Mulation exhibited a viability of 93?.8 compared to 55?.7 following treatment with an equivalent concentration of free D-luciferin (9 M). The microcapsules also enhanced the cytotoxicity of the bioluminescence/PS system while decreasing the cell viability from 96 to 88 via rose Bengal-PDT or to 71 with hypericin-PDT (both PSs were used at the equivalent concentrations of 0.03 mg.mL-1). To further reduce the toxicity, Hsu et al. proposed the use of a self-illuminated bioluminescent Rluc8-QD, which was previously synthesized by So et al., [58] to activate a PS [59]. Rluc8 is an enzyme that oxidizes a substrate, coelenterazine, to produce a characteristic fluorescence emission at 480nm. This energy can be transferred to quantum dots, (QD) by Forster Resonant Energy Transfer (FRET) or by Bioluminescent FRET (BRET), leading to their excitation. The QD energy relaxation is accompanied by an emission of luminescence centered in the red (655 nm) that can be used to activate a PS. Thus, BRET allows for shifting of the emission of the bioluminescent molecule, coelenterazine, to a wavelength that could excite a conjugated PS. In their study, Hsu et al. SB 202190 web observed a 5-fold reduction in tumor volume compared to a control that used Foscan?loaded micelles as a PS self-activated by the conjugated coelenterazine/QD compound. In a more recent study, Kim et al. [60] used the same bioluminescent probe, Rluc8-QD, to activate a different PS, chlorin e6 (Ce6), to induce PDT. Besides reporting the efficacy of their conjugated system on cell viability and tumor growth suppression in vitro and in vivo respectively, the authors evaluated the equivalent light dose required to reach a similar killing efficiency as with laser activated PDT. This dose was estimated to be 2.2 mW.cm-2. In addition, a detailed in vivo study showed the impact of the RLuc8-QD mediated excitation of Ce6 on the reduction of cancer cells spread to sentinel and secondary lymph nodes, potentially indicating a promising new modality to decrease likelihood of distant metastases. Taken together, these results demonstrate a promising new method to induce PDTmediated cytotoxicity at depth. Even though the light dose produced by chemi- or bio-luminescent probes is lower than that usually required for PDT, this method still efficiently induces cytotoxicity, emphasizing the complexity and the limited understanding of the processes involved. Further investigations and thorough validations on the efficiency of these probes (either as free probes or in nanoparticle form) are required prior to clinical translation.NIR radiation to induce PDT in deeper tissueThe most direct approach to excite PS for PDT in deeper tissue is to use radiation that lies within the NIR optical window (Fig. 1). Following absorption of a single photon, the excitation energy is below the production threshold of singlet oxygen molecules, allowing only type I PDT or oxygen purchase TAPI-2 independent-PDT [3]. Two photon (TP) processes use longer excitation wavelengths and can also be considered to excite the PS and induce photodynamic tissue damage. In the following sections we will discuss various options for PDT in deeper tissues using NIR radiation. These options are also illustrated in Fig 5A.TP excited PSUsing TP processes to excite a PS would not only enhance the penetration depth but also localize the illumination area. Indeed, TP excitation is a non-linear process whose efficiency quadratically increases with the laser intensity [61]. Th.Mulation exhibited a viability of 93?.8 compared to 55?.7 following treatment with an equivalent concentration of free D-luciferin (9 M). The microcapsules also enhanced the cytotoxicity of the bioluminescence/PS system while decreasing the cell viability from 96 to 88 via rose Bengal-PDT or to 71 with hypericin-PDT (both PSs were used at the equivalent concentrations of 0.03 mg.mL-1). To further reduce the toxicity, Hsu et al. proposed the use of a self-illuminated bioluminescent Rluc8-QD, which was previously synthesized by So et al., [58] to activate a PS [59]. Rluc8 is an enzyme that oxidizes a substrate, coelenterazine, to produce a characteristic fluorescence emission at 480nm. This energy can be transferred to quantum dots, (QD) by Forster Resonant Energy Transfer (FRET) or by Bioluminescent FRET (BRET), leading to their excitation. The QD energy relaxation is accompanied by an emission of luminescence centered in the red (655 nm) that can be used to activate a PS. Thus, BRET allows for shifting of the emission of the bioluminescent molecule, coelenterazine, to a wavelength that could excite a conjugated PS. In their study, Hsu et al. observed a 5-fold reduction in tumor volume compared to a control that used Foscan?loaded micelles as a PS self-activated by the conjugated coelenterazine/QD compound. In a more recent study, Kim et al. [60] used the same bioluminescent probe, Rluc8-QD, to activate a different PS, chlorin e6 (Ce6), to induce PDT. Besides reporting the efficacy of their conjugated system on cell viability and tumor growth suppression in vitro and in vivo respectively, the authors evaluated the equivalent light dose required to reach a similar killing efficiency as with laser activated PDT. This dose was estimated to be 2.2 mW.cm-2. In addition, a detailed in vivo study showed the impact of the RLuc8-QD mediated excitation of Ce6 on the reduction of cancer cells spread to sentinel and secondary lymph nodes, potentially indicating a promising new modality to decrease likelihood of distant metastases. Taken together, these results demonstrate a promising new method to induce PDTmediated cytotoxicity at depth. Even though the light dose produced by chemi- or bio-luminescent probes is lower than that usually required for PDT, this method still efficiently induces cytotoxicity, emphasizing the complexity and the limited understanding of the processes involved. Further investigations and thorough validations on the efficiency of these probes (either as free probes or in nanoparticle form) are required prior to clinical translation.NIR radiation to induce PDT in deeper tissueThe most direct approach to excite PS for PDT in deeper tissue is to use radiation that lies within the NIR optical window (Fig. 1). Following absorption of a single photon, the excitation energy is below the production threshold of singlet oxygen molecules, allowing only type I PDT or oxygen independent-PDT [3]. Two photon (TP) processes use longer excitation wavelengths and can also be considered to excite the PS and induce photodynamic tissue damage. In the following sections we will discuss various options for PDT in deeper tissues using NIR radiation. These options are also illustrated in Fig 5A.TP excited PSUsing TP processes to excite a PS would not only enhance the penetration depth but also localize the illumination area. Indeed, TP excitation is a non-linear process whose efficiency quadratically increases with the laser intensity [61]. Th.
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