Response surface methodological approach for optimization of photodynamic therapy of onychomycosis using chlorin e6 loaded nail penetration enhancer vesicles.

Antimicrobial photodynamic inactivation (aPDI) has a tremendous potential as an alternative therapeutic modality to conventional antifungals in treatment of onychomycosis, yet the nail barrier properties and the deep-seated nature of fungi within the nails remain challenging. Therefore, the aim of this study was to prepare, optimize, and characterize Chorin e6 (Ce6) nail penetration enhancer containing vesicles (Ce6-nPEVs) and evaluate their photodynamic mediated effect against Trichophyton rubrum (T.rubrum); the main causative agent of onychomycosis. Optimization of the particle size and encapsulation efficiency of nPEVs was performed using a four-factor two-level full factorial design. The transungual delivery potential of the selected formulation was assessed in comparison with the free drug. The photodynamic treatment conditions for T.rubrum aPDI by free Ce6 was optimized using response surface methodology based on Box-Behnken design, and the aPDI effect of the selected Ce6-nPEVs was evaluated versus the free Ce6 at the optimized condition. Results showed that formulations exhibited high encapsulation efficiency for Ce6 ranging from 79.4 to 98%, particle sizes ranging from 225 to 859 nm, positive zeta potential values ranging from +30 to +70 mV, and viscosity ranging from 1.26 to 3.43 cP. The predominant parameters for maximizing the encapsulation efficiency and minimizing the particle size of Ce6-nPEVs were identified. The selected formulation showed 1.8-folds higher nail hydration and 2.3 folds improvement in percentage of Ce6 up-taken by nails compared to the free drug. Results of the microbiological study confirmed the reliability and adequacy of the Box-Behnken model, and delineated Ce6 concentration and incubation time as the significant model terms. Free Ce6 and Ce6-nPEVs showed an equipotent in vitro fungicidal effect on T.rubrum at the optimized conditions, however Ce6-nPEVs is expected to show a differential effect at the in vivo level where the advantage of the enhanced nail penetration feature will be demonstrated.

ABCG2-mediated suppression of chlorin e6 accumulation and photodynamic therapy efficiency in glioblastoma cell lines can be reversed by KO143.

BACKGROUND: Photodynamic therapy (PDT) of malignant brain tumors is a promising adjunct to standard treatment, especially if tumor stem cells thought to be responsible for tumor progression and therapy resistance were also susceptible to this kind of treatment. However, some photosensitizers have been reported to be substrates of ABCG2, one of the membrane transporters mediating resistance to chemotherapy. Here we investigate, whether inhibition of ABCG2 can restore sensitivity to photosensitizer chlorin e6-mediated PDT. METHODS: Accumulation of chlorin e6 in wild type U87 and doxycycline-inducible U251 glioblastoma cells with or without induction of ABCG2 expression or ABCG2 inhibition by KO143 was analyzed using flow cytometry. In U251 cells, ABCG2 was inducible by doxycycline after stable transfection with a tet-on expression plasmid. Tumor sphere cultivation under low attachment conditions was used to enrich for cells with stem cell-like properties. PDT was done on monolayer cell cultures by irradiation with laser light at 665nm. RESULTS: Elevated levels of ABCG2 in U87 cells grown as tumor spheres or in U251 cells after ABCG2 induction led to a 6-fold lower accumulation of chlorin e6 and the light dose needed to reduce cell viability by 50% (LD50) was 2.5 to 4-fold higher. Both accumulation and PDT response can be restored by KO143, an efficient non-toxic inhibitor of ABCG2. CONCLUSION: Glioblastoma stem cells might escape phototoxic destruction by ABCG2-mediated reduction of photosensitizer accumulation. Inhibition of ABCG2 during photosensitizer accumulation and irradiation promises to restore full susceptibility of this crucial tumor cell population to photodynamic treatment.

Bacterial keratitis: Photodynamic inactivation reduced experimental inflammation.

The successful treatment of bacterial keratitis remains an unsolved clinical problem. The current study aimed to establish a murine keratitis model and to investigate the effect of chlorin e6 (Ce6) and photodynamic inactivation (PDI) on corneal inflammation. The cornea of anesthetized mice was scratched and covered with a bacterial suspension of Pseudomonas aeruginosa. A paste containing Ce6 was applied to the cornea with subsequent exposure to specified light. Two days later the animals were sacrificed, and the globes were processed for light microscopy. Evaluation parameters were the maximal corneal thickness and the severity of the hypopyon. The maximal corneal thickness of 290±16 µm in the infected and untreated group was significantly reduced to 220±8 µm in the infected and treated group (P<0.05). In addition, the hypopyon was less severe in the infected and treated group. In conclusion, the present study indicates that PDI using Ce6 may be a potential approach to treat patients suffering with severe bacterial keratitis.

Chlorin e6 mediated photodynamic inactivation for multidrug resistant Pseudomonas aeruginosa keratitis in mice in vivo.

Following corneal epithelium scratches, mouse corneas were infected with the multidrug resistant (MDR) P. aeruginosa strain PA54. 24 hours later, 0% (for control group), 0.01%, 0.05% or 0.1% Chlorin e6 (Ce6), a second generation photosensitizer derived from chlorophyll, was combined with red light, for photodynamic inactivation (PDI). 1 hour or 2 days later, entire mouse eyes were enucleated and homogenized for counting colony forming units (CFU) of P. aeruginosa. For comparison, 0.1% Ce6 mediated PDI was started at 12 hours post infection, and 0.005% methylene blue mediated PDI 24 hours post infection. Clinical scores of corneal manifestation were recorded daily. Compared to the control, CFU 1 hour after PDI started 24 hours post infection in the 0.01% Ce6 and 0.05% Ce6 groups were significantly lower (more than one log10 reduction), the CFU 2 days post PDI higher in the 0.1% Ce6 group, clinical score lower in the 0.1% Ce6 group at 1 day post PDI. These findings suggest that PDI with Ce6 and red light has a transient efficacy in killing MDR-PA in vivo, and repetitive PDI treatments are required to fully resolve the infection. Before its clinical application, the paradoxical bacterial regrowth post PDI has to be further studied.

Photodynamic inactivation of multidrug-resistant Staphylococcus aureus by chlorin e6 and red light (λ=670nm).

Multidrug-resistant Staphylococcus aureus (MDR-SA) are a frequent cause of antibiotic treatment refractory bacterial corneal infections. Photodynamic therapy (PDT) is being discussed as a putative treatment option to cure this type of bacterial infection. Here we tested the in vitro susceptibility of a set of 12 clinically derived MDR-SA isolates with differing genetic backgrounds and antibiotic resistance profiles against photodynamic inactivation (PDI) by the porphyrin chlorin e6 (Ce6) and red light (λ=670nm). All tested clinical isolates displayed a 5-log10 reduction in viable cells by Ce6 and red light, when cells were preincubated with the photosensitizer at concentrations ≥128µM for 30min in the dark, and a subsequent irradiation with light at λ=670nm (power density: 31mW/cm(2), absorbed dose: 18,6J/cm(2)) was applied. Similarly, cells of the laboratory strain Newman required the same Ce6 pre-incubation and light dose for a 5-log10 reduction in cell viability. Inactivation of crtM in strain Newman, which interferes with pigment production in S. aureus, rendered the mutant more susceptible to this PDT procedure, indicating that the level of resistance of S. aureus to this therapy form is affected by ability of the pathogen to produce the carotenoid pigment staphyloxanthin. Incubation of freshly explanted porcine corneas with a 0.5% Ce6 gel demonstrated that the photosensitizer can diffuse into and accumulate within the stroma of the cornea in concentrations found to be sufficient to yield a 5-log10 reduction of the S. aureus cell pool in vitro. These data suggest that PDI with Ce6 and red light might be a promising new option for the treatment of MDR-SA induced corneal infections.

Pharmacokinetics of the photosensitizers aminolevulinic acid and aminolevulinic acid hexylester in oro-facial tumors embedded in the chorioallantois membrane of a hen's egg.

BACKGROUND: Oral squamous-cell carcinoma is a frequent form of cancer in the head and neck region. The survival rate is poor. Therapy success is highly dependent on the stage of cancer development at which diagnosis is made. The disease is mostly diagnosed at a late stage. Photodynamic diagnosis is a new tool for screening examinations. This technique calls for reliable photosensitizers, such as aminolevulinic acid (ALA) and aminolevulinic acid hexylester (h-ALA). ALA and h-ALA are the source material for the synthesis of protoporphyrin IX in tumor cells. Protoporphyrin IX has a high detection rate for tumor tissue within a reasonable period of time. METHODS: Tumor specimens were harvested from oral carcinomas and basaliomas of the face. The vital cells of the specimens and the human tumor cell line (CLS- 354) were cultured in a 90% RPMI and 10% fetal bovine serum medium. A constant number of 50,000 cells from each specimen and the cell line were transferred to an in vivo model on the hen's egg model. The grown specimens were tested for tumor fluorescence with ALA and h-ALA. The intensity of tumor fluorescence during the following 24 hours was measured spectroscopically as the degree of concentration of protoporphyrin IX within the cells. RESULTS: All tumors showed higher protoporphyrin IX enrichment and fluorescence, compared to healthy tissue. Using h-ALA, the peak concentration of protoporphyrin IX was achieved 20%-25% more quickly with 3- or 6-mM solutions than with ALA. The highest contrast between tumorous and healthy tissue achieved owing to fluorescence was 1:11 using h-ALA, compared to 1:5 using ALA with the peak concentrations of protoporphyrin IX. CONCLUSIONS: Using h-ALA, the peak concentration of protoporphyrin IX, compared to ALA, is achieved 20% percent more quickly and with twice as much contrast between tumorous and healthy tissue (1:11 compared and 1:5, respectively). This facilitates a faster, better discrimination between tumorous and healthy tissue.