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(Figure 2). J/cm2, and 630-nm light (red upwards triangles) at 65 mW/cm2 for 230 J/cm2. Error bars represent standard error of the mean. Cells were treated with the following concentrations of TLD1433 before exposure to 532 nm light of 0.01 μM, 0.05 μM, 0.1 μM, 0.3 μM and 0.5 μM. Similarly, cells were treated with TLD1433 concentrations of 2 μM, 10 μM, 20 μM, and 40 μM before 630 nm light illumination. TLD1433 only (no exposure to light) was assessed at higher concentrations of TLD1433: 0.1 μM, 0.3 Pharmaceuticals 2020, 13, 137 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 30 μM, and 60 μM. In vitro experiments were completed in triplicate. 4 of 9 Pharmaceuticals 2020, 13, x 4 of 9 Figure 2. Relative cell viability of A549 cells treated with 630-nm (150 J/cm2) or 532-nm (7 J/cm2) laser light with and without TLD1433. Cell viability was evaluated with rezasurin cell assay. In vitro experiments were completed in triplicate. The two light conditions exhibited a different irradiance dependence (Figure 2), and similar photocytotoxicity could be obtained between the wavelengths by changing the concentration of TLD1433, the fluence, and the irradiance. Increasing the irradiance to 150 mW/cm2 at 630 nm with laserlightwith2 andwithoutTLD1433.Cellviabilitywasevaluatedwithrezasurincellassay.Invitro with 14 μM (J/cm ), with no significant toxicity observed when cells were treated with light alone 22 4500μFiMgur(Je/c2m. R)eilnatdivuececdellavsiaimbiliiltayropfhAo5t4o9cycteolltsotxriecaiteydawsi5th3263n0m-nmus(i1n5g0aJ/ncmirr)aodria5n32c-enomf(278J/mcmW)/cm experiments were completed in triplicate. 2.2. In Vitro PDT with OSA Illumination 2.2. In Vitro PDT with OSA Illumination A549 cells, seeded in a 96-well plate, were treated with TLD1433 then illuminated either directly A549 cells, seeded in a 96-well plate, were treated with TLD1433 then illuminated either directly with the OSA or with tissue mimicking phantoms present (Figure 3). Areas of cell viability at the with the OSA or with tissue mimicking phantoms present (Figure 3). Areas of cell viability at the surface of the OSA were similar between plates treated with or without the 15-mm backscatter surface of the OSA were similar between plates treated with or without the 15-mm backscatter phantom. After passage through 3- or 5-mm phantoms, cell viability was lower in multiwall plates phantom. After passage through 3- or 5-mm phantoms, cell viability was lower in multiwall plates treated with the 15-mm backscatter phantom versus the plates treated without a backscatter phantom treated with the 15-mm backscatter phantom versus the plates treated without a backscatter phantom (Figures S1 and S2). The light simulations suggest that an increased irradiance occurred when the (Figure S1 and S2). The light simulations suggest that an increased irradiance occurred when the 15- 15-mm phantom acting as a backscatter was placed below the OSA. mm phantom acting as a backscatter was placed below the OSA. 22 Figure 3. (A) A photo of a 3-mm phantom over the optical surface applicator (OSA) with two 2-cm Figure 3. (A) A photo of a 3-mm phantom over the optical surface applicator (OSA) with two 2-cm radial radial diffusers used for the in vitro light administration. Schematic configuration of the OSA, the 96- diffusers used for the in vitro light administration. Schematic configuration of the OSA, the 96-well well plate and phantoms are shown in (B) where 96-well plate was placed on top of the OSA silicon plate and phantoms are shown in (B) where 96-well plate was placed on top of the OSA silicon beads, beads, (C) where 96-well plate was placed on 3- or 5-mm phantoms atop the OSA and (D) same as C, (C) where 96-well plate was placed on 3- or 5-mm phantoms atop the OSA and (D) same as (C), where the OSA was placed on a 15-mm phantom (backscatter phantom). where the OSA was placed on a 15-mm phantom (backscatter phantom). The 3- and 5-mm phantoms atop of the OSA attenuated the light and resulted in higher cell viability when compared to no phantom. However, there was still an effective therapy observed with the 3-mm phantom. In Figure 4, we demonstrate the relationship between cell viability and light irradiance and fluence. The light was delivered through the OSA with a 3-mm phantom on top and the 15-mm backscatter below. Figure 4A depicts a color map of the A549 cell viability, and Figure 4BPDF Image | TLD1433-Mediated Photodynamic Therapy Lung Cancer Cells
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