We compared the efficacy of PDT mediated by the porphyrin-based photosensitizer TMP-1363 with that of the phenothiazine MB against C. albicans in vitro
. As shown in , upon irradiation at a fixed fluence of 2.4 J cm−2
, photosensitization with TMP-1363 resulted in greater than three logs more killing of C. albicans
than MB at the same incubation concentration. This representation, however, may underestimate the photodynamic efficacy of TMP-1363 because, as illustrated in , it is excited less efficiently by our irradiation source than MB. Based on the output spectrum of the irradiation light source and the absorption spectra of the sensitizers, we used Equation 1
to determine that, for equivalent durations of irradiation, the number of photons absorbed by TMP-1363 was ~ 10-fold lower than that absorbed by the equivalent concentration of MB. Because both photosensitizers generate reactive oxygen species at high yields, it is likely that differences in cellular uptake and/or intracellular localization account for the significantly greater PDT efficacy of TMP-1363.
Comparison of methylene blue (MB) and TMP-1363 in PDT of C. albicans in vitro
Our previous in vivo
imaging study with GFP-labeled C. albicans
in the infected mouse ear model revealed that the morphologies of C. albicans
range from yeast to multicellular hyphal filaments [22
]. Therefore, a rigorous evaluation of photosensitizer localization demanded that the uptake of TMP-1363 by C. albicans
be examined in both the yeast and the hyphal form of the organism. TMP-1363 was incubated with both of these forms of C. albicans
and its subcellular localization imaged using confocal fluorescence microscopy. As shown in , bright intracellular fluorescence from TMP-1363 was observed after a 10-minute incubation with a concentration of 10 μg mL−1
. A series of 0.8 μm-thick optical sections through a field of C. albicans
illustrated in , confirmed that the sensitizer fluorescence did not originate solely with cell-surface-bound-material but was distributed throughout the cell. The punctate appearance of intracellular fluorescence, most evident in , suggests a degree of organelle compartmentalization of TMP-1363 in C. albicans
. These findings in vitro
provided strong rationale for the further evaluation of TMP-1363 in vivo
TMP-1363 fluorescence in C. albicans in vitro
illustrates TMP-1363 localization in the C. albicans-infected mouse ear pinna model. To begin the investigation, TMP-1363 was solubilized in a glycerol:water (2:3) vehicle. The addition of glycerol to water-soluble TMP-1363 was intended to enhance the association of the photosensitizer with the pinna. TMP-1363 was applied topically to the entire ear. We evaluated TMP-1363 fluorescence using large field of view stereofluorescence imaging, in vivo spectroscopy, and confocal fluorescence imaging. As shown in the stereofluorescence image of , we observed strong fluorescence in selected areas distributed over large areas of the ear, consistent with specific localization to infected areas. To confirm that the fluorescence signal originated from TMP-1363, we used a bifurcated fiber bundle attachment to a fluorometer to perform in vivo fluorescence spectroscopy of the infected ear. At 420 nm excitation, the fluorescence emission spectrum, an example of which is shown in , was dominated overwhelmingly by TMP-1363. In order to visualize the distribution of TMP-1363 in the infected tissue at high spatial resolution, the mouse ear was imaged in vivo using confocal microscopy. Images like that shown in revealed that the intense fluorescence from TMP-1363 appeared highly localized to individual C. albicans cells. This highly selective localization was persistent up to at least 6 hours after the topical application.
TMP-1363 localization in the C. albicans-infected mouse ear pinna model
In a recently reported study [22
], we used a C. albicans
strain transfected with yeast GFP [27
] placed under the control of the constitutively active ADH1
promoter to image C. albicans
morphogenesis during the progression of an intradermal infection in the ear in vivo
. Using this model system, we rigorously evaluated the extent of selectivity of TMP-1363 for C. albicans
following topical application in vivo
. GFP fluorescence from C. albicans
was excited efficiently by 488 nm light, and in the absence of TMP-1363 there was no detectable endogenous fluorescence under conditions of 639 nm excitation (data not shown). TMP-1363 fluorescence was not detected with excitation at 488 nm but was excited well at 639 nm. show in vivo
images from identical fields of view in an infected ear that are comprised exclusively of GFP or TMP-1363 fluorescence when excited by 488 or 639 nm light, respectively. is a merged image of the two channels. Strong pixel co-localization of GFP and TMP-1363 fluorescence is depicted by the yellow/orange color. The panel of images in demonstrates on a magnified scale the extent of co-localization between the GFP and TMP-1363 fluorescence in a region of interest indicated by the box superimposed on the image shown in . In the merged image of , a high degree of overlap is evident between pixels that display both GFP fluorescence and TMP-1363 labeling.
Selectivity of TMP-1363 for C. albicans in vivo
Analysis of the data shown in using the Mander’s coefficient approach as described in Methods and summarized in Equation (2)
yielded co-localization coefficients Mred
= 0.99 and Mgreen
= 0.89. Thus, 99% of pixels exhibiting red TMP-1363 fluorescence also contained a green signal from GFP-expressing C. albicans
, and 89% of the pixels positive for GFP had a TMP-1363 signal, as well. Taken together, the data suggest that TMP-1363 exhibits high affinity for C. albicans
relative to host tissue and may serve not only as a selective PDT agent but also as an excellent fluorescent contrast agent for detection of C. albicans
Based on the highly selective staining of C. albicans
with TMP-1363 in the context of infection, we subjected C. albicans
-infected tissue to PDT. One of the challenges in the topical administration of photosensitizer is the barrier posed by stratum corneum. Recent studies by Boiy et al.
] investigated the penetration of the photosensitizer hypericin into mouse skin using different vehicles, and reported significant, vehicle-dependent differences in penetration depths. Based on our own initial findings and their results, we evaluated PDT efficacy in vivo
following the topical administration of 50 μL of 0.3 mg mL−1
TMP-1363 in two vehicles:glycerol:water (2:3) and ethanol:glycerol:water (2:2:1), each applied once for 30 min. Infected ears were then irradiated at 514 nm for 90 J cm−2
with an irradiance of 50 mW cm−2
. Shown in , the statistical analysis of organism levels recovered from infected control animals demonstrated a consistent level of infection with C. albicans,
as reflected by a low standard deviation in CFU recovered from tissue homogenates. Significantly reduced organism levels (p
< 0.05) were observed following PDT using the ethanol:glycerol:water formulation compared to untreated controls, resulting in an ~ 50-fold reduction in CFU/ear relative to untreated control mice. PDT using TMP-1363 in ethanol:glycerol:water resulted in a significant reduction in organism burden (p
< 0.05) compared to PDT using the photosensitizer in the glycerol:water formulation.
PDT of C. albicans infection in vivo using topical TMP-1363
To address the issue of potential phototoxicity to host tissue, we monitored an infected mouse ear pinna subjected to PDT with TMP-1363 in ethanol:glycerol:water using the same treatment parameters. Again, the TMP-1363 formulation was applied topically to the dorsal surface of the ear, and the entire dorsal surface was irradiated. The panel of photographs shown in taken over 19 days post-PDT demonstrated initial scar formation and subsequent healing following PDT, including recovery of hair growth.
Mouse ear pinna following PDT using topical TMP-1363
In summary, we demonstrated that tetra-cationic, porphyrin-based photosensitizer TMP-1363 displayed substantial efficacy against C. albicans both in vitro and in vivo. The in vitro efficacy of TMP-1363 in PDT of early stationary phase yeast forms is particularly striking in comparison to MB, especially given the relatively inefficient excitation of TMP-1363 under the conditions of our experiments. In vivo, fluorescence microscopy and image analysis demonstrated that TMP-1363 had a remarkably strong selectivity for C. albicans in the context of infection. We suggest that the phototoxicity of TMP-1363 for C. albicans, coupled with selectivity for the fungus, resulted in significant organism reduction using a PDT dose that allowed apparently normal healing of infected tissue. These collective observations indicate that topical application of the photosensitizer TMP-1363 in the appropriate formulation could be effective in PDT of focal C. albicans infections in humans.