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  • Teicoplanin br The UV vis and fluorescence DNA titrations da

    2020-08-18


    The UV–vis and fluorescence DNA titrations data for Pc1 are quite different as Pc1 has a higher tendency to form H-aggregates both in water and buffer solutions. Indeed, during the DNA titration experi-ment, we observed increasing intensity and red-shifting of the Q-band for all three DNA structures tested. This increase in intensity was ac-companied by a decrease of the Teicoplanin intensity of the H-aggregate of this compound. Thus, we speculate that during DNA binding with Pc1, this photosensitizer's H-aggregates convert into the monomeric form of the Pc which binds to DNA. As the monomeric form of the Pc1
    Fig. 4. Frontier molecular orbitals of Pc1 and Pc2.
    Fig. 5. TDDFT-predicted UV–vis spectra of Pc1 and Pc2. The band units are represented by nm (cm−1).
    Fig. 6. Singlet oxygen production UV–vis spectra for Pc1 and Pc2 in DMSO using DPBF.
    has higher DNA-binding affinity (because of the less demanding steric restrictions), and since H-aggregates of Pcs are non-fluorescent in nature [64,65], it was not surprising that an initial decrease in fluor-escence intensity was observed (Fig. 7) during titration experiments. However, once non-fluorescent H-aggregates monomerized and interact with DNA, the overall fluorescence of the DNA-Pc1 assemblies in-creases as expected (Fig. 7). Overall, these DNA titration experiments suggest strong interactions between both Pc1 and Pc2 photosensitizers and all three DNA structures investigated, consistent with non-specific interactions with the phosphodiester backbone. Therefore, there is promise in a broad spectrum of pharmacological activity of Pc1 and Pc2 photosensitizers with a variety of DNA structures beyond those tested. 
    3.5. Photosensitizer/cell interaction study
    To evaluate the potential of Pc2 as a photosensitizer for photo-dynamic therapy as compared to Pc1, the changes in cell morphology, nucleolar compaction, and DNA damage (via histone H2AX P139S phosphorylation) in MCF-7 cells that were treated with Pc1 or Pc2 both pre- and post-irradiation were compared.
    As a reference and control, the top two rows of Fig. 9 show healthy, adherent cells with normal nuclei and only a background signal from the DNA damage marker. As expected, no change is observed if these cells are not irradiated at the appropriate wavelength that excites the dyes. In contrast, cells treated with Pc1 (Fig. 9) and irradiated show cellular rounding, nuclear compaction and detachment from the
    Fig. 7. UV–vis and fluorescence spectra of the DNA titrations involving Pc1.
    extracellular matrix, consistent with apoptotic cells [66], and DNA damage, a hallmark of PDT [67,68]. Cells that were not irradiated ap-pear healthy but do have a markedly higher background signal from the DNA damage marker than the untreated cells. Direct detection of Pc1 using the CY5 light filters in the microscope proved troublesome; while some of the dye can be observed by our microscope in the non-irra-diated sample, fluorescence is not observed post-irradiation. When Pc1 was observed pre-irradiation, it appears to be diffuse and in the cyto-plasm with some cells having compact granules containing Pc1 present in their nuclei. The lower photostability of the Pc1 compared to Pc2 is not surprising as its DFT-predicted HOMO energy is significantly higher than the HOMO energy in Pc2, which makes Pc1 more prone to oxi-dative degradation and is in agreement with earlier observations on phthalocyanines bearing electron-donating groups at the α-positions of 
    Pc2 treated cells showed a similar response to irradiation as Pc1 treated cells: detachment, rounding, nuclear compaction, and DNA damage markers were observed (Fig. 9). Unlike Pc1, Pc2 could be observed both pre- and post-irradiation. Like Pc1, Pc2 is mostly diffuse and cytoplasmic before irradiation. Post-irradiation, fluorescence from Pc2 is localized to the cell nuclei. Of note, is that the background-level signal from the DNA damage marker is significantly less in the non-irradiated Pc2 treated cells compared to the non-irradiated Pc1 treated cells. Since mutagenesis of healthy cells that may take up a PDT pho-tosensitizing agent is a concern, the reduced background DNA damage signal from Pc2 in the non-irradiated cells may indicate it to be of less risk to healthy cells.
    Next, we followed the timeline of cell death after irradiating the Pc1
    Fig. 8. UV–vis and fluorescence spectra of the DNA titrations involving Pc2.
    or Pc2 treated cells by live cell imaging. Remarkably, with only 1 min of irradiation, changes in cell morphology only 5 min into the experi-ment were observed for the Pc2 treated cells (Fig. 10A).
    Concurrent with the beginning of cell rounding, Pc2 foci were ob-served and began to appear in the nucleus. Over the next 25 min, Pc2 continued to accumulate in the nucleus, which proceeded to round and compact. At the final 30-minute time point, essentially all cells ap-peared apoptotic and Pc2 formed the same compact foci that were observed in the compacted nuclei of the fixed cells (Fig. 9). Pc1-treated cells only showed marginal change in morphology after 15 min, albeit by the 30-minute time point, the Pc1 treated cells are in the same state as the Pc2 treated cells (Fig. 10b).