Correlation between PDT Effects and DpO2 in CAM Samples

1. Experimental procedure to study the correlation between the PDT effects and DpO2 t - oxymetry For PDT Delayed fluorescence lifetime measurements were performed during and at the end of the illumination of Chick chorioAllantoic Membranes (CAM) Samples preparation: • Fertilized eggs were incubated during 11 days (membrane development). • 20 µl of ALA (0.15 M) were administered topically, 4h before PDT and the DF measurements PDT illumination conditions: • Light source: CW diode laser @ 405 nm; 2.5 mW/cm2; up to 2.25 J/cm2. • Circular spot produced by a frontal light distributor from Medlight SA: 8 mm diameter. 1) CAM 2) Protective drop of aqueous serum CAM membrane at embryonic development day 11 3) 0.19 mm cover-glass 4) PDT fiber 1) Egg 2) Laser 3) PDT fiber 5) DF measurement fiber (1 mm2 probed area) 4) DF Spectrometer 5) DF measurement fiber 6) DF measurement fiber holder 6) DF measurement fiber holder Piffaretti et al., JBO, 2012

2. Experimental procedure (cont.) t - oxymetry For PDT 80μm Vascular damage assessment • 17 h after PDT the eggs were taken out of the incubator and vascular damage were examined with an epifluorescence microscope (Ex. 450 – 490 nm, Em. > 520 nm). • 20 µl of a 150 µM solution of fluorescein isothiocyanate dextran (150 KD) were administered intravenously and 0.1 ml of india ink was injected under the CAM to reduce the background fluorescence. • The diameter of the largest closed vessel was measured and used as an index of vascular damage. (method described in: Lange et al., IOVS 2001) 4X objective 200 µm 10X objective Piffaretti et al., JBO, 2012

3. t - oxymetry For PDT Delayed fluorescence lifetime measurement setup Piffaretti et al., JBO, 2012

4. t - oxymetry For PDT Tissular oxygen depletion during PDT N = 17 16 Bars: SEM Different symbols: different measurement series • The pO2 decreases monotonically as the PDT illumination increase. • An horizontal asymptotic value is reached after ~ 500 sec (~1.3 J/cm2) Piffaretti et al., JBO, 2012

5. t - oxymetry For PDT Vascular damage index observed after PDT versusthe reciprocal lifetime differences (before/after PDT) N = 20 Bars: SEM • An excellent correlation is observed between the vessel damages and the pO2 reduction. Piffaretti et al., JBO, 2012

6. - oxymetry Exo. probe Setup for micro-imaging and photo-treatment FITC-dextran angiographies 24 h post irradiation - Assessment of the vascular damages based on the diameter of the largest closed vessel. - The index of vascular damage (Ivd) range assessment between “0” (no detectable photodamage) and “5” (total closure of all blood vessels in the irradiated area).

7. Phototoxicity and leakage of Ru(Phen)32+ versus PdTCPP • - oxymetry Exo. probe 4 eggs / condition (PdTCPP) 1 min after i.v. injection 4 eggs / condition (PdTCPP) - Phototoxic threshold: 1 mg/kg; 10 J/cm2 @ 420 + 20 nm Lethal dose - Dark toxicity after iv injection of 10, 50 and 100 mg/kg: 100 % survival after 24 h (6 eggs/condition) Huntosova et al., Metallomics, 2014 10 min after i.v. injection Phototoxicity of Ru(Phen)32+ << PdTCPP Huntosova et al., JBO, 2014

8. - oxymetry Exo. probe Luminescence lifetime measurement setup CAM were subjected to different pO2 conditions in a gas chamber. Detection was performed 10 min after 1mg/kg b.w. Ru(Phen)32+ 20 l injection. Light dose < 120 mJ/cm2 Much smaller than the phototoxic threshold 10 J/cm2 & 1 mg/kg

9. - oxymetry Exo. probe pO2 dependence of the Ru(Phen)32+ reciprocal lifetime Measurements performed 10 min after iv injection of a 20 l solution of Ru(Phen)32+ (1 mg/kg of b.w.). CAM were subject to different environmental pO2 in a gas controlled and temperature stabilized (25 °C) chamber. 6 eggs; 4 measurements / condition. (0.9% NaCl isotonic solution) Light dose @ 420 nm necessary to perform one pO2 measurement: < 120 mJ/cm2 Much smaller than the phototoxic threshold: 10 J/cm2 Huntosova et al., Metallomics, 2014 Huntosova et al., JBO, 2014