therefore, in order to obtain a reliable therapeutic effect, we examined the relationship between photothermal therapy (PTT) parameters and therapeutic effects. We found that the maximum temperature of the tumor during NIR irradiation was most strongly correlated with the therapeutic effect.

Outline of the experimental design
An intradermal tumor model was used to verify the anti-tumor effect of PTT using ICG lactosome on a tumor. In the tumor model, the only tissue between the tumor and the laser was epidermal tissue. Based on the results of our previous experiments, ICG lactosome was administered 48 h before NIR laser irradiation. Fluorescence images 8 were obtained by an in vivo imaging system (IVIS; PerkinElmer) before NIR laser irradiation. The temperature of the tumor during NIR irradiation was monitored in real time by using an infrared radiation thermometer (FT-H10; Keyence). On day 21, the three-dimensional size of the tumor was measured by using a digital caliper.

Specific accumulation of ICG lactosome in the tumor
In order to determine whether ICG lactosome accumulates specifically in tumor tissue, the consistency between the fluorescence and the actual tumor location from ICG lactosome was confirmed using a Colon26 cell line transfected with Nano-lantern luminescent protein (NLC26), which degrades the luminescent substrate coelenterazine h (Wako Pure Chemical Industries Ltd., Osaka, Japan). Figure 1A shows the consistency among the white image (WLI), the bioluminescent image emitted from a luminescent substrate degraded in tumor cells (BLI), and the fluorescent image from ICG lactosome (FLI). These images were taken at 48 hours after injection by the IVIS.
Fluorescence images were taken at 5 min, 30 min, 4 h, 12 h, 24 h and 48 h after intravenous injection of ICG lactosome for observing tumor selectivity (Fig. 1B). At 12 h after ICG lactosome injection, the margin of the tumor started to become clear in the 9 fluorescent image. After that, the tumor/non-tumor tissue fluorescence intensity ratio increased, and the ratio was more than 12-times higher at 48 h.
Since fluorescence from ICG lactosome consistent with luminescence from the tumor was observed, ICG lactosome had excellent selective accumulation in the tumor.

Correlation of temperature in PTT with antitumor effect.
Intradermal tumor model mice were used to investigate changes in the tumor temperature and anti-tumor effect with changes in the parameters of NIR irradiation.
First, we changed the fluence rate to observe the time courses of tumor temperature and antitumor effect. Fluence rates were set at 250, 500, 750 and 1000 mW/cm 2 . The irradiation time was set at 1000 s and the dose of ICG lactosome was 8.8 mg/kg. When the NIR laser was turned on, the temperature at the surface of the skin covering the tumor reached a peak at about 120 s at all fluence rates ( Fig. 2A). After reaching the peak, the temperature remained constant or gradually decreased. When the fluence rate was increased from 250 to 1000 mW/cm 2 , the temperature during NIR irradiation increased ( Fig. 2A); however, there was no statistically significant difference in tumor temperature between NIR irradiation with 750 mW/cm 2 and that with 1000 mW/cm 2 .
After NIR irradiation, we investigated how the fluence rate changes antitumor effect 1 0 (Fig. 2B). Significant differences were observed between a fluence rate of 1000 mW/cm 2 and fluence rates of 0, 250 mW/cm 2 , and between a fluence rate of 750 mW/cm 2 and 0 mW/cm 2 . Although tumor growth seemed to be suppressed in proportion to the fluence rate, there were no significant differences among the three fluence rates  Figure 3 shows that the two tumors that did not disappear were included in the groups of 750 and 1000 mW/cm 2 , and both cases were irradiated for 111 s. 1 1 The results suggested that tumor cure depends on the tumor temperature during NIR irradiation. We have developed a thermal sensor circuit-based NIR laser irradiation system using a non-contact thermometer in order to keep the temperature of the irradiated target constant during irradiation (Nomura et al. , 2017). We carried out a NIR irradiation experiment using the temperature-feedback laser system. Using this system, tumors were irradiated at 40, 41, 42 or 43°C for 333 s, and tumor size was measured up to 21 days after irradiation. The results showed that the tumor was not cured at a temperature below 41°C. However, 3 of 5 tumors were cured at 42°C and all of the tumors were cured at 43°C (Table 1).

Negligible increase in temperature at the irradiated non-tumor site
To confirm that ICG lactosome does not accumulate in tissue other than the tumor tissue and that the increase in temperature at non-tumor sites is negligible, temperatures of the tumor site and non-tumor site were measured during NIR laser irradiation (Fig.   4A). The increases in temperature (means ± SD) at the tumor site from the initial temperature to maximum temperature at 250, 500, 750 and 1000 mW/cm 2 were 5.6 ±1.0, 10.6 ± 1.2, 13.6 ± 3.1, 19.0 ± 1.9°C, respectively, while those at the non-tumor site were 0.2 ± 0.1, 1.6 ± 0.2, 2.7 ± 0.4, 4.6 ± 0.3°C, respectively (Fig. 4B): the higher the fluence 1 2 rate was, the larger was the difference in temperature increase. We also compared the differences in temperature increases at non-tumor sites with and without injection of ICG lactosome. In the case of ICG lactosome injection, the non-tumor site was irradiated at 48 hours after the injection. A slight increase in temperature during NIR irradiation was observed, and the increase in temperature was correlated with increase in fluence rate (Fig. 4C, red circles). However, the increase in temperature at the non-tumor site in the case of ICG lactosome injection was not significantly different from that at the non-tumor site in the case of no ICG lactosome injection (Fig. 4C, black circles). These results suggest that there is little accumulation of ICG lactosome at sites other than the tumor site.

DISCUSSION
This study showed that ICG lactosome selectively accumulated in the tumor, and it was also found that the photothermal reaction generated by the combination of ICG lactosome and NIR light suppressed the tumor growth. However, it was found that fluence rate and time of laser irradiation were not determinants for tumor cure. It was shown that tumors disappeared when the maximum temperature reached or exceeded 1 3 43°C during NIR irradiation. In addition, the increase in temperature due to NIR irradiation was negligible at non-tumor sites even when ICG lactosome had been injected. Thus, ICG lactosome facilitates a photothermal effect only at the tumor site without increasing the temperature around the tumor (non-tumor sites).
The results suggest that thermal dosimetry during NIR irradiation is a more useful index than fluence rate or irradiation time for prediction of tumor cure. We verified this concept by combining ICG lactosome and a temperature-controlled NIR laser system Thermal dosimetry-assisted PTT may be useful for clinical use not only to effectively cure tumors but also to not damage normal tissues. It would be convenient for clinicians to predict the therapeutic effect during irradiation. Generally, the protocols of laser therapies including photodynamic therapy are set before irradiation. Thus, even if However, in different types of tumor, the tissue structure, heat tolerance and therapeutic threshold temperature might be different from those in other cancer types.

Conclusions
In conclusion, ICG lactosome was highly accumulated in the intradermal tumor model and when the surface temperature of the tumor exceeded 43°C, the tumor disappeared in this model regardless of the fluence rate or irradiation time. PTT using ICG lactosome with thermal dosimetry will be a novel and useful theranostic tool for cancer strategy and provide clinicians with more reliable cancer treatment. 1 6

Tumor-targeted photo-absorbent
The ICG lactosome is a micelle-based agent that was synthesized as previously reported

Animals
Female Balb/c mice at 6 weeks of age (Japan SLC, Hamamatsu, Japan) were fed under specific pathogen-free conditions. All animal procedures followed the guidelines approved by the National Defense Medical College Animal Care and Use Committee. 1 8 A murine Colon26 cell line (NLC26) stably expressing Nano-lantern (Saito et al. , 2012) was established as previously described (Maday et al. , 2008). The cells were (Antibiotic-Antimycotic, Life Technologies) at 37°C in 5% CO2 with 95% humidity.

Temperature controlled NIR laser irradiation experiments.
Since the results obtained by PTT suggested that the peak of temperature of the tumor during NIR irradiation is the most reliable index to predict tumor shrinkage, we conducted animal experiments using a system that can control the temperature of the tumor. In order to keep the temperature constant, we used a NIR laser system for which the irradiation power is modulated by a temperature feedback control circuit that we previously reported (Nomura et al., 2017). The temperature was set 40, 41, 42 or 43°C (n = 5 in each). The irradiation time was set at 333 s.

Statistical methods
Data are presented as the means ± standard deviation (SD) in Fig. 2B    irradiation. Each plot is divided into two groups ("cured" and "not cured"). Fluence rate (mW/cm 2 ) is shown in each graph. The results indicate that tumors are cured at a temperature above 43°C regardless of the fluence rate.  Temperature was preset at 40, 41, 42, or 43°C (n=5 each). The "measured temperature"

Figure Legends
was the value just before the end of irradiation. The thermal measurement was at a refresh rate of 10/s.