Irradiation and fluorescence parameters for each photosensitizer.

<p>Irradiation and fluorescence parameters for each photosensitizer.</p

Molecular features that could affect APDI effectiveness.

a<p>As the conjugate has 37 primary amino groups, the number of charges depends of pH and microenviroment.</p>b<p>Average extinction coefficient over range 400–700-nm.</p

Effect of laccase enzyme on photodynamic inactivation of <i>C. neoformans</i> ATCC 208820 (laccase positive strain, black squares), and ATCC 208819 (laccase negative strain, open circles).

<p>(A) methylene blue, (B) rose bengal, (C) EtNBSe, (D) BB6, (E) pL-ce6 were used as photosensitizers at 10 µM in PBS for 30 min followed by a wash and illumination with the wavelengths specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054387#pone-0054387-t002" target="_blank">Table 2</a>. Data are means and bars are the standard deviation. * P<0.05; ** P<0.01; *** P<0.001 for survival of 208820 vs 208820.</p

Protection of <i>C. neoformans</i> against APDI by melanin.

<p><i>C. neoformans</i> ATCC 208820 (laccase positive strain) were grown in a minimal medium with or without L-dopa and subjected to APDI with pL-ce6 (10 µM in PBS for 30 min followed by a wash and illumination with 665-nm). Data are means and bars are the standard deviation. * P<0.05; ** P<0.01; *** P<0.001 for survival of +L-dopa vs no L-dopa.</p

Chemical structures of the photosensitizers used in this study.

<p>(A) Methylene blue; (B) Rose Bengal; (C) Selenium Nile blue derivatrive (EtNBSe); (D) Tris-cationic fullerene (BB6); poly-L-lysine chlorin (e6) conjugate (pL-ce6).</p

Confocal microscopy image of <i>C. neoformas</i> KN99α.

<p>Cells were treated with APDI mediated by pL-ce6 (10 µM) and then incubated with FITC-annexin V and PI. Green represents fluorescence of externalized phosphatidylserine that is correlated to the initial steps of apoptosis, and red corresponds to fluorescence of PI (advanced apoptosis/necrosis). We present three pictures of the same field: Transmittance in column A, green and red fluorescence in columns B and C respectively. The first line of figures is the stained samples before APDI (0J/cm²), the second line is following an irradiation of 10J/cm² and the last one was irradiated with fluence of 40J/cm². Scale bars 8µm.</p

Confocal fluorescence microscopy images of <i>C. neoformans</i> strains.

<p>Cells were incubated with 10 µM photosensitizers for 30 min as described (methylene blue, rose bengal, EtNBSe, and pL-ce6) and then labeled with MitoTracker Green™ (for methylene blue, EtNBSe, and pL-ce6) or MitoTracker Red™ (for rose bengal). All photosensitizers emitted red fluorescence, except rose bengal that emits green fluorescence. We present one picture of the superimposed images for each strain incubated with each photosensitizer. Scale bars 8µm.</p

Effect of (A) capsule (KN99α and <i>cap59),</i> and (B) laccase enzyme (208820 and 208819) on photosensitize uptake.

<p>Each strain was incubated with photosensitizers at 10 µM for 30 min, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054387#s2" target="_blank">material and methods</a>. Data are means and bars are the standard deviation. * p<0.05 between the positive and negative strains, for each photosensitizer.</p

Effect of capsule on photodynamic inactivation of <i>C. neoformans</i> KN99α (black squares) and <i>cap59</i> (open squares).

<p>(A) methylene blue, (B) rose bengal, (C) EtNBSe, (D) BB6, (E) pL-ce6 were used as photosensitizers at 10 µM in PBS for 30 min followed by a wash and illumination with the wavelengths specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054387#pone-0054387-t002" target="_blank">Table 2</a>. Data are means and bars are the standard deviation. * P<0.05; ** P<0.01; *** P<0.001 for survival of KN99α vs <i>cap59.</i></p