**8. Photodynamic therapy —** *In vitro* **tests in species which cause Tegumentary Leishmaniasis**

The effectiveness of PDT on CL treatment was first conducted in humans and in animal models. *In vitro* tests began less than ten years ago to allow testing of PDT parameters like the efficiency of different types of photosensitizers, their respective uptakes and concentrations and accumulation sites.

Sujoy Dutta *et al*. began *in vitro* studies with the New World specie, *L. amazonensis* in 2005 [69]. The first part of that work evaluated *Leishmania* transfectants expressing GFPs. The PS tested was aluminum phthalocyanine chloride (AlPhCl) in different concentrations. The principal factor tested was the light-mediated cytolysis when cells were in the presence or pre-incubated with the AlPhCl. In the dark there was no phototoxicity for both promastigote and amastigote forms of the parasite. The opposite effect occurred when the photosensitizer received red light illumination, showing that promastigotes appear to be more sensible than amastigote forms. In addition, the loss of fluorescence of the GFP parasites indicated cell death. On the second part of the study, J774 cells (cell line immortalized murine Balb⁄ c monocyte ⁄ macrophage) were tested at the same conditions reported above. The authors observed that they were 10-20 fold more resistant than promatigotes. According to the authors, the photosensitized *Leishma‐ nia* cells are susceptible to cytolysis, probably due to the generation of reactive oxidative species after illumination, an indicative of inefficiency of their antioxidant mechanisms. ALA did not induce protoporphyrin IX (PpIX) production in the *Leishmania* cells, because of a deficiency in the heme biosynthetic pathway in this parasite [57, 70].

Tests with other phthalocyanines were developed by Pinto *et al.* using species of Old and New world *Leishmanias*, *L. major* and *L. braziliensis*. The parasites were incubated with aluminum phthalocyanine tetrasulfonate (AlPcS4) at different concentrations and irradiated with a GaAlAs diode laser (λ= 659 nm, 40 mW). The experiments indicated a significant reduction of viable parasites in both species compared to controls, however *L. braziliensis* demonstrated higher mortality than *L. major* [71].

In Brazil, Song *et al*. performed tests to understand mechanism of action of PDT using MB in a case report. Promastigotes of *L. amazonensis* were incubated with different concentrations of MB, washed with PBS and illuminated using a home-built LED light source with a wavelength of maximum emission at λ= 650 nm. After the irradiation, cell survival was determined with MTT assay that detected cell toxicity after irradiation of light in the presence MB. There was an increase in the phototoxicity with the increase in the MB concentration indicating a concentration-dependent response [27]. Differently from PPIX induced by ALA and MAL, MB has parasite intracellular target. In fact, PS seems to be localized in mitochondria (Figure 4).

This brief account of the use of PDT for the treatment of CL demonstrates the ability of this therapeutic modality and encourages its use. It also stimulates research in the pur‐ suit of new protocols with new PS, which could ensure not only healing but also clinical

Details of parameters from scientific studies using PDT on the treatment of Old World and

The effectiveness of PDT on CL treatment was first conducted in humans and in animal models. *In vitro* tests began less than ten years ago to allow testing of PDT parameters like the efficiency of different types of photosensitizers, their respective uptakes and concentrations and

Sujoy Dutta *et al*. began *in vitro* studies with the New World specie, *L. amazonensis* in 2005 [69]. The first part of that work evaluated *Leishmania* transfectants expressing GFPs. The PS tested was aluminum phthalocyanine chloride (AlPhCl) in different concentrations. The principal factor tested was the light-mediated cytolysis when cells were in the presence or pre-incubated with the AlPhCl. In the dark there was no phototoxicity for both promastigote and amastigote forms of the parasite. The opposite effect occurred when the photosensitizer received red light illumination, showing that promastigotes appear to be more sensible than amastigote forms. In addition, the loss of fluorescence of the GFP parasites indicated cell death. On the second part of the study, J774 cells (cell line immortalized murine Balb⁄ c monocyte ⁄ macrophage) were tested at the same conditions reported above. The authors observed that they were 10-20 fold more resistant than promatigotes. According to the authors, the photosensitized *Leishma‐ nia* cells are susceptible to cytolysis, probably due to the generation of reactive oxidative species after illumination, an indicative of inefficiency of their antioxidant mechanisms. ALA did not induce protoporphyrin IX (PpIX) production in the *Leishmania* cells, because of a deficiency in

Tests with other phthalocyanines were developed by Pinto *et al.* using species of Old and New world *Leishmanias*, *L. major* and *L. braziliensis*. The parasites were incubated with aluminum phthalocyanine tetrasulfonate (AlPcS4) at different concentrations and irradiated with a GaAlAs diode laser (λ= 659 nm, 40 mW). The experiments indicated a significant reduction of viable parasites in both species compared to controls, however *L. braziliensis* demonstrated

In Brazil, Song *et al*. performed tests to understand mechanism of action of PDT using MB in a case report. Promastigotes of *L. amazonensis* were incubated with different concentrations of MB, washed with PBS and illuminated using a home-built LED light source with a wavelength of maximum emission at λ= 650 nm. After the irradiation, cell survival was determined with MTT assay that detected cell toxicity after irradiation of light in the presence MB. There was

New World tegumentary leishmaniasis in humans are described in Table 2.

**8. Photodynamic therapy —** *In vitro* **tests in species which cause**

and parasitological cure of these patients.

404 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

**Tegumentary Leishmaniasis**

the heme biosynthetic pathway in this parasite [57, 70].

higher mortality than *L. major* [71].

accumulation sites.

**Figure 4.** Top: Rhodamine 123 and Methylene blue fluorescences in promastigote parasites of *L. amazonensis.* Bot‐ tom: transmission image and colocalization of R123, MB and transmission images.

Other researchers have investigated the susceptibility of *L. amazonensis* regarding PDT. In order to verify the lethality of phenothiazine's derivatives on the promastigote forms, Barbosa *et al*[72] tested TBO (toluidine blue O), MB and a TBO/MB solutions. Irradiation was performed with a diode laser (λ= 660 nm, P= 40 mW). They tested different PS incubation time (5 and 60 min) and two energy densities (4.2 and 8.4 J/cm2 ). The results showed a representative decrease on the viability of *L.amazonensis* promastigotes for all treated groups in comparison to their controls. The authors did not find statistical differences between the dyes, but reported that the best result was observed with TBO.

Dutta *et al*. published an article that described the use of a combination of photosensitizers. Uroporphyrin (URO1) and aluminum phthalocyanine chloride (AlPhCl) were used in uroporphyrinogenic mutants of *L*. *amazonensis* (RAT/ BA/ 74 /LV78) 12-1 clone, transfected with pX-alad and p6,5-PBGD [73]. This transfected *Leishmania* is able to absorb ALA and turns it into URO 1. The authors evaluated the combination of both drugs into promastigotes with and without irradiation of red light. Results showed photolysis of the irradiated parasites with both photosensitizers whereas non-irradiated parasites showed no damage.

Hernández *et al*. published another study that compared encapsulated chloroaluminum phthalocyanine (CLAlPc) in liposomes (UDL-CLAlPc) and free in solution. The experiments were conducted with two species of New World *Leishmania* in promastigote and amastigote forms and in THP1 cells. The experiments tried to verify the ability of the photosensitizer in reaching the *Leishmania* inside THP1 host cell. According to the authors, the UDL-ClAlPc photosensitizer was almost 10 times more photoactive than free ClAlPc on THP-1 cells as well as on promastigotes and with intracellular amastigotes of *L. chagasi* and *L. panamensis* [74].
