Fractionated photothermal therapy in a murine tumor model: comparison with single dose

Abstract

Purpose: Photothermal therapy (PTT) exploits the light-absorbing properties of nanomaterials such as silica-gold nanoshells (NS) to inflict tumor death through local hyperthermia. However, in in vivo studies of PTT, the heat distribution is often found to be heterogeneous throughout the tumor volume, which leaves parts of the tumor untreated and impairs the overall treatment outcome. As this challenges PTT as a one-dose therapy, this study here investigates if giving the treatment repeatedly, ie, fractionated PTT, increases the efficacy in mice bearing subcutaneous tumors. Methods: The NS heating properties were first optimized in vitro and in vivo. Two fractionated PTT protocols, consisting of two and four laser treatments, respectively, were developed and applied in a murine subcutaneous colorectal tumor model. The efficacy of the two fractionated protocols was evaluated both by longitudinal monitoring of tumor growth and, at an early time point, by positron emission tomography (PET) imaging of ¹⁸F-labeled glucose analog ¹⁸F-FDG. Results: Overall, there were no significant differences in tumor growth and survival between groups of mice receiving single-dose PTT and fractionated PTT in our study. Nonetheless, some animals did experience inhibited tumor growth or even complete tumor disappearance due to fractionated PTT, and these animals also showed a significant decrease in tumor uptake of ¹⁸F-FDG after therapy. Conclusion: This study only found an effect of giving PTT to tumors in fractions compared to a single-dose approach in a few animals. However, many factors can affect the outcome of PTT, and reliable tools for optimization of treatment protocol are needed. Despite the modest treatment effect, our results indicate that ¹⁸F-FDG PET/CT imaging can be useful to guide the number of treatment sessions necessary.

Keywords: cancer; fractionated therapy; hyperthermia; nanoparticle; photothermal therapy; positron emission tomography.

Figure 1

Heating of aqueous solution of NS under NIR light. (A) Thermographic imaging and temperature elevation as a function of time and laser intensity of a 5×10⁹ NS/mL aqueous solution of NS. The dashed line represents the top of the sample. (B) Temperature elevation of a 5×10⁹ NS/mL aqueous solution of NS irradiated in four cycles at a laser intensity of 1.5 W/cm². Inset shows a TEM image of intact NS after second cycle of heating. (C) Absorption spectrum of NS measured in water.

Figure 2

Reduction of unspecific heating using glycerol. (A) Temperature elevation on the tumor surface in the absence of glycerol as a function of time and tumor size. (B) Temperature elevation on the tumor surface in the presence of glycerol as a function of time and tumor size. Temperatures on the last time point (300 s) were compared between the 500 mm³ and both smaller groups. ** Denotes a p value <0.01. Abbreviation: ns, non-significant.

Figure 3

Temperature response and treatment outcome using two doses of laser irradiation. (A) Experimental timeline, which includes a standard protocol receiving one laser treatment on day 0 (dark blue) and a two-dose protocol receiving laser treatment on day 0 and day 2 (light blue). Both protocols consist of NS-laden tumors (group receiving one dose: NS1, n=5; group receiving two doses: NS2, n=6), saline groups (group receiving one dose: Saline1, n=8; group receiving two doses: Saline2, n=6) and a sham group (group receiving NS but no laser treatment, n=6). All animals are ¹⁸FDG PET/CT scanned the day before the first PTT and 1 day after their last laser treatment. (B) Temperature elevation during the first laser dose of all animals. The maximum temperatures reached after 5 minutes were compared. For the first treatment, both NS groups together were compared against both saline groups. **** p<0.0001, *** p<0.001. (C) Temperature elevation during the second laser dose. Maximum temperatures for NS2 and Saline2 were compared. (D) Tumor growth after treatment and (E) overall survival for all four groups. Tumor growth is plotted until n≥3 and data shown are mean±SEM.

Figure 4

PET-based treatment evaluation. (A) Representative ¹⁸F-FDG PET/CT images of NS, saline, and sham animals at baseline and after treatment. Arrows point to the tumors. 1T=1 treatment, 2T=2 treatments. (B) The mean tumor ¹⁸F-FDG uptake at baseline and at day 1 or day 3 (NS group receiving one dose of irradiation: NS1, n=5; NS group receiving two doses of irradiation: NS2, n=6; saline group receiving one dose of irradiation: Saline1, n=8; saline group receiving two doses of irradiation: Saline2, n=6; sham group receiving NS but no irradiation: Sham, n=6). * p<0.05, *** p<0.001. Data shown are mean±SEM. (C) Correlation between ¹⁸F-FDG uptake ratio (baseline/day 3) for animals in the NS2 and Saline2 groups.

Figure 5

Temperature response and treatment outcome using four doses of laser irradiation. (A) Experimental timeline where the protocol consists of two groups receiving either NS or saline which were laser treated four times with 1 day in between treatments. All animals were baseline scanned the day before PTT and 1 day after their last PTT. (B) Temperature elevation during all four laser treatments in NS-laden tumors (NS, n=6) and the control group (Saline, n=7). The maximum temperatures reached at the last time point (300 s) were compared. * p<0.05, ** p<0.01, and *** p<0.001. (C) Tumor growth after treatment, and (D) overall survival for both groups. Day 60 was considered the end of the study. Tumor growth is plotted until n≥3 and data shown are mean±SEM.

Figure 6

PET-based treatment evaluation. (A) Representative ¹⁸F-FDG PET/CT images of NS and saline-treated animals at baseline and after treatment. Arrows point to the tumors. (B) The mean ¹⁸F-FDG tumor uptake at baseline and day 7 (NS group, n=6; saline group, n=6). Data shown are mean±SEM. (C) Correlation between ¹⁸F-FDG uptake ratio (baseline/day 7) and survival for animals in the NS and saline groups.