(P 298) EVALUATING THE PHOTOTHERMAL AND CYTOTOXIC PROPERTIES OF INDOCYANINE GREEN IN VITRO FOR INTRAOPERATIVE PHOTOTHERMAL THERAPY IN SARCOMA

Objective: The main objective of this project is to evaluate the heat producing and cytotoxic profile of Indocyanine Green (ICG) when exposed to infrared wavelengths of light. The basis of this investigation is to consider ICG as a photothermal agent for interoperative photothermal therapy (PTT) for sarcoma; performing as adjunct to kill remaining deposits or locoregional micro metastasis of malignant cells after resection. The secondary objectives of this project are: To establish the dark toxicity profile of ICG to confirm the effect of light and demonstrate its acknowledged safety. To briefly analyse how the parameters of duration of infrared radiation and light intensity impact the efficacy of PTT to help establish an optimal protocol.

Methods: CCK-8 Assay
Chondrosarcoma cell line HT1080 was seeded overnight into 96-well plates at 3.2 x 104 cells per well; following data collected in a seeding density study, ensuring cells were in a consistent growth phase. Plates were treated under a heat lamp bulb from Philips for either: 20 minutes; 10 minutes; 10+10 minutes with a 5-minute intermission; or not treated but left in ambient light. A dimmer switch was used to change intensity of the lamp to halfway or full output. Figure 1. depicts the layout of the experiments. Each plate contained cells exchanged with fresh media after pre-treatment with 0, 0.01. 0.1, 1, 10, 25, 50 or 100uM of ICG for 30 minutes, with 6 replicates at each concentration. For each group of treatment conditions separate plates were analysed at 0h, 24h, 48h and 72h post treatment with a CCK-8 proliferation assay. After an hour incubation with CCK-8 solution absorbance values were measured with an Omega plate reader. For each concentration of ICG absorbance values were compared with the absorbance values for 0uM within the same plate using unpaired t-tests.
Temperature Studies
Solutions of ICG and complete HT1080 media at 0, 0.01. 0.1, 1, 10, 25, 50 or 100uM concentration were placed in a bead bath set to 37⁰C for an hour. Using the set-up from figure 1, an infrared thermometer gun was used to take surface temperature measurements of different concentrations of ICG loaded into 6-well plates. Measurements were taken between 0-30 minutes every 5 minutes during full intensity radiation of the heat lamp with each concentration being measured at separate 30-minute intervals. At each time point measurements were recorded three times.

Results: CCK-8 solution reacts with lactate dehydrogenase (LDH) produced by viable cells inducing a colorimetric change proportional to the level of LDH activity in the well increasing the absorbance. CCK-8 absorbance values measured directly after PTT show a slight drop in ICG treated cells compared with 0uM. At 72h post treatment there are more striking differences in absorbance. Figure 2 shows the mean absorbance values collected at 72h for each concentration of ICG. Asterisks denote the significance derived from unpaired t-tests compared against absorbance values for 0uM in the respective plate. * = P ≤ 0.05. ** = P ≤ 0.01 *** = P ≤ 0.001. Noticeably, cells treated at different concentrations of ICG but only left in ambient light do not share the same trend of overt decrease in absorption.
Temperature recordings from two separate temperature studies taken on separate days can be seen in Figures 3&4. Temperatures during PDT increased with time and ICG concentration in both studies. At 0uM there is still a substantial increase in temperature due to infrared radiation from the heat lamp. It is important to note that there was a ~15⁰C temperature increase between the weather on the day of the first experiment and the second.

Conclusion: Although this is study is only a preliminary exploration into the effects of PTT with small samples sizes and one cell line; the results are promising and indicate that ICG could be used as an effective photothermal agent for interoperative PTT for sarcoma after further testing.