Enhanced efficacy of photodynamic therapy via a sequential targeting protocol

Abstract

This study was designed to examine determinants of the discovery that low-dose lysosomal photodamage (lyso-PDT) could potentiate the efficacy of subsequent low-dose mitochondrial photodamage (mito-PDT). The chlorin NPe6 and the benzoporphyrin derivative (BPD) were used to separately target lysosomes and mitochondria, respectively, in murine hepatoma cells. Lyso-PDT (LD(5) conditions) followed by mito-PDT (LD(15) conditions) enhanced the loss of the mitochondrial membrane potential, activation of procaspases-3/7 and photokilling. Reversing the sequence was less effective. The optimal sequence did not enhance reactive oxygen species formation above that obtained with low-dose mito-PDT. In contrast, alkalinization of lysosomes with bafilomycin also enhanced low-dose mito-PDT photokilling, but via a different pathway. This involves redistribution of iron from lysosomes to mitochondria leading to enhanced hydroxyl radical formation, effects not observed after the sequential procedure. Moreover, Ru360, an inhibitor of mitochondrial calcium and iron uptake, partially suppressed the ability of bafilomycin to enhance mito-PDT photokilling without affecting the enhanced efficacy of the sequential protocol. We conclude that sequential PDT protocol promotes PDT efficacy by a process not involving iron translocation, but via promotion of the pro-apoptotic signal that derives from mitochondrial photodamage.

Figure 1

Excitation spectra of NPe6 and BPD (10 μM) in ethanol.

Figure 2

Cells were treated with NPe6 and BPD (see legend to Fig. 1), and an image of BPD fluorescence acquired (EM = 400 nm, EX = 680–700 nm). After irradiation on the microscope stage (660 nm, 75 mJ/cm sq), a second image of the same field was was acquired. Magnification = 1000 x.

Figure 3

Singlet oxygen production following irradiation. Cultures were incubated with DADB (10 μM) + 0.5 μM BPD or 40 μM NPe6 for 60 min, and then refed with fresh medium and irradiated at 690 nm (BPD, 37.5 mJ/sq cm), or at 660 nm (NPe6, 75 mJ/sq cm), or sequentially at both wavelengths. Additional cultures were incubated with only DADB. Images of DADB fluorescence were captured in (A) untreated control cultures, or directly after: (B) low-dose BPD PDT, (C) low-dose NPe6 PDT, (D) sequential NPe6-BPD PDT. Panel (E) shows pixel brightness analyses of ~100 cells per field. Note that DADB fluorescence is lost upon interaction with singlet oxygen. *Significantly less than either A or C (p<0.05). There was no significant difference between A and C, or B and D.

Figure 4

Effects of PDT on maintenance of the mitochondrial membrane potential. Panels A–E: cultures were loaded with NPe6 and BPD and irradiated as described in Fig. 2. Panels F–J: cultures treated with Ru360 (10 μM) during photosensitization and after washing and refeeding with fresh medium. MTO (2 μM) was added immediately after irradiation and imaging was carried out 10 minutes later. A,F = untreated control cells, B, G = irradiation at 690 nm (37.5 mJ/cm sq), C,H = irradiation at 660 nm (75 mJ/sq cm), D, I = irradiation at 660 nm before 690 nm, E,J = irradiation at 690 nm before 660 nm.

Figure 5

Fluorescence co-localization of iron and mitochondrial or lysosomal loci shortly after irradiation. Cultures were loaded with sensitizers and irradiated as described in Fig. 2. RN1 (red fluorescence) or Baf was added to cultures 1 h prior to irradiation. The mitochondrial fluorescence probe MTG (A–D, green) or the lysosomal fluorescence probe LTG (E–H, green) were added directly after irradiation, and imaged 10 min later. Treatments were: (A,E) no treatment; (B,F) in the presence of Baf; (C,G) after NPe6-PDT; and (D,H) after NPe6-PDT followed by BPD-PDT.

Figure 6

Fluorescence co-localization analyses of iron and mitochondrial loci 60 min after irradiation. Cultures were incubated with RN1 and NPe6 ± BPD, washed, refed, and irradiated as in Fig. 4. Fluorescence co-localization of RN-1 (red) and MTG (green) was performed as described in Figure 4 except that images were captured 60 after irradiation. Treatments were: (A) low-dose NPe6 PDT; (B) low-dose NPe6 PDT followed by low-dose BPD PDT.

Figure 7

Effects of Bafilomycin on PDT-induced hydroxyl radical formation. Cultures were incubated for 60 min with 2.5 μM APF and (as specified) 50 μM Baf, 0.5 μM BPD, or 40 μM NPe6 before being washed and refed. Treatments were: (A) untreated control, (B) Baf alone, (C) low-dose BPD PDT, (D) low-dose NPe6 PDT, (E) NPe6 PDT → BPD PDT, (F) Baf → BPD PDT. Panel (G) shows the pixel brightness analyses of 100 cells per treatment. *Significantly greater fluorescence than in untreated controls, BAF alone, and low dose NPe6 PDT, p<0.05. ^†Significantly greater than all other treatment groups, p< 0.05.