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Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes—In vitro study
Accepted Manuscript Title: Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes − in vitro study Author: Juliana Guerra Pinto Andr´e Henrique Correia Pereira Marco Antonio de Oliveira Cristina Kurachi Leandro Jos´e Raniero Juliana Ferreira-Strixino PII: DOI: Reference:
S1572-1000(16)30043-6 http://dx.doi.org/doi:10.1016/j.pdpdt.2016.04.014 PDPDT 771
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
20-12-2015 12-4-2016 22-4-2016
Please cite this article as: Pinto Juliana Guerra, Pereira Andr´e Henrique Correia, de Oliveira Marco Antonio, Kurachi Cristina, Raniero Leandro Jos´e, Ferreira-Strixino Juliana.Chlorin E6 phototoxicity in L.major and L.braziliensis promastigotes minus in vitro study.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2016.04.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes - in vitro study Juliana Guerra Pintoa*[email protected], André Henrique Correia Pereiraa, Marco Antonio de Oliveirab, Cristina Kurachic, Leandro José Ranierod, Juliana Ferreira-Strixinoa a
Laboratório de Terapia Fotodinâmica – Instituto de Pesquisa e desenvolvimento. Univap –
Universidade do Vale do Paraíba. Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, SP, Brazil b
Laboratório de Parasitologia e Biotecnologia - Instituto de Pesquisa e desenvolvimento. Univap –
Universidade do Vale do Paraíba. Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, SP, Brazil c
Laboratório de Biofotônica – Instituto de Física. USP - Universidade de São Paulo. USP São Carlos -
Campus 1 - Avenida Trabalhador São-Carlense, 400 - Parque Arnold Schimidt, São Carlos - SP, 13566 d
Laboratório de Nanossensores - Instituto de Pesquisa e desenvolvimento. Univap – Universidade do
Vale do Paraíba. Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, SP, Brazil
Highlights
• The chlorin is located in the cytosol and seems to have affinity to flagellar membrane.
• PDT with chlorin was decrease viability and the affect parasites morphology. • Both species of Leishmania had the morphology affected by the PDT.
Abstract Background: Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. Conventional treatments are long and aggressive, and they trigger a diversity of side effects. Photodynamic Therapy was originally proposed as an alternative treatment for cancer, and it appears to be a promising alternative therapy for local treatment with fewer side effects. Methods: This study aimed to evaluate Chlorin e6 internalization by Leishmania major and Leishmania braziliensis promastigotes and its viability and effects on mitochondrial activity. Control groups were kept in the dark, while PDT groups received a fluency of 10 J/cm² (660 nm). Chlorin internalization was evaluated using confocal microscopy after one hour of incubation for both species. Results: The mitochondrial activity was evaluated by MTT assay, and viability was measured by the Trypan blue exclusion test. Giemsa staining was used to observe morphological changes. PS was internalized in both species and mitochondrial activity changed in all groups. However, the obtained MTT and Trypan results indicated that despite the change in mitochondrial activity in the dark groups, their viability was not affected, whereas the PDT treated groups had significantly reduced viability. Morphology was drastically altered in PDT treated groups, while groups kept in the dark exhibited the standard morphology. Conclusions: This study demonstrates that Chlorin has great potential for being used in PDT as a treatment for cutaneous leishmaniasis, although more studies are needed to determine in vivo application protocols. Keywords Leishmania major, Leishmania braziliensis, Chlorin e6, Photodynamic therapy
Introduction Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. The protozoan parasites mononuclear phagocyte system cells (MPS) and begins to multiply inside the phagocytes, installing an infectious process [1,2]. It is considered by the World Health Organization (WHO) as a public health problem, present in over 80 countries [3]. The biological cycle of the disease has two evolutionary forms, the amastigote that resides in interior of MPS cells, and promastigote found inside the insect vector [4]. The first line treatment for cutaneous leishmaniasis is pentavalent antimony, followed by pentamidines, and the antibiotics Amphotericin B and Rifanpicine. The choice of drug or combination of more than one line of treatment is in accordance with the patent’s response to treatment [5,6]. Although conventional treatment is effective if conducted properly, the long treatment, the need for patient hospitalization and severe side effects reinforce the evident need for alternative therapies, with less systemic impact for the patient. In such context, Photodynamic therapy (PDT) arises as an alternative therapy. PDT requires the interaction of a photosensitizer (PS), light at the PS absorption wavelength, and tissue oxygen, to generate reactive oxygen species that induces cell death[7,8]. Initially proposed as a cancer therapy, PDT has been widely studied for dermatological treatments, keratoses, and microbiological control [9–11]. Although the use of PDT in cutaneous leishmaniasis treatment has been widely studied, the parameters are varied. The photosensitizers used are varieties of phthalocyanines, protoporphyrin, uroporphyrins, precursors such as aminolevulinic acid and its derivatives, and fluencies used also vary from 0.8 J/cm² to 100 J/cm² [12–15]. These studies validate PDT as a promising treatment for cutaneous leishmaniasis, but also expose the need for protocol standardization before in vivo applications. Neverthelses, the search remains for higher efficiency photosensitizers, which would have greater impact on parasites viability, thereby, increasing performance of the overall cutaneous leishmaniasis treatment. This study evaluated Chlorine e6 internalization by promastigotas of Leishmania major and Leishmania braziliensis as well as its viability and effects on mitochondrial activity after PDT.
Materials and methods Parasites culture The Leishmania major strain was kindly provided by Dr. Ângela K. Cruz (Universidade de São Paulo) and the Leishmania braziliensis strain by Dr. Mario Steindel (Universidade de Santa Catarina,
Departamento de Microbiologia e Parasitologia). Both strains were maintained in LIT medium (Liver infusion tryptose, Sigma ®) supplemented with Fetal Bovine Serum (Sigma®), 2.5 µg/ml hemin (Sigma®), and maintained at 28 °C. The strains were subcultured weekly by transferring an aliquot (10%) in fresh supplemented medium. Chlorin e6 Chlorin e6 was provided by Biophotonics laboratory - Institute of Physics, USP - São Carlos [16]. It was first diluted in DMSO (dimethyl sulfoxide – Sigma®), 5 % in PBS (Phosphate Buffer Solution – Sigma®) in stock solution at 1.0 mg/ml. Assay concentrations were applied in serial dilutions
(1:2) starting from 400 µg/ml up to 6.25 µg /ml, diluted in PBS (Sigma®) in a final DMSO concentration of 0,2%. Internalization of Chlorin e6 into L. major and L. braziliensis promastigotes. In order to verify internalization of Chlorin into both promastigotes species, they were incubated with Chlorin E6 at the concentration of 125 µg /mL. After one-hour incubation, the medium with the PS was removed. The incubation time was previously determined by the cytotoxic tests. The assay with confocal microscopy demonstrated that after one-hour incubation the Chlorin was internalized by the promastigotes. The concentration of 125 µg /ml was chosen because the purpose of the experiment was to verify the Chlorin endocitose, and higher concentrations lead to an accumulation that prevents determination of PS location and overrides the DAPI (Sigma®) signal. Parasites were fixed with 4% PBS diluted paraformaldehyde and then adhered on circular coverslips previously treated with poly-1lysine. Coverslips were prepared with Prolong Gold antifading, containing DAPI (4',6-diamidino-2phenylindole, dihydrochloride) for DNA labeling. All processing was done in the dark, and the slides were examined under confocal microscope Zeiss LSM 700®. The excitation laser was tuned to 488 nm for Chlorin e6, with emission over 500 nm, and tuned to 358 nm for DAPI, with emission at 461 nm.
Chlorin e6 Incubation Protocol All tests were performed in triplicate with promastigotes at the stationary phase. The experimental groups were divided into "dark" (control, wherein all chlorin concentrations were tested and not irradiated) and "PDT" (irradiated control named LED and PDT, which was tested at all chlorin concentrations). PDT groups received fluency of 10 J/cm² from a Biotable LED (Biopdi, 450 nm). Serial dilutions (1:2) were applied from 400 µg/mL to 6.25 µg/mL. Promastigotes were counted and subsequently transferred to 96-well plates at 1x106 parasites per well concentration, along with chlorin or culture medium (control groups). After one hour of incubation with the PS, the medium was removed and replace for fresh medium before irradiation. The incubation time was the same for the dark controls and for the PDT treatments. For the mitochondrial activity, Trypan blue exclusion method and Morphological analysis were performed eighteen hours after treatments. The same period was applied to the dark controls. Mitochondrial Activity Mitochondrial activity was assessed through MTT test, which consists in the MTT salt ([3- (4,5dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide]) degradation by the mitochondria forming Formazan crystals. Eighteen hours after the treatments, as described in the incubation protocol above, 50 µL of MTT solution (5 mg/ml) were added to the dark and irradiated groups, followed by 4 hours incubation at 28 ºC. After this time, the medium and MTT solution were removed, and 200 µL of DMSO added to dissolve the formed crystals. Absorbance was measured using a Microplate Spectrophotometer (BioTek® Synergy HT) with a 570 nm filter. Cellular viability analysis of parasites through the Trypan Blue exclusion method The viability test in concern, Trypan Blue exclusion, assumes that non-viable cells are stained while cells with intact membrane function remain discolored. For the Trypan Blue exclusion method, eighteen hours after the treatments, as described in the incubation protocol above, 10 µl of parasites were added to 90 uL of Trypan Blue (Sigma®). After gently mixing, an aliquot of 20 ul was transferred to a Neubauer chamber, and the number of living and dead parasites were counted with a Leica® microscope (Leica DM 2500). Morphological analysis – staining The same conditions used to the MTT and Trypan blue test were applied to morphological analysis. Eighteen hours after treatments, one aliquot of each treatment group was taken, and smears were made
from them. The smears were dried for a period of 12 hours and stained. For Giemsa staining, the samples were initially covered with May Grunwald for a minute. Then the same volume of water was added and removed after a minute. Giemsa was subsequently added. Samples were diluted in phosphate buffer and kept for 15 minutes. Then, the excess dye was removed, coverslips were rinsed in water, dried, and the permanent slides were assembled for microscopic morphology analysis. Statistical analysis: All tests were performed in three independent experiments, each one in triplicate. MTT and Trypan blue data were submitted to One - way ANOVA test using the BioEstat 5.0 software.
Results Chlorin e6 internalization After one hour incubation with chlorin at 125 µg /mL concentration, an accumulation was observed in the cytosol, forming agglomerations, possibly endocytic vesicles. Both species, L. major (Figure 1) and L. braziliensis (Figure 2), exhibited diffuse accumulation in the cytosol and small points of PS accumulation. An accumulation of chlorin in the scourges of L. braziliensis was also noted (Figure 2).
Mitochondrial activity evaluation and exclusion with Blue Trypan after Chlorin E6 treatment Mitochondrial activity evaluation of L. major and L. braziliensis, after interaction with Chlorin e6, in the dark, showed similar patterns for both species. There was a reduction in activity compared to the control group (Figure 3A), wherein L. braziliensis showed lower values than L. major. After PDT, however, mitochondrial activity values were even lower than the in the dark values (Figure 3B), for both strains, which showed less than 10% of mitochondrial activity.
Trypan test revealed, although the mitochondrial activity of the groups treated with Chlorin e6 was affected in the dark, this reduction did not directly affect parasites viability (Figure 3C). After PDT application, viability was affected in all groups. The most efficient concentrations for L. major were of 400 µg/mL, 200 µg /mL, 100 µg /mL, 50 µg /mL, and 25 µg /mL (Figure 3D), while lower concentrations did not show the same outcome. The most effective concentrations for L. braziliensis were of 400 µg /mL, which led to 100% death, and then 200 µg /mL, 100 µg /mL, 50 µg /mL. Morphological analysis after Chlorin E6 treatment Morphological changes were observed in both species after PDT treatment. L. major species presented its characteristic morphology in the control (Figure 4A) and irradiated (Figure 4I) groups, as well as in groups treated with chlorin and kept in the dark (Figure 4B to 4H). After PDT, a significant change in morphology was observed in L. major (Figure 4D to 4J), at all concentrations tested. L. braziliensis also had morphology affected after PDT with Chlorin e6 (Figure 5J to 5P) in all tested concentrations. At higher concentrations, the changes were even more significant and the complete loss of spindle shape was detected at 400 µg/ml concentration (Figure 5J). The dark control group (Figure 5A), LED control (Figure 5I), and all groups treated with Chlorin E6 and kept in the dark showed characteristic morphology. Discussion This study demonstrated that Chlorin E6 effected viability and mitochondrial activity of L. major and L. braziliensis promastigotes. In addition, Chlorin E6 remained inside the parasites after an hour of incubation. Gardner and colleagues [17], using PDT with acenaphthene porphyrins in L. tarentolae and L. panamensis, noted the affinity of the drug for cell membranes, and the fact that employing acenaphthene porphyrin liposomes enhanced the drug internalization, compared to the free drug. Dutta and colleagues [15] conducted in vitro studies with different phthalocyanines in L. amazonensis for the purpose of describing drug internalization. They observed that different phthalocyanine compositions bring about different intracellular localization and accumulation patterns, as well as therapy effect. In another study, Dutta and colleagues [14] performed PDT, with uroporphyrin Chlorin and aluminum phthalocyanine in L. amazonenis, and correlated phase-contrast and fluorescence microscopy techniques. They observed that phthalocyanines have higher affinity for cell membranes while uroporphyrins were found diffused in the cytosol, accumulated in the scourge and endocytic
vacuoles. Chlorin was present in the cytosol, accumulated in apparent endocytic vesicles, and showed an affinity for the flagellar membrane. Both species have similar response pattern to the mitochondrial activity test, in dark and PDT groups. However, Trypan Blue tests showed that the chlorin exposure was not toxic in the dark, while PDT treatment was capable of significantly reducing viability. Several studies have used MTT assays to determine the percentage of viable cells; however, it is necessary to take into consideration that MTT test does not always provide accurate information on cell viability [8,14,15,17–20]. Taylor et al. [20] also used MTT test to determine the viability of diethyl and dimethyl carbaporphyrin. They observed the toxicity of the compounds in the dark, but the evaluation was made only through enzymatic activity. A decrease in mitochondrial activity does not necessarily mean a reduction of viability, as was demonstrated in this study. For example, the interaction between promastigotes and chlorin, in the dark, caused a meaningful decrease in mitochondrial activity in all tested concentrations, but associating Tripan test data, found that there was no mortality in these groups. Thus, we can infer that the test with trypan blue provides reliable information to assess the effectiveness of in vitro treatment, because it is a vital dye. Unviable cells are stained in blue, while cells whose membrane is intact are not stained. Pinto et al. [21] performed a PDT study with Aluminium tetrasulfonated phthalocyanine and evaluated it with the trypan blue test. They found the treatment was able to reduce the viability, and verified L. braziliensis was more receptive to treatment than L. major. In this study, PDT with a concentration of 400 ug/mL, reduced the viability of L. braziliensis by 100%, while L. major showed 90% reduced viability, which demonstrates an increased susceptibility of L. braziliensis with this treatment protocol. Morphological alterations were observed in all PDT treated groups, with the most expressive changes occurring in treatments at the higher concentrations. Moreover, the control group in the dark, at all concentrations, showed no significant morphological change. Hernandez et al. [18] also pointed out morphological changes in amastigotes after PDT treatment with aluminum phthalocyanine chloride. Regardless of amastigotes being internalized by macrophages, PDT was able to cause changes in the parasite. In the current study, after PDT with chlorin was performed, loss of the parasites fusiform shape was observed, nucleus and kinetoplast were not always visible, and in some cases the scourge was absent. L. major species presented large morphological changes after PDT in higher concentrations, exhibiting rounded parasites, and loss of fusiform shape. L. braziliensis also showed expressive morphological changes after PDT, particularly at the three highest concentrations. Morphologic trypanosomatid studies have shown that below the Leishmania cell membrane there is a structure composed of microtubules, which ensures the parasite’s characteristic shape and limits molecules internalization, which mostly occurs through the flagellar pocket [22,23]. The morphological changes demonstrated in this study may be related to this microtubules layer, which can be somehow affected by PDT with chlorin. Conclusion The results of this study indicate that PDT may be a substitute treatment for cutaneous leishmaniasis. Chlorin remains internalized by all tested Leishmania species, passed one hour of incubation. Mitochondrial activity was found to be affected by interaction with chlorin, in the dark, and after PDT. However, changes in mitochondrial activity of incubated groups with the chlorin in the dark did not lead to cellular death. Only the groups treated with PDT had their viability affected. Further tests with infected macrophages are still needed, in order to stipulate protocols for future in vivo experiments. Acknowledgements This work was supported by Fundação de amparo à pesquisa do Estado de São Paulo – FAPESP (2013/12284-3) and Fundação Valeparaibana de Ensino. We also thank Alene AlderRangel for reviewing the English in the manuscript. References [1]
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Figure 1: Location of chlorin inside L. major after one-hour incubation. (A) Nucleus (Arrow head) and kinetoplast (arrow) evidenced by marking with DAPI; (B) Fluorescence of Chlorin E6 in the cytosol; (C) Overlapping signals from DAPI and Chlorin E6;
Figure 2: Location of chlorin inside L. braziliensis: (A) Nucleus (Arrow head) and kinetoplast (arrow) evidenced by marking with DAPI; (B) Fluorescence of chlorin in the cytosol; (C) Overlapping signals from DAPI and chlorin;
Figure 3: (A) Percentage of mitochondrial activity for both species, after Chlorin E6 incubation in the dark. (B) Percentage of mitochondrial activity of both species, after PDT with Chlorin e6, and the control group treated with a LED fluency of 10 J/cm². (C) Promastigotes viability of both species determined by the trypan blue exclusion test, and the control group treated with Chlorin e6 kept in the dark. (D) Viability of promastigotes given by Trypan blue test for both species after PDT with Chlorin E6, and the control group treated only with a LED fluence of 10 J/cm² (• p <0.01) (* p <0.01)(° p<0,05).
Figure 4: L. major morphology in all the experimental groups after Chlorin E6 treatment and controls. A- control in the dark, B- 400 µg/ml in the dark, C- 200 µg/ml in the dark, D- 100 µg/ml in the dark, E- 50 µg/ml in the dark, F- 25 µg/ml in the dark, G- 12.5 µg/ml in the dark, H- 6.25 µg/ml in the dark, I- LED (10 J/cm²), J- PDT 400 µg/ml PDT, K- 200 µg/ml, L- PDT 100 µg/ml, M- PDT 50 µg/ml, N-PDT 25 µg/ml, O- PDT 12.5 µg/ml, and P- PDT 6.25 µg/ml.
Figure 5: L. braziliensis morphology in all the experimental groups after Chlorin E6 treatment and controls. A- control in the dark, B- 400 µg/mL in the dark, C- 200 µg/mL in the dark, D- 100 µg/mL in the dark, E- 50 µg/mL in the dark, F- 25 µg/mL in the dark, G- 12.5 µg/mL in the dark, H- 6.25 µg/mL in the dark, I- LED (10J / cm²), J- PDT 400 µg/mL, K- PDT 200 µg/mL, L- PDT 100 µg/mL, M- PDT 50 µg/ml, N- PDT 25 µg/mL, O- PDT 12.5 µg/mL, and P- PDT 6.25 µg/mL.
S1572-1000(16)30043-6 http://dx.doi.org/doi:10.1016/j.pdpdt.2016.04.014 PDPDT 771
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
20-12-2015 12-4-2016 22-4-2016
Please cite this article as: Pinto Juliana Guerra, Pereira Andr´e Henrique Correia, de Oliveira Marco Antonio, Kurachi Cristina, Raniero Leandro Jos´e, Ferreira-Strixino Juliana.Chlorin E6 phototoxicity in L.major and L.braziliensis promastigotes minus in vitro study.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2016.04.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chlorin E6 phototoxicity in L. major and L. braziliensis promastigotes - in vitro study Juliana Guerra Pintoa*[email protected], André Henrique Correia Pereiraa, Marco Antonio de Oliveirab, Cristina Kurachic, Leandro José Ranierod, Juliana Ferreira-Strixinoa a
Laboratório de Terapia Fotodinâmica – Instituto de Pesquisa e desenvolvimento. Univap –
Universidade do Vale do Paraíba. Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, SP, Brazil b
Laboratório de Parasitologia e Biotecnologia - Instituto de Pesquisa e desenvolvimento. Univap –
Universidade do Vale do Paraíba. Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, SP, Brazil c
Laboratório de Biofotônica – Instituto de Física. USP - Universidade de São Paulo. USP São Carlos -
Campus 1 - Avenida Trabalhador São-Carlense, 400 - Parque Arnold Schimidt, São Carlos - SP, 13566 d
Laboratório de Nanossensores - Instituto de Pesquisa e desenvolvimento. Univap – Universidade do
Vale do Paraíba. Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, SP, Brazil
Highlights
• The chlorin is located in the cytosol and seems to have affinity to flagellar membrane.
• PDT with chlorin was decrease viability and the affect parasites morphology. • Both species of Leishmania had the morphology affected by the PDT.
Abstract Background: Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. Conventional treatments are long and aggressive, and they trigger a diversity of side effects. Photodynamic Therapy was originally proposed as an alternative treatment for cancer, and it appears to be a promising alternative therapy for local treatment with fewer side effects. Methods: This study aimed to evaluate Chlorin e6 internalization by Leishmania major and Leishmania braziliensis promastigotes and its viability and effects on mitochondrial activity. Control groups were kept in the dark, while PDT groups received a fluency of 10 J/cm² (660 nm). Chlorin internalization was evaluated using confocal microscopy after one hour of incubation for both species. Results: The mitochondrial activity was evaluated by MTT assay, and viability was measured by the Trypan blue exclusion test. Giemsa staining was used to observe morphological changes. PS was internalized in both species and mitochondrial activity changed in all groups. However, the obtained MTT and Trypan results indicated that despite the change in mitochondrial activity in the dark groups, their viability was not affected, whereas the PDT treated groups had significantly reduced viability. Morphology was drastically altered in PDT treated groups, while groups kept in the dark exhibited the standard morphology. Conclusions: This study demonstrates that Chlorin has great potential for being used in PDT as a treatment for cutaneous leishmaniasis, although more studies are needed to determine in vivo application protocols. Keywords Leishmania major, Leishmania braziliensis, Chlorin e6, Photodynamic therapy
Introduction Cutaneous leishmaniasis is a zoonosis caused by protozoa of the genus Leishmania. The protozoan parasites mononuclear phagocyte system cells (MPS) and begins to multiply inside the phagocytes, installing an infectious process [1,2]. It is considered by the World Health Organization (WHO) as a public health problem, present in over 80 countries [3]. The biological cycle of the disease has two evolutionary forms, the amastigote that resides in interior of MPS cells, and promastigote found inside the insect vector [4]. The first line treatment for cutaneous leishmaniasis is pentavalent antimony, followed by pentamidines, and the antibiotics Amphotericin B and Rifanpicine. The choice of drug or combination of more than one line of treatment is in accordance with the patent’s response to treatment [5,6]. Although conventional treatment is effective if conducted properly, the long treatment, the need for patient hospitalization and severe side effects reinforce the evident need for alternative therapies, with less systemic impact for the patient. In such context, Photodynamic therapy (PDT) arises as an alternative therapy. PDT requires the interaction of a photosensitizer (PS), light at the PS absorption wavelength, and tissue oxygen, to generate reactive oxygen species that induces cell death[7,8]. Initially proposed as a cancer therapy, PDT has been widely studied for dermatological treatments, keratoses, and microbiological control [9–11]. Although the use of PDT in cutaneous leishmaniasis treatment has been widely studied, the parameters are varied. The photosensitizers used are varieties of phthalocyanines, protoporphyrin, uroporphyrins, precursors such as aminolevulinic acid and its derivatives, and fluencies used also vary from 0.8 J/cm² to 100 J/cm² [12–15]. These studies validate PDT as a promising treatment for cutaneous leishmaniasis, but also expose the need for protocol standardization before in vivo applications. Neverthelses, the search remains for higher efficiency photosensitizers, which would have greater impact on parasites viability, thereby, increasing performance of the overall cutaneous leishmaniasis treatment. This study evaluated Chlorine e6 internalization by promastigotas of Leishmania major and Leishmania braziliensis as well as its viability and effects on mitochondrial activity after PDT.
Materials and methods Parasites culture The Leishmania major strain was kindly provided by Dr. Ângela K. Cruz (Universidade de São Paulo) and the Leishmania braziliensis strain by Dr. Mario Steindel (Universidade de Santa Catarina,
Departamento de Microbiologia e Parasitologia). Both strains were maintained in LIT medium (Liver infusion tryptose, Sigma ®) supplemented with Fetal Bovine Serum (Sigma®), 2.5 µg/ml hemin (Sigma®), and maintained at 28 °C. The strains were subcultured weekly by transferring an aliquot (10%) in fresh supplemented medium. Chlorin e6 Chlorin e6 was provided by Biophotonics laboratory - Institute of Physics, USP - São Carlos [16]. It was first diluted in DMSO (dimethyl sulfoxide – Sigma®), 5 % in PBS (Phosphate Buffer Solution – Sigma®) in stock solution at 1.0 mg/ml. Assay concentrations were applied in serial dilutions
(1:2) starting from 400 µg/ml up to 6.25 µg /ml, diluted in PBS (Sigma®) in a final DMSO concentration of 0,2%. Internalization of Chlorin e6 into L. major and L. braziliensis promastigotes. In order to verify internalization of Chlorin into both promastigotes species, they were incubated with Chlorin E6 at the concentration of 125 µg /mL. After one-hour incubation, the medium with the PS was removed. The incubation time was previously determined by the cytotoxic tests. The assay with confocal microscopy demonstrated that after one-hour incubation the Chlorin was internalized by the promastigotes. The concentration of 125 µg /ml was chosen because the purpose of the experiment was to verify the Chlorin endocitose, and higher concentrations lead to an accumulation that prevents determination of PS location and overrides the DAPI (Sigma®) signal. Parasites were fixed with 4% PBS diluted paraformaldehyde and then adhered on circular coverslips previously treated with poly-1lysine. Coverslips were prepared with Prolong Gold antifading, containing DAPI (4',6-diamidino-2phenylindole, dihydrochloride) for DNA labeling. All processing was done in the dark, and the slides were examined under confocal microscope Zeiss LSM 700®. The excitation laser was tuned to 488 nm for Chlorin e6, with emission over 500 nm, and tuned to 358 nm for DAPI, with emission at 461 nm.
Chlorin e6 Incubation Protocol All tests were performed in triplicate with promastigotes at the stationary phase. The experimental groups were divided into "dark" (control, wherein all chlorin concentrations were tested and not irradiated) and "PDT" (irradiated control named LED and PDT, which was tested at all chlorin concentrations). PDT groups received fluency of 10 J/cm² from a Biotable LED (Biopdi, 450 nm). Serial dilutions (1:2) were applied from 400 µg/mL to 6.25 µg/mL. Promastigotes were counted and subsequently transferred to 96-well plates at 1x106 parasites per well concentration, along with chlorin or culture medium (control groups). After one hour of incubation with the PS, the medium was removed and replace for fresh medium before irradiation. The incubation time was the same for the dark controls and for the PDT treatments. For the mitochondrial activity, Trypan blue exclusion method and Morphological analysis were performed eighteen hours after treatments. The same period was applied to the dark controls. Mitochondrial Activity Mitochondrial activity was assessed through MTT test, which consists in the MTT salt ([3- (4,5dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide]) degradation by the mitochondria forming Formazan crystals. Eighteen hours after the treatments, as described in the incubation protocol above, 50 µL of MTT solution (5 mg/ml) were added to the dark and irradiated groups, followed by 4 hours incubation at 28 ºC. After this time, the medium and MTT solution were removed, and 200 µL of DMSO added to dissolve the formed crystals. Absorbance was measured using a Microplate Spectrophotometer (BioTek® Synergy HT) with a 570 nm filter. Cellular viability analysis of parasites through the Trypan Blue exclusion method The viability test in concern, Trypan Blue exclusion, assumes that non-viable cells are stained while cells with intact membrane function remain discolored. For the Trypan Blue exclusion method, eighteen hours after the treatments, as described in the incubation protocol above, 10 µl of parasites were added to 90 uL of Trypan Blue (Sigma®). After gently mixing, an aliquot of 20 ul was transferred to a Neubauer chamber, and the number of living and dead parasites were counted with a Leica® microscope (Leica DM 2500). Morphological analysis – staining The same conditions used to the MTT and Trypan blue test were applied to morphological analysis. Eighteen hours after treatments, one aliquot of each treatment group was taken, and smears were made
from them. The smears were dried for a period of 12 hours and stained. For Giemsa staining, the samples were initially covered with May Grunwald for a minute. Then the same volume of water was added and removed after a minute. Giemsa was subsequently added. Samples were diluted in phosphate buffer and kept for 15 minutes. Then, the excess dye was removed, coverslips were rinsed in water, dried, and the permanent slides were assembled for microscopic morphology analysis. Statistical analysis: All tests were performed in three independent experiments, each one in triplicate. MTT and Trypan blue data were submitted to One - way ANOVA test using the BioEstat 5.0 software.
Results Chlorin e6 internalization After one hour incubation with chlorin at 125 µg /mL concentration, an accumulation was observed in the cytosol, forming agglomerations, possibly endocytic vesicles. Both species, L. major (Figure 1) and L. braziliensis (Figure 2), exhibited diffuse accumulation in the cytosol and small points of PS accumulation. An accumulation of chlorin in the scourges of L. braziliensis was also noted (Figure 2).
Mitochondrial activity evaluation and exclusion with Blue Trypan after Chlorin E6 treatment Mitochondrial activity evaluation of L. major and L. braziliensis, after interaction with Chlorin e6, in the dark, showed similar patterns for both species. There was a reduction in activity compared to the control group (Figure 3A), wherein L. braziliensis showed lower values than L. major. After PDT, however, mitochondrial activity values were even lower than the in the dark values (Figure 3B), for both strains, which showed less than 10% of mitochondrial activity.
Trypan test revealed, although the mitochondrial activity of the groups treated with Chlorin e6 was affected in the dark, this reduction did not directly affect parasites viability (Figure 3C). After PDT application, viability was affected in all groups. The most efficient concentrations for L. major were of 400 µg/mL, 200 µg /mL, 100 µg /mL, 50 µg /mL, and 25 µg /mL (Figure 3D), while lower concentrations did not show the same outcome. The most effective concentrations for L. braziliensis were of 400 µg /mL, which led to 100% death, and then 200 µg /mL, 100 µg /mL, 50 µg /mL. Morphological analysis after Chlorin E6 treatment Morphological changes were observed in both species after PDT treatment. L. major species presented its characteristic morphology in the control (Figure 4A) and irradiated (Figure 4I) groups, as well as in groups treated with chlorin and kept in the dark (Figure 4B to 4H). After PDT, a significant change in morphology was observed in L. major (Figure 4D to 4J), at all concentrations tested. L. braziliensis also had morphology affected after PDT with Chlorin e6 (Figure 5J to 5P) in all tested concentrations. At higher concentrations, the changes were even more significant and the complete loss of spindle shape was detected at 400 µg/ml concentration (Figure 5J). The dark control group (Figure 5A), LED control (Figure 5I), and all groups treated with Chlorin E6 and kept in the dark showed characteristic morphology. Discussion This study demonstrated that Chlorin E6 effected viability and mitochondrial activity of L. major and L. braziliensis promastigotes. In addition, Chlorin E6 remained inside the parasites after an hour of incubation. Gardner and colleagues [17], using PDT with acenaphthene porphyrins in L. tarentolae and L. panamensis, noted the affinity of the drug for cell membranes, and the fact that employing acenaphthene porphyrin liposomes enhanced the drug internalization, compared to the free drug. Dutta and colleagues [15] conducted in vitro studies with different phthalocyanines in L. amazonensis for the purpose of describing drug internalization. They observed that different phthalocyanine compositions bring about different intracellular localization and accumulation patterns, as well as therapy effect. In another study, Dutta and colleagues [14] performed PDT, with uroporphyrin Chlorin and aluminum phthalocyanine in L. amazonenis, and correlated phase-contrast and fluorescence microscopy techniques. They observed that phthalocyanines have higher affinity for cell membranes while uroporphyrins were found diffused in the cytosol, accumulated in the scourge and endocytic
vacuoles. Chlorin was present in the cytosol, accumulated in apparent endocytic vesicles, and showed an affinity for the flagellar membrane. Both species have similar response pattern to the mitochondrial activity test, in dark and PDT groups. However, Trypan Blue tests showed that the chlorin exposure was not toxic in the dark, while PDT treatment was capable of significantly reducing viability. Several studies have used MTT assays to determine the percentage of viable cells; however, it is necessary to take into consideration that MTT test does not always provide accurate information on cell viability [8,14,15,17–20]. Taylor et al. [20] also used MTT test to determine the viability of diethyl and dimethyl carbaporphyrin. They observed the toxicity of the compounds in the dark, but the evaluation was made only through enzymatic activity. A decrease in mitochondrial activity does not necessarily mean a reduction of viability, as was demonstrated in this study. For example, the interaction between promastigotes and chlorin, in the dark, caused a meaningful decrease in mitochondrial activity in all tested concentrations, but associating Tripan test data, found that there was no mortality in these groups. Thus, we can infer that the test with trypan blue provides reliable information to assess the effectiveness of in vitro treatment, because it is a vital dye. Unviable cells are stained in blue, while cells whose membrane is intact are not stained. Pinto et al. [21] performed a PDT study with Aluminium tetrasulfonated phthalocyanine and evaluated it with the trypan blue test. They found the treatment was able to reduce the viability, and verified L. braziliensis was more receptive to treatment than L. major. In this study, PDT with a concentration of 400 ug/mL, reduced the viability of L. braziliensis by 100%, while L. major showed 90% reduced viability, which demonstrates an increased susceptibility of L. braziliensis with this treatment protocol. Morphological alterations were observed in all PDT treated groups, with the most expressive changes occurring in treatments at the higher concentrations. Moreover, the control group in the dark, at all concentrations, showed no significant morphological change. Hernandez et al. [18] also pointed out morphological changes in amastigotes after PDT treatment with aluminum phthalocyanine chloride. Regardless of amastigotes being internalized by macrophages, PDT was able to cause changes in the parasite. In the current study, after PDT with chlorin was performed, loss of the parasites fusiform shape was observed, nucleus and kinetoplast were not always visible, and in some cases the scourge was absent. L. major species presented large morphological changes after PDT in higher concentrations, exhibiting rounded parasites, and loss of fusiform shape. L. braziliensis also showed expressive morphological changes after PDT, particularly at the three highest concentrations. Morphologic trypanosomatid studies have shown that below the Leishmania cell membrane there is a structure composed of microtubules, which ensures the parasite’s characteristic shape and limits molecules internalization, which mostly occurs through the flagellar pocket [22,23]. The morphological changes demonstrated in this study may be related to this microtubules layer, which can be somehow affected by PDT with chlorin. Conclusion The results of this study indicate that PDT may be a substitute treatment for cutaneous leishmaniasis. Chlorin remains internalized by all tested Leishmania species, passed one hour of incubation. Mitochondrial activity was found to be affected by interaction with chlorin, in the dark, and after PDT. However, changes in mitochondrial activity of incubated groups with the chlorin in the dark did not lead to cellular death. Only the groups treated with PDT had their viability affected. Further tests with infected macrophages are still needed, in order to stipulate protocols for future in vivo experiments. Acknowledgements This work was supported by Fundação de amparo à pesquisa do Estado de São Paulo – FAPESP (2013/12284-3) and Fundação Valeparaibana de Ensino. We also thank Alene AlderRangel for reviewing the English in the manuscript. References [1]
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Figure 1: Location of chlorin inside L. major after one-hour incubation. (A) Nucleus (Arrow head) and kinetoplast (arrow) evidenced by marking with DAPI; (B) Fluorescence of Chlorin E6 in the cytosol; (C) Overlapping signals from DAPI and Chlorin E6;
Figure 2: Location of chlorin inside L. braziliensis: (A) Nucleus (Arrow head) and kinetoplast (arrow) evidenced by marking with DAPI; (B) Fluorescence of chlorin in the cytosol; (C) Overlapping signals from DAPI and chlorin;
Figure 3: (A) Percentage of mitochondrial activity for both species, after Chlorin E6 incubation in the dark. (B) Percentage of mitochondrial activity of both species, after PDT with Chlorin e6, and the control group treated with a LED fluency of 10 J/cm². (C) Promastigotes viability of both species determined by the trypan blue exclusion test, and the control group treated with Chlorin e6 kept in the dark. (D) Viability of promastigotes given by Trypan blue test for both species after PDT with Chlorin E6, and the control group treated only with a LED fluence of 10 J/cm² (• p <0.01) (* p <0.01)(° p<0,05).
Figure 4: L. major morphology in all the experimental groups after Chlorin E6 treatment and controls. A- control in the dark, B- 400 µg/ml in the dark, C- 200 µg/ml in the dark, D- 100 µg/ml in the dark, E- 50 µg/ml in the dark, F- 25 µg/ml in the dark, G- 12.5 µg/ml in the dark, H- 6.25 µg/ml in the dark, I- LED (10 J/cm²), J- PDT 400 µg/ml PDT, K- 200 µg/ml, L- PDT 100 µg/ml, M- PDT 50 µg/ml, N-PDT 25 µg/ml, O- PDT 12.5 µg/ml, and P- PDT 6.25 µg/ml.
Figure 5: L. braziliensis morphology in all the experimental groups after Chlorin E6 treatment and controls. A- control in the dark, B- 400 µg/mL in the dark, C- 200 µg/mL in the dark, D- 100 µg/mL in the dark, E- 50 µg/mL in the dark, F- 25 µg/mL in the dark, G- 12.5 µg/mL in the dark, H- 6.25 µg/mL in the dark, I- LED (10J / cm²), J- PDT 400 µg/mL, K- PDT 200 µg/mL, L- PDT 100 µg/mL, M- PDT 50 µg/ml, N- PDT 25 µg/mL, O- PDT 12.5 µg/mL, and P- PDT 6.25 µg/mL.