Red and blue light in antitumor photodynamic therapy with chlorin-based photosensitizers: a comparative animal study assisted by optical imaging modalities

Abstract

The goal of this study is a comparative analysis of the efficiency of the PDT protocols for CT26 tumor model treatment in Balb/c mice employing red and blue light with both topical and intravenous administration of chlorin-based photosensitizers (PSs). The considered protocols include the doses of 250 J/cm² delivered at 660 nm, 200 J/cm² delivered at 405 nm, and 250 J/cm² delivered at both wavelengths with equal energy density contribution. Dual-wavelength fluorescence imaging was employed to estimate both photobleaching efficiency, typical photobleaching rates and the procedure impact depth, while optical coherence tomography with angiography modality (OCT-A) was employed to monitor the tumor vasculature response for up to 7 days after the procedure with subsequent histology inspection. Red light or dual-wavelength PDT regimes with intravenous PS injection were demonstrated to provide the most pronounced tumor response among all the considered cases. On the contrary, blue light regimes were demonstrated to be most efficient among topical application and irradiation only regimes. Tumor size dynamics for different groups is in good agreement with the tumor response predictions based on OCT-A taken in 24h after exposure and the results of histology analysis performed in 7 days after the exposure.

Fig. 1.

Autofluorescence images of a Balb/c mouse leg with inoculated CT26 tumor (a,c) and fluorescence images of the murine leg covered with a reflecting tape in 30 minutes after PS topical application and removal of excessive PS (b,d) acquired at the excitation wavelengths of 405 (a,b) and 660 (c,d) nm. Dashed contour shows the position of the mouse leg.

Fig. 2.

Fluorescence images of a Balb/c mouse leg with inoculated CT26 tumor in 3(a,e), 15 (b,f), 60 (c,g) and 120 (d,h) minutes after intravenous PS injection acquired at the excitation wavelengths of 450 (a-d) and 660 (e-h) nm and corresponding PS accumulation dynamics within the tumor ROI after intravenous injection (i).

Fig. 3.

Typical photobleaching kinetics registered by dual-wavelength (@405nm and @660 nm) fluorescence monitoring: normalized intensity of PS fluorescence in the course of PDT procedures with red (a) and blue (b) light regimes. Dots correspond to the measured fluorescence intensity. The bi-exponential fit (solid line for intravenous injection and dashed line for topical administration) calculated by Eq.(4) is given for each measured kinetics.

Fig. 4.

Photobleaching rate constants d⁽¹⁾ (a) and d⁽²⁾ (b), photobleaching efficiency PE (c) and fluorescence signal ratio R_λ (d) registered upon fluorescence excitation by blue (@405 nm) and red (@660 nm) light in different intravenous (i) and topical application (t) regimes of the PDT procedure according to Table 1. Asterisk shows statistically significant difference.

Fig. 5.

Temperature increase $\mathrm{\Delta }$T as the result of PDT and irradiation for different exposure regimes.

Fig. 6.

Photos of an untreated tumor, tumor after red light exposure without PS application (IRR_r250), and tumors undergone red light PDT protocol after topical administration (PDT_r250-t) and intravenous injection (PDT_r250-i) prior to (a), immediately after (b), in 1 (c), 4 (d), and 7 (e) days after exposure at λ = 660 nm with the light dose of 250 J/cm². Millimeter scale is given for reference.

Fig. 7.

Tumor growth dynamics after PDT procedures with topical PS administration (a) and intravenous PS injection (b) for red (r) and blue (b) light procedures and combined action (rb). Untreated (control) tumor growth dynamics and the results for irradiation without PS administration are given for reference.

Fig. 8.

Tumor growth inhibition factor (TGI) for different treatment regimes in 4 (a) and 7 (b) days after the procedure.

Fig. 9.

Results of angiographic OCT monitoring of natural tumor growth (Control), tumor after IRR_r250 exposure without PS administration, after PDT with PS topical administration (PDT_r250-t) and intravenous injection (PDT_r250-i) at λ  = 660 nm with dose of 250 J/cm² prior to (a), immediately after (b), in 1(c), 4(d), and 7(e) days after exposure. The bottom row corresponding to PDT_r250-i protocol demonstrates the interruption of tumor microcirculatory activity observed by OCT-angiography.

Fig. 10.

Histological images of tumors in 7 days after red light exposure (a - PDT_r250-i, d - PDT_r250-t, g - IRR_r250), blue light exposure (b - PDT_b200-i, e - PDT_ b200-t, h - IRR_b200), dual-wavelength exposure (c - PDT_rb250-i, f - PDT_rb250-t, i - IRR_rb250), and an untreated tumor (j). Typical observed changes are outlined in the figures: (1) dystrophic cell alterations and necrobiosis, (2) vessel destruction, (3) increased mitotic activity and formation of giant cells, (4) inflammatory infiltration, (5) hyalinosis of tumor stroma.