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Progress in the photodynamic therapy treatment of Leishmaniasis

Abstract

Leishmaniasis is a serious and endemic infectious disease that has been reported in more than 90 countries and territories. The classical treatment presents a series of problems ranging from difficulty in administration, development of resistance, and a series of side effects. Photodynamic therapy (PDT) has already shown great potential for use as a treatment for leishmaniasis that is effective and non-invasive, with very minor side effects. PDT can also be inexpensive and easy to administer. In this review, we will report the most recent developments in the field, starting with the chemical diversity of photosensitizers, highlighting important mechanistic aspects, and noting information that may assist in designing and developing new and promising photosensitizer molecules.

Photodynamic therapy; Photosensitizer; Cutaneous leishmaniasis


Introduction

Leishmaniasis is a group of neglected diseases caused by parasites of the genus Leishmania. More than 20 species are found worldwide, while the main species found in Brazil are Leishmania (Viannia) braziliensis, L. (V.) guyanensis, L. (V.) lainsoni, L. (V) naiffi, L. (V.) lindenberg, L. (V.) shawi, L. (Leishmania) amazonensis, and L. (L.) infantum chagasi. These parasites belong to the Trypanosomatidae family and have two forms: the promastigote (flagellated form found in the vector's digestive tract) and the amastigote (intracellular spherical non-flagellated form lodged in macrophages) (11. Drugs for Neglected Diseases initiative, America Latina. Leishmanioses. Available at <https://www.dndial.org/doencas/leishmanioses/> [accessed March 16, 2021]
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).

Transmission occurs through the bites of female sandflies, and the reservoirs of the parasites are humans, wild rodents, marsupials, and domestic dogs. There are three main forms of the disease: cutaneous, visceral or kala-azar, and mucocutaneous (11. Drugs for Neglected Diseases initiative, America Latina. Leishmanioses. Available at <https://www.dndial.org/doencas/leishmanioses/> [accessed March 16, 2021]
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).

It is estimated that one billion people live in endemic areas, and more than 80 economically vulnerable countries are affected by visceral leishmaniasis (VL). With one million new cases of cutaneous leishmaniasis (CL) annually, the most affected countries are Brazil, India, Ethiopia, Kenya, South Sudan, Somalia, and Sudan (11. Drugs for Neglected Diseases initiative, America Latina. Leishmanioses. Available at <https://www.dndial.org/doencas/leishmanioses/> [accessed March 16, 2021]
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).

The main symptoms of CL include single or multiple ulcerous or nodular lesions on the skin, which can become chronic or heal spontaneously six months after the sandfly bite. The condition is not fatal but it can be disabling and leave permanent scars (11. Drugs for Neglected Diseases initiative, America Latina. Leishmanioses. Available at <https://www.dndial.org/doencas/leishmanioses/> [accessed March 16, 2021]
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).

In VL or kala-azar, a systemic parasite infection occurs, with symptoms such as enlarged organs, especially the liver and spleen, as well as prolonged fever, weight loss, and anemia. VL can also affect the lymph nodes and bone marrow, leading to death when left untreated. A major problem with this form is the risk of co-infection, for example, with HIV. Such co-infections have already been reported in 35 endemic countries worldwide (11. Drugs for Neglected Diseases initiative, America Latina. Leishmanioses. Available at <https://www.dndial.org/doencas/leishmanioses/> [accessed March 16, 2021]
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).

Despite the toxicity, high cost, and difficult administration, treatment is still carried out with pentavalent antimonials, amphotericin B, pentamidines, and miltefosine. It is therefore necessary to find effective, safe, low-cost, and short-term treatment, of which photodynamic therapy (PDT) is a promising candidate (11. Drugs for Neglected Diseases initiative, America Latina. Leishmanioses. Available at <https://www.dndial.org/doencas/leishmanioses/> [accessed March 16, 2021]
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).

PDT is a technique used in the treatment of several diseases. It uses electronic excitation of a photosensitive compound (photosensitizer) to produce a variety of reactive oxidant species (ROS), including excited states, free radicals, and strong oxidants (55. Allison RR, Downie GH, Cuenca R, Hu XH, Childs CJH, Sibata CH. Photosensitizers in clinical PDT. Photodiagnosis Photodyn Ther 2004; 1: 27-42, doi: 10.1016/S1572-1000(04)00007-9.
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). It is one of the most promising strategies for local anti-microbial therapy and killing drug-resistant microorganisms (99. Tardivo JP, Baptista MS, Correa JA, Adami F, Pinhal MAS. Development of the tardivo algorithm to predict amputation risk of diabetic foot. PLoS One 2015; 10: e0135707, doi: 10.1371/journal.pone.0135707.
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). Compared with conventional therapies, PDT is a low-cost, minimally invasive technique with minor side effects (99. Tardivo JP, Baptista MS, Correa JA, Adami F, Pinhal MAS. Development of the tardivo algorithm to predict amputation risk of diabetic foot. PLoS One 2015; 10: e0135707, doi: 10.1371/journal.pone.0135707.
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). Since CL is a local infection, PDT has been studied by several researchers and clinical groups as a treatment alternative (1010. Gardlo K, Horska Z, Enk CD, Rauch L, Megahed M, Ruzicka T, et al. Treatment of cutaneous leishmaniasis by photodynamic therapy. J Am Acad Dermatol 2003; 48: 893-896, doi: 10.1067/mjd.2003.218.
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,1111. Aureliano DP, Lindoso JAL, de Castro Soares SR, Takakura CFH, Pereira TM, Ribeiro MS. Cell death mechanisms in Leishmania amazonensis triggered by methylene blue-mediated antiparasitic photodynamic therapy. Photodiagnosis Photodyn Ther 2018; 23: 1-8, doi: 10.1016/j.pdpdt.2018.05.005.
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).

PDT has three essential components - the photosensitizer, the light, and the molecular oxygen. The photosensitization process (Figure 1) starts with the absorption of light by the photosensitizer, raising it to the singlet excited state (S1), which can return to the ground state by losing heat or emitting light (fluorescence). S1 state may suffer changes in the electron spin (intersystem crossing), forming an excited triplet state (T1) with a longer lifetime. T1 has time to react and diffuse, either by transferring energy to molecular oxygen and forming singlet oxygen (Type II reaction) or by transferring electrons (or hydrogen) to a substrate (Type I reaction). The electron transfer reaction forms radicals and produces ROS after a subsequent reaction with oxygen, such as the superoxide radical anion, hydrogen peroxide, and the hydroxyl radical (1212. Foote CS. Mechanisms of photosensitized oxidation. There are several different types of photosensitized oxidation which may be important in biological systems. Science 1968; 162: 963-970, doi: 10.1126/science.162.3857.963.
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,1313. Baptista MS, Cadet J, Di Mascio P, Ghogare AA, Greer A, Hamblin MR, et al. Type I and type II photosensitized oxidation reactions: guidelines and mechanistic pathways. Photochem Photobiol 2017; 93: 912-919, doi: 10.1111/php.12716.
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).

Figure 1
Photosensitization process. PS: ground state photosensitizer; PS (S1): singlet state photosensitizer; PS (T1): triplet state photosensitizer; ISC: intersystem crossing; orange box: type I reaction; yellow box: type II reaction.

Photosensitized oxidations, which are reactions triggered by the interaction of light with photosensitizer (PS) molecules, act by inducing damage in cytoplasmic or organelle membranes. These are key to modulating the mechanism and overall efficiency of regulated cell death (1414. Martins WK, Santos NF, Rocha CS, Bacellar IOL, Tsubone TM, Viotto AC, et al. Parallel damage in mitochondria and lysosomes is an efficient way to photoinduce cell death. Autophagy 2019; 15: 259-279, doi: 10.1080/15548627.2018.1515609.
https://doi.org/10.1080/15548627.2018.15...
). Type I reactions consist of the direct oxidation of biological targets (direct-contact reactions), while those of type II involve oxidation mediated by diffusing species (independent-contact reactions), mainly the singlet oxygen. In direct-contact reactions, the damage occurs at the exact point where the excited species are generated. In contrast, singlet oxygen or other diffusing species can carry oxidation potentials hundreds of nanometers or micrometers away from the point of light absorption (1515. Bacellar IOL, Baptista MS. Mechanisms of photosensitized lipid oxidation and membrane permeabilization. ACS Omega 2019; 4: 21636-21646, doi: 10.1021/acsomega.9b03244.
https://doi.org/10.1021/acsomega.9b03244...
). Nevertheless, the detailed molecular steps leading to biological injury remain largely uncharacterized, and the level of precision in the spatial damage caused by photosensitized oxidation reactions remains unclear.

For a PS to fully compromise membrane function, it needs to engage in electron transfer reactions with either the lipid double bond or the lipid hydroperoxide. This process forms peroxyl and alkoxyl radicals within the membranes that undergo Beta-scission and generate lipid-truncated aldehydes, which cause membrane leakage (1616. Bacellar IOL, Oliveira MC, Dantas LS, Costa EB, Junqueira HC, Martins WK, et al. Photosensitized membrane permeabilization requires contact-dependent reactions between photosensitizer and lipids. J Am Chem Soc 2018; 140: 9606-9615, doi: 10.1021/jacs.8b05014.
https://doi.org/10.1021/jacs.8b05014...
). Therefore, cellular damage occurs precisely at the PS locus. This highlights the importance of finding molecule-specific oxidation-induced photodamage. Since the efficiency of membrane leakage results from an electron transfer reaction that usually causes photobleaching, PS regeneration should be exploited as an effective tool to develop improved PDT photosensitizers (1616. Bacellar IOL, Oliveira MC, Dantas LS, Costa EB, Junqueira HC, Martins WK, et al. Photosensitized membrane permeabilization requires contact-dependent reactions between photosensitizer and lipids. J Am Chem Soc 2018; 140: 9606-9615, doi: 10.1021/jacs.8b05014.
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,1717. Bacellar IOL, Tsubone TM, Pavani C, Baptista MS. Photodynamic efficiency: From molecular photochemistry to cell death. Int J Mol Sci 2015; 16: 20523-20559, doi: 10.3390/ijms160920523.
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).

Many series of PSs that are candidates for PDT are being tested as potential treatments for CL. Understanding how each class works will facilitate the design and development of the PS and improve PDT results. A growing body of literature has examined the use of various PSs in treating leishmaniasis, other infectious diseases (55. Allison RR, Downie GH, Cuenca R, Hu XH, Childs CJH, Sibata CH. Photosensitizers in clinical PDT. Photodiagnosis Photodyn Ther 2004; 1: 27-42, doi: 10.1016/S1572-1000(04)00007-9.
https://doi.org/10.1016/S1572-1000(04)00...
,1818. Baptista MS, Wainwright M. Photodynamic antimicrobial chemotherapy (PACT) for the treatment of malaria, leishmaniasis and trypanosomiasis. Braz J Med Biol Res 2011; 44: 1-10, doi: 10.1590/S0100-879X2010007500141.
https://doi.org/10.1590/S0100-879X201000...

19. Aureliano DP, Ribeiro MS, Lindoso JAL, Pogliani FC, Sellera FP, Song D, et al. Treatment and control of leishmaniasis using photodynamic therapy. Leishmaniasis - Trends Epidemiol Diagnosis Treat 2014; 1: 394-412, doi: 10.5772/57456.
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-2020. Chang KP, Kolli BK, Batchu RB, Chen HW, Chow LMC, Elliott R, et al. New “light” for one-world approach toward safe and effective control of animal diseases and insect vectors from leishmaniac perspectives. Parasit Vectors 2016; 9: 1-13, doi: 10.1186/s13071-015-1291-6.
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), and cancer (2121. Li H, Zhao Y, Jia Y, Qu C, Li J. Covalently assembled dopamine nanoparticle as an intrinsic photosensitizer and pH-responsive nanocarrier for potential application in anticancer therapy. Chem Commun 2019; 55: 15057-15060, doi: 10.1039/C9CC08294H.
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-22. Li H, Jia Y, Peng H, Li J. Recent developments in dopamine-based materials for cancer diagnosis and therapy. Adv Colloid Interface Sci 2018; 252: 1-20, doi: 10.1016/j.cis.2018.01.001.
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2323. Cao H, Wang L, Yang Y, Li J, Qi Y, Li Y, et al. An assembled nanocomplex for improving both therapeutic efficiency and treatment depth in photodynamic therapy. Angew Chemie 2018; 130: 7885-7889, doi: 10.1002/ange.201802497.
https://doi.org/10.1002/ange.201802497...
). In this review, we aim to describe and discuss the main achievements and challenges of using PDT to treat CL by examining recent results and a contemporary view of the mechanisms of the major series of photosensitizers, including phenothiazinium salts, delta-aminolevulinic acid (ALA) and ALA derivatives, phthalocyanine, porphyrin, and phenothiazinium (Figure 2).

Figure 2
Structure of presented photosensitizers. I-IV, delta-aminolevulinic acid (ALA) and ALA derivatives; V-IX, porphyrins and derivatives; X and XI, phthalocyanines; XII, phenothiazinium salt.

ALA-PDT

Leishmania species are known to use tetrapyrroles to promote the growth of promastigotes and to transform amastigotes into promastigote forms. Tetrapyrroles are not acquired from heme biosynthesis since Leishmania spp. entirely lack or are deficient in seven of the eight enzymes in the heme biosynthetic pathway (2424. Chin Shen Chang, Kwang-Poo Chang. Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis. Mol Biochem Parasitol 1985; 16: 267-276, doi: 10.1016/0166-6851(85)90069-6.
https://doi.org/10.1016/0166-6851(85)900...
,2525. Sah JF, Ito H, Kolli BK, Peterson DA, Sassa S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem 2002; 277: 14902-14909, doi: 10.1074/jbc.M200107200.
https://doi.org/10.1074/jbc.M200107200...
).

A study involving Leishmania (L.) amazonensis showed that the parasites become sensitive to UV irradiation due to the presence of uroporphyrinogen I (URO), which is a by-product of hydroxymethylbilane (heme group synthesis) inside the parasitic cells that causes a loss of motility in Leishmania parasites (2525. Sah JF, Ito H, Kolli BK, Peterson DA, Sassa S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem 2002; 277: 14902-14909, doi: 10.1074/jbc.M200107200.
https://doi.org/10.1074/jbc.M200107200...
). These findings showed the potential of porphyrins as photosensitizers in Leishmania PDT.

The first step in heme biosynthesis is the formation of ALA, which is caused by the condensation of glycine and succinyl-CoA in a process catalyzed by the enzyme ALA synthase (ALAS) (2626. Kaneko JJ. Porphyrins and the porphyrias. Clin Biochem Domest Anim 2008; 241-258, doi: 10.1016/B978-0-12-370491-7.00008-8.
https://doi.org/10.1016/B978-0-12-370491...
-27. Moore MR, Disler PB. Chemistry and biochemistry of the porphyrins and porphyrias. Clin Dermatol 1985; 3: 7-23, doi: 10.1016/0738-081X(85)90032-X.
https://doi.org/10.1016/0738-081X(85)900...
2828. Chiabrando D, Mercurio S, Tolosano E. Heme and erythropoieis: more than a structural role. Haematologica 2014; 99: 973-983, doi: 10.3324/haematol.2013.091991.
https://doi.org/10.3324/haematol.2013.09...
). The latter is a mitochondrial enzyme that undergoes negative feedback from heme as it can induce or inhibit the enzyme once present, interrupting the production of ALA (2828. Chiabrando D, Mercurio S, Tolosano E. Heme and erythropoieis: more than a structural role. Haematologica 2014; 99: 973-983, doi: 10.3324/haematol.2013.091991.
https://doi.org/10.3324/haematol.2013.09...
,2929. Granick S. The induction in vitro of the synthesis of delta-aminolevulinic acid synthetase in chemical porphyria: a response to certain drugs, sex hormones, and foreign chemicals. J Biol Chem 1966; 241: 1359-1375, doi: 10.1016/S0021-9258(18)96783-9.
https://doi.org/10.1016/S0021-9258(18)96...
).

Although ALA is not a photosensitizer, it is the first precursor in the biosynthesis of the heme group and the universal precursor of tetrapyrroles like porphyrins (2626. Kaneko JJ. Porphyrins and the porphyrias. Clin Biochem Domest Anim 2008; 241-258, doi: 10.1016/B978-0-12-370491-7.00008-8.
https://doi.org/10.1016/B978-0-12-370491...
,3030. Kennedy JC, Pottier RH. New trends in photobiology. Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy. J Photochem Photobiol B 1992; 14: 275-292, doi: 10.1016/1011-1344(92)85108-7.
https://doi.org/10.1016/1011-1344(92)851...
,3131. Kosaka S, Akilov OE, O'Riordan K, Hasan T. A mechanistic study of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis [6]. J Invest Dermatol 2007; 127: 1546-1549, doi: 10.1038/sj.jid.5700719.
https://doi.org/10.1038/sj.jid.5700719...
). It became the target of PDT studies after it was discovered to be converted into protoporphyrin IX (PpIX) in heme synthesis, accumulating in some cells and acting as a photosensitizer (3030. Kennedy JC, Pottier RH. New trends in photobiology. Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy. J Photochem Photobiol B 1992; 14: 275-292, doi: 10.1016/1011-1344(92)85108-7.
https://doi.org/10.1016/1011-1344(92)851...
,3232. Fukuda H, Casas A, Batlle A. Aminolevulinic acid: from its unique biological function to its star role in photodynamic therapy. Int J Biochem Cell Biol 2005; 37: 272-276, doi: 10.1016/j.biocel.2004.04.018.
https://doi.org/10.1016/j.biocel.2004.04...
). The synthesis of PpIX is determined by the amount of ALA produced, which is regulated in turn by the concentration of the free heme group. However, this feedback mechanism can be circumvented by the administration of exogenous ALA that induces the production and accumulation of PpIX or by certain mutations in the ALAS, which also cause PpIX accumulation (3030. Kennedy JC, Pottier RH. New trends in photobiology. Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy. J Photochem Photobiol B 1992; 14: 275-292, doi: 10.1016/1011-1344(92)85108-7.
https://doi.org/10.1016/1011-1344(92)851...
,3333. Whatley SD, Ducamp S, Gouya L, Grandchamp B, Beaumont C, Badminton MN, et al. C-terminal deletions in the ALAS2 gene lead to gain of function and cause X-linked dominant protoporphyria without anemia or iron overload. Am J Hum Genet 2008; 83: 408-414, doi: 10.1016/j.ajhg.2008.08.003.
https://doi.org/10.1016/j.ajhg.2008.08.0...
).

Akilov et al. (3434. Akilov OE, Kosaka S, O'riordan K, Hasan T. Parasiticidal effect of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis is indirect and mediated through the killing of the host cells. Exp Dermatol 2007; 16: 651-660, doi: 10.1111/j.1600-0625.2007.00578.x.
https://doi.org/10.1111/j.1600-0625.2007...
) performed a well-documented study on the action of PDT using ALA (termed ALA-PDT) in CL and a cellular assay. In the in vivo studies, the authors observed the formation of inflammation and necrosis, indicating damage to vascularized areas. Furthermore, ALA-PDT was found to decrease the parasitic burden 24-fold in the ear lesion. However, in the in vitro studies, the results showed that the amount of PpIX that L. (L.) major was able to obtain from the host cell was not sufficient to produce a photodynamic action (500-fold less than the amount needed to kill metacyclic forms). The results suggest that the success of the in vivo ALA-PDT is due to indirect action such as immune modulation since the PDT was not able to kill parasites.

As mentioned above, Leishmania spp. needs a supplementary source of tetrapyrroles (2424. Chin Shen Chang, Kwang-Poo Chang. Heme requirement and acquisition by extracellular and intracellular stages of Leishmania mexicana amazonensis. Mol Biochem Parasitol 1985; 16: 267-276, doi: 10.1016/0166-6851(85)90069-6.
https://doi.org/10.1016/0166-6851(85)900...
,2525. Sah JF, Ito H, Kolli BK, Peterson DA, Sassa S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem 2002; 277: 14902-14909, doi: 10.1074/jbc.M200107200.
https://doi.org/10.1074/jbc.M200107200...
), meaning that producing PpIX from ALA may not be possible (3131. Kosaka S, Akilov OE, O'Riordan K, Hasan T. A mechanistic study of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis [6]. J Invest Dermatol 2007; 127: 1546-1549, doi: 10.1038/sj.jid.5700719.
https://doi.org/10.1038/sj.jid.5700719...
).

In agreement with the results previously found by Akilov et al. (3434. Akilov OE, Kosaka S, O'riordan K, Hasan T. Parasiticidal effect of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis is indirect and mediated through the killing of the host cells. Exp Dermatol 2007; 16: 651-660, doi: 10.1111/j.1600-0625.2007.00578.x.
https://doi.org/10.1111/j.1600-0625.2007...
), another study was performed using ALA and the derivative MAL (methyl-5-aminolevulinate) (3535. Mateus JE, Valdivieso W, Hernández IP, Martínez F, Páez E, Escobar P. Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment. Biomedica 2014; 34: 589-597, doi: 10.7705/biomedica.v34i4.2272.
https://doi.org/10.7705/biomedica.v34i4....
). Although these exhibited an internalization of the PS, no photodamage was observed in the promastigote forms, confirming the inability of Leishmania spp. in converting ALA to PpIX. However, exogenous PpIX was able to provoke phototoxicity in parasites.

In contrast to results found for the promastigotes, infected cells produced PpIX but not in sufficient amounts to promote damage on intracellular amastigotes (3535. Mateus JE, Valdivieso W, Hernández IP, Martínez F, Páez E, Escobar P. Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment. Biomedica 2014; 34: 589-597, doi: 10.7705/biomedica.v34i4.2272.
https://doi.org/10.7705/biomedica.v34i4....
), consistent with previous findings (3434. Akilov OE, Kosaka S, O'riordan K, Hasan T. Parasiticidal effect of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis is indirect and mediated through the killing of the host cells. Exp Dermatol 2007; 16: 651-660, doi: 10.1111/j.1600-0625.2007.00578.x.
https://doi.org/10.1111/j.1600-0625.2007...
). Despite MAL having an extra alkyl chain in the structure, no difference was found in the internalization or photoactivity between the two compounds (3535. Mateus JE, Valdivieso W, Hernández IP, Martínez F, Páez E, Escobar P. Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment. Biomedica 2014; 34: 589-597, doi: 10.7705/biomedica.v34i4.2272.
https://doi.org/10.7705/biomedica.v34i4....
).

As previously seen, ALA-PDT may act through an immune modulation mechanism when applied directly to the lesion (3434. Akilov OE, Kosaka S, O'riordan K, Hasan T. Parasiticidal effect of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis is indirect and mediated through the killing of the host cells. Exp Dermatol 2007; 16: 651-660, doi: 10.1111/j.1600-0625.2007.00578.x.
https://doi.org/10.1111/j.1600-0625.2007...
). This hypothesis was supported by some results that demonstrated a decrease in the parasitic load following an increase in interferon-gamma (INF-γ) levels after ALA-PDT of infected mouse paws (3636. Souza DM, Alves PM, Silva MLF, Paulino TP, Coraspe HO, Mendonça MMS, et al. 5-ALA-mediated photodynamic therapy reduces the parasite load in mice infected with Leishmania braziliensis. Parasite Immunol 2017; 39: e12403, doi: 10.1111/pim.12403.
https://doi.org/10.1111/pim.12403...
). The increase in INF-γ levels is described as causing resistance to infection by L. (L.) major due to the development of protective immunity (3737. Romão PRT, Santiago HC, Ramos CDL, De Oliveira CFE, Monteiro MC, Cunha FQ, et al. Mast cell degranulation contributes to susceptibility to Leishmania major. Parasite Immunol 2009; 31: 140-146, doi: 10.1111/j.1365-3024.2008.01084.x.
https://doi.org/10.1111/j.1365-3024.2008...
).

Silva et al. (3838. Silva MLF, Alves PM, Souza DM, Silva MV, Dos Santos JP, PaulinoTP, et al. Analysis of macrophage activation markers in an experimental model of cutaneous leishmaniasis treated with photodynamic therapy mediated by 5-aminolevulinic acid. Photobiomodul Photomed Laser Surg 2019; 37: 298-304, doi: 10.1089/photob.2018.4574.
https://doi.org/10.1089/photob.2018.4574...
) studied macrophage modulation in mice infected by L. (V.) braziliensis, the action of ALA-PDT in the lesions, and whether ALA-PDT could alter heme oxygenase 1 (Hmox-1), an enzyme responsible for the persistence of infection and inflammatory response activation (3939. Luz NF, Andrade BB, Feijó DF, Araújo-Santos T, Carvalho GQ, Andrade D, et al. Heme oxygenase-1 promotes the persistence of leishmania chagasi infection. J Immunol 2012; 188: 4460-4467, doi: 10.4049/jimmunol.1103072.
https://doi.org/10.4049/jimmunol.1103072...
).

As seen in the previous study (3636. Souza DM, Alves PM, Silva MLF, Paulino TP, Coraspe HO, Mendonça MMS, et al. 5-ALA-mediated photodynamic therapy reduces the parasite load in mice infected with Leishmania braziliensis. Parasite Immunol 2017; 39: e12403, doi: 10.1111/pim.12403.
https://doi.org/10.1111/pim.12403...
), the parasite burden decreased significantly compared to the untreated group, and Hmox-1 levels were not significantly altered, indicating no association of the enzyme with the persistence of the infection or any decrease in the parasite load (3939. Luz NF, Andrade BB, Feijó DF, Araújo-Santos T, Carvalho GQ, Andrade D, et al. Heme oxygenase-1 promotes the persistence of leishmania chagasi infection. J Immunol 2012; 188: 4460-4467, doi: 10.4049/jimmunol.1103072.
https://doi.org/10.4049/jimmunol.1103072...
).

However, a notable increase in iNOS (nitric oxide synthase) and iNOS/arginine ratio was observed. This could be associated with the ability of ALA-PDT to kill parasites since iNOS catalyzes the production of NO (nitric oxide), which is a leishmanicidal agent. Together with the earlier study, these findings show an association between ALA-PDT and the activation of macrophages that release leishmanicidal mediators. Taken together, these effects increase parasite mortality and decrease the rate of parasitism (3636. Souza DM, Alves PM, Silva MLF, Paulino TP, Coraspe HO, Mendonça MMS, et al. 5-ALA-mediated photodynamic therapy reduces the parasite load in mice infected with Leishmania braziliensis. Parasite Immunol 2017; 39: e12403, doi: 10.1111/pim.12403.
https://doi.org/10.1111/pim.12403...
,3939. Luz NF, Andrade BB, Feijó DF, Araújo-Santos T, Carvalho GQ, Andrade D, et al. Heme oxygenase-1 promotes the persistence of leishmania chagasi infection. J Immunol 2012; 188: 4460-4467, doi: 10.4049/jimmunol.1103072.
https://doi.org/10.4049/jimmunol.1103072...
).

ALA is not a PS and cannot inhibit the parasite itself since it is a prodrug, and the parasite is incapable of converting ALA to the photoactive product, porphyrin. However, it is an excellent option for use with PDT directly on the lesion as it causes an alteration in the immune response and decreases parasite load.

Although ALA does not kill the parasite, the structure can be used to develop a new PS, exploiting the capacity of ALA to be converted into photosensitive porphyrins. Porphyrin can be used as a PS in PDT against Leishmania spp. successfully.

Porphyrins

Porphyrins have been described as an excellent choice for PSs since their effectiveness against Leishmania has already been demonstrated during PDT studies (2525. Sah JF, Ito H, Kolli BK, Peterson DA, Sassa S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem 2002; 277: 14902-14909, doi: 10.1074/jbc.M200107200.
https://doi.org/10.1074/jbc.M200107200...
,3535. Mateus JE, Valdivieso W, Hernández IP, Martínez F, Páez E, Escobar P. Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment. Biomedica 2014; 34: 589-597, doi: 10.7705/biomedica.v34i4.2272.
https://doi.org/10.7705/biomedica.v34i4....
). One PDT study used exogenous PpIX as a positive control and tested against L. (L.) infantum and L. (V.) panamensis (3535. Mateus JE, Valdivieso W, Hernández IP, Martínez F, Páez E, Escobar P. Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment. Biomedica 2014; 34: 589-597, doi: 10.7705/biomedica.v34i4.2272.
https://doi.org/10.7705/biomedica.v34i4....
). Although PpIX was only used as a control, it showed promising results against the promastigote forms of L. (L.) infantum and L. (V.) panamensis.

Before this, Bristow et al. (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
) studied four types of cationic porphyrin PSs as potential photodynamic anti-Leishmania agents. The cationic PSs were chosen due to the negative character of the membrane of Leishmania spp. and were tested against the promastigote forms of Leishmania (L.) major. Macrophages and keratinocytes were used to simulate amastigote intracellular forms of Leishmania and the healthy tissue around the skin lesion, respectively (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
). PSs were tested and showed very different results, which was thought to be due to the differences in the membrane charge of the three cell lines studied. As noted, the cell membrane of the Leishmania spp. has an anionic character (4141. Zhang K, Beverley SM. Phospholipid and sphingolipid metabolism in Leishmania. Mol Biochem Parasitol 2010; 170: 55-64, doi: 10.1016/j.molbiopara.2009.12.004.
https://doi.org/10.1016/j.molbiopara.200...
).

Compounds 1 (phosphorous-centered cationic porphyrin) and 3 (nitrogen-centered cationic porphyrin) were photoactivated against the promastigote forms at a low concentration. However, compound 1 showed no activity while compound 3 inhibited macrophages with a similar LD50 for L. (L.) major promastigotes (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
). Meanwhile, compound 1 was active against keratinocytes at a concentration equivalent to almost half that needed to kill promastigotes, but compound 3 reached inhibition at a concentration 10 times higher than the one established for promastigotes. Since compound 3 had the best results, a dose adjustment was considered so that it could be used to kill promastigote and amastigote forms without causing damage in uninfected tissue (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
).

In 2018, Andrade et al. (4242. Andrade CG, Figueiredo RCBQ, Ribeiro KRC, Souza LIO, Sarmento-Neto JF, Rebouças JS, et al. Photodynamic effect of zinc porphyrin on the promastigote and amastigote forms of: Leishmania braziliensis. Photochem Photobiol Sci 2018; 17: 482-490, doi: 10.1039/C7PP00458C.
https://doi.org/10.1039/C7PP00458C...
) studied the effect of zinc porphyrin (ZnP), a cationic porphyrin, to verify the effect of the charge and the zinc on membranes. ZnP was active in both tested concentrations of about 65 and 90%. Furthermore, an analysis of the parasitic cells after 24 h of PDT showed that cells were incapable of replicating. These results were successful because the PS concentration was low, the incubation time was only 10 min, and the irradiation time was short (4242. Andrade CG, Figueiredo RCBQ, Ribeiro KRC, Souza LIO, Sarmento-Neto JF, Rebouças JS, et al. Photodynamic effect of zinc porphyrin on the promastigote and amastigote forms of: Leishmania braziliensis. Photochem Photobiol Sci 2018; 17: 482-490, doi: 10.1039/C7PP00458C.
https://doi.org/10.1039/C7PP00458C...
). This treatment exhibited high permeability in the parasitic membrane. The microscopic alterations included a shortening and rounding of the parasitic cells, shrinkage of the plasmatic membrane, and vacuolization. ZnP was less cytotoxic than compounds 1 and 3 described by Bristow et al. (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
) Approximately 70% of human cells remained viable after ZnP PDT, while about 50% of cells showed viability after a cationic porphyrin assay (4242. Andrade CG, Figueiredo RCBQ, Ribeiro KRC, Souza LIO, Sarmento-Neto JF, Rebouças JS, et al. Photodynamic effect of zinc porphyrin on the promastigote and amastigote forms of: Leishmania braziliensis. Photochem Photobiol Sci 2018; 17: 482-490, doi: 10.1039/C7PP00458C.
https://doi.org/10.1039/C7PP00458C...
).

Both studies (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
,4242. Andrade CG, Figueiredo RCBQ, Ribeiro KRC, Souza LIO, Sarmento-Neto JF, Rebouças JS, et al. Photodynamic effect of zinc porphyrin on the promastigote and amastigote forms of: Leishmania braziliensis. Photochem Photobiol Sci 2018; 17: 482-490, doi: 10.1039/C7PP00458C.
https://doi.org/10.1039/C7PP00458C...
) were important demonstrations of the effectiveness of porphyrins in PDT against Leishmania. It was possible to observe that cationic porphyrin is a promising PS for use against Leishmania spp. However, the former study (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
) made it clear that the positive charge is not the only factor to be considered. Meanwhile, the presence of the metal in the second study (4242. Andrade CG, Figueiredo RCBQ, Ribeiro KRC, Souza LIO, Sarmento-Neto JF, Rebouças JS, et al. Photodynamic effect of zinc porphyrin on the promastigote and amastigote forms of: Leishmania braziliensis. Photochem Photobiol Sci 2018; 17: 482-490, doi: 10.1039/C7PP00458C.
https://doi.org/10.1039/C7PP00458C...
) indicates that this strategy may be relevant in structure activity studies.

Carbaporphyrin ketals are porphyrin derivatives, called porphyrinoids, in which the pyrrole ring of porphyrin is replaced by a ketal-substituted indene ring. This class of compounds was tested in vitro and in vivo against Leishmania (L.) amazonensis, L. (L.) infantum, and L. (V.) panamensis (4343. Taylor VM, Cedeão DL, Muãoz DL, Jones MA, Lash TD, Young AM, et al. In vitro and in vivo studies of the utility of dimethyl and diethyl carbaporphyrin ketals in treatment of cutaneous leishmaniasis. Antimicrob Agents Chemother 2011; 55: 4755-4764, doi: 10.1128/AAC.00671-11.
https://doi.org/10.1128/AAC.00671-11...
). Such a study allows verification of whether the alteration in the activity is caused by the changes in compound structure.

The carbaporphyrin dimethyl ketal (CKOMe) and carbaporphyrin diethyl ketal (CKOEt) had high activity levels against axenic and intracellular amastigotes of studied Leishmania. When used in a liposomal formulation, PDT had a stronger effect against L. (L.) amazonensis for both compounds. However, similarly to the case of cationic porphyrins (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
), carbaporphyrins were toxic to human cells, although the toxicity of CKOMe to PMH cells (peritoneal macrophages obtained from hamsters) decreased when administered in a liposomal formulation (4343. Taylor VM, Cedeão DL, Muãoz DL, Jones MA, Lash TD, Young AM, et al. In vitro and in vivo studies of the utility of dimethyl and diethyl carbaporphyrin ketals in treatment of cutaneous leishmaniasis. Antimicrob Agents Chemother 2011; 55: 4755-4764, doi: 10.1128/AAC.00671-11.
https://doi.org/10.1128/AAC.00671-11...
). Even though CKOEt is more hydrophobic than CKOMe, the latter showed better results, even when solubilized in DMSO or a liposome, showing that something other than hydrophobicity is important. The size of the molecule may be important here, as CKOEt has two ethyl chains that could cause a steric effect that would affect activity. The study also shows that altering the porphyrin backbone is useful for increasing activity against Leishmania species.

Phthalocyanine

Phthalocyanine (Pc) is a synthetic dye that consists of four isoindole rings connected by four nitrogen atoms (4444. Santos KLM, Barros RM, Lima DPS, Nunes AMA, Sato MR, Faccio R, et al. Prospective application of phthalocyanines in the photodynamic therapy against microorganisms and tumor cells: a mini-review. Photodiagnosis Photodyn Ther 2020; 32: 102032, doi: 10.1016/j.pdpdt.2020.102032.
https://doi.org/10.1016/j.pdpdt.2020.102...
). Pc is an alternative to porphyrins since PSs do not absorb in the 400-600 nm range, having no phototoxicity to the skin. Unlike porphyrins, Pc has an absorption and fluorescence wavelengths in the range of 650-800 nm and high production of singlet oxygen. However, like porphyrins, Pc can form metal complexes that further increase the ability to produce singlet oxygen (4444. Santos KLM, Barros RM, Lima DPS, Nunes AMA, Sato MR, Faccio R, et al. Prospective application of phthalocyanines in the photodynamic therapy against microorganisms and tumor cells: a mini-review. Photodiagnosis Photodyn Ther 2020; 32: 102032, doi: 10.1016/j.pdpdt.2020.102032.
https://doi.org/10.1016/j.pdpdt.2020.102...
,4545. Lo PC, Rodríguez-Morgade MS, Pandey RK, Ng DKP, Torres T, Dumoulin F. The unique features and promises of phthalocyanines as advanced photosensitisers for photodynamic therapy of cancer. Chem Soc Rev 2020; 49: 1041-1056, doi: 10.1039/C9CS00129H.
https://doi.org/10.1039/C9CS00129H...
).

When exposed to aluminum phthalocyanine chloride (AlPhCl) and light, promastigote forms of L. (L.) amazonensis were rapidly killed, while axenic amastigotes underwent structural alterations. Both groups exhibited loss of fluorescence, indicating cell lysis. It is important to note that neither the light nor AlPhCl was toxic when administered alone, and both the dose of light and the Pc concentration used were low. In addition, AlPhCl reduced the number of macrophages and amastigote forms in an infected culture (4646. Dutta S, Ray D, Kolli BK, Chang KP. Photodynamic sensitization of Leishmania amazonensis in both extracellular and intracellular stages with aluminum phthalocyanine chloride for photolysis in vitro. Antimicrob Agents Chemother 2005; 49: 4474-4484, doi: 10.1128/AAC.49.11.4474-4484.2005.
https://doi.org/10.1128/AAC.49.11.4474-4...
).

Escobar and colleagues (4747. Escobar P, Hernández IP, Rueda CM, Martínez F, Páez E. Photodynamic activity of aluminium (III) and zinc (II) phthalocyanines in Leishmania promastigotes. Biomedica 2006; 26: 49-56, doi: 10.7705/biomedica.v26i1.1499.
https://doi.org/10.7705/biomedica.v26i1....
) analyzed the photoactivity of aluminum chloride and zinc phthalocyanines (AlPc and ZnPc) against promastigote forms of L. (L.) chagasi and L. (V.) panamensis. AlPc had a dose-response activity and was more phototoxic to L. (L.) chagasi than L. (V.) panamensis (30- to 50-fold). AlPc also caused greater photosensitization than ZnPc in both parasitic species, and none of the PSs presented phototoxicity in the dark. The differences found between the two PSs could be related to the fact that ZnPc is very hydrophobic, which could make it difficult to enter the cell, unlike the amphiphilic AlPc (4747. Escobar P, Hernández IP, Rueda CM, Martínez F, Páez E. Photodynamic activity of aluminium (III) and zinc (II) phthalocyanines in Leishmania promastigotes. Biomedica 2006; 26: 49-56, doi: 10.7705/biomedica.v26i1.1499.
https://doi.org/10.7705/biomedica.v26i1....
).

To facilitate entry into infected cells, decrease the formation of dimers, improve photoactivity, and reduce phototoxic effects, Hernández et al. (4848. Hernández IP, Montanari J, Valdivieso W, Morilla MJ, Romero EL, Escobar P. In vitro phototoxicity of ultradeformable liposomes containing chloroaluminum phthalocyanine against New World Leishmania species. J Photochem Photobiol B Biol 2012; 117: 157-163, doi: 10.1016/j.jphotobiol.2012.09.018.
https://doi.org/10.1016/j.jphotobiol.201...
) reported the activity of ultradeformable liposomes containing chloroaluminum phthalocyanine (UDL-ClAlPc) and free ClAlPc against L. (L.) chagasi and L. (V.) panamensis promastigotes and amastigotes. UDL-ClAlPc was more phototoxic than free ClAlPc in both species and parasitic forms. As previously reported, ClAlPc showed no selectivity for the parasitic intracellular form compared to host cells (3434. Akilov OE, Kosaka S, O'riordan K, Hasan T. Parasiticidal effect of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis is indirect and mediated through the killing of the host cells. Exp Dermatol 2007; 16: 651-660, doi: 10.1111/j.1600-0625.2007.00578.x.
https://doi.org/10.1111/j.1600-0625.2007...
,4646. Dutta S, Ray D, Kolli BK, Chang KP. Photodynamic sensitization of Leishmania amazonensis in both extracellular and intracellular stages with aluminum phthalocyanine chloride for photolysis in vitro. Antimicrob Agents Chemother 2005; 49: 4474-4484, doi: 10.1128/AAC.49.11.4474-4484.2005.
https://doi.org/10.1128/AAC.49.11.4474-4...
). This result confirmed that the death of the parasite could occur due to a secondary mechanism.

The use of genetically modified Leishmania to produce and accumulate URO has been successfully described (2525. Sah JF, Ito H, Kolli BK, Peterson DA, Sassa S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem 2002; 277: 14902-14909, doi: 10.1074/jbc.M200107200.
https://doi.org/10.1074/jbc.M200107200...
). Therefore, Dutta and colleagues decided to use this technique along with AlPhCl to increase parasitic photodynamic efficiency (4949. Dutta S, Waki K, Chang KP. Combinational sensitization of leishmania with uroporphyrin and aluminum phthalocyanine synergistically enhances their photodynamic inactivation in vitro and in vivo. Photochem Photobiol 2012; 88: 620-625, doi: 10.1111/j.1751-1097.2012.01076.x.
https://doi.org/10.1111/j.1751-1097.2012...
).

First of all, URO and AlPhCl alone showed a very different localization into cellular structures. As expected and described (2525. Sah JF, Ito H, Kolli BK, Peterson DA, Sassa S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem 2002; 277: 14902-14909, doi: 10.1074/jbc.M200107200.
https://doi.org/10.1074/jbc.M200107200...
,4747. Escobar P, Hernández IP, Rueda CM, Martínez F, Páez E. Photodynamic activity of aluminium (III) and zinc (II) phthalocyanines in Leishmania promastigotes. Biomedica 2006; 26: 49-56, doi: 10.7705/biomedica.v26i1.1499.
https://doi.org/10.7705/biomedica.v26i1....
,5050. Dutta S, Kolli BK, Tang A, Sassa S, Chang KP. Transgenic Leishmania model for delta-aminolevulinate-inducible monospecific uroporphyria: Cytolytic phototoxicity initiated by singlet oxygen-mediated inactivation of proteins and its ablation by endosomal mobilization of cytosolic uroporphyrin. Eukaryot Cell 2008; 7: 1146-1157, doi: 10.1128/EC.00365-07.
https://doi.org/10.1128/EC.00365-07...
), the more hydrophobic AlPhCl showed a greater accumulation in structures such as the cell membrane, while the hydrophilic URO was more widespread in the cytoplasm (4949. Dutta S, Waki K, Chang KP. Combinational sensitization of leishmania with uroporphyrin and aluminum phthalocyanine synergistically enhances their photodynamic inactivation in vitro and in vivo. Photochem Photobiol 2012; 88: 620-625, doi: 10.1111/j.1751-1097.2012.01076.x.
https://doi.org/10.1111/j.1751-1097.2012...
). Promastigotes photosensitized with URO or AlPhCl alone showed a reduction in viability. Also, a total loss of cell viability was only possible after photosensitizing the cells with both PSs (URO and AlPhCl). This loss of viability was maintained after five days of culture, showing that the synergism technique may have photoinhibition applications. Notably, infected macrophages were not affected by this treatment, although all the parasitic intracellular burden was eliminated (4949. Dutta S, Waki K, Chang KP. Combinational sensitization of leishmania with uroporphyrin and aluminum phthalocyanine synergistically enhances their photodynamic inactivation in vitro and in vivo. Photochem Photobiol 2012; 88: 620-625, doi: 10.1111/j.1751-1097.2012.01076.x.
https://doi.org/10.1111/j.1751-1097.2012...
).

The fact that the photosensitizers were used together and the host cells were not affected is an important outcome (4949. Dutta S, Waki K, Chang KP. Combinational sensitization of leishmania with uroporphyrin and aluminum phthalocyanine synergistically enhances their photodynamic inactivation in vitro and in vivo. Photochem Photobiol 2012; 88: 620-625, doi: 10.1111/j.1751-1097.2012.01076.x.
https://doi.org/10.1111/j.1751-1097.2012...
), as previous studies (3434. Akilov OE, Kosaka S, O'riordan K, Hasan T. Parasiticidal effect of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis is indirect and mediated through the killing of the host cells. Exp Dermatol 2007; 16: 651-660, doi: 10.1111/j.1600-0625.2007.00578.x.
https://doi.org/10.1111/j.1600-0625.2007...
,4646. Dutta S, Ray D, Kolli BK, Chang KP. Photodynamic sensitization of Leishmania amazonensis in both extracellular and intracellular stages with aluminum phthalocyanine chloride for photolysis in vitro. Antimicrob Agents Chemother 2005; 49: 4474-4484, doi: 10.1128/AAC.49.11.4474-4484.2005.
https://doi.org/10.1128/AAC.49.11.4474-4...
,4848. Hernández IP, Montanari J, Valdivieso W, Morilla MJ, Romero EL, Escobar P. In vitro phototoxicity of ultradeformable liposomes containing chloroaluminum phthalocyanine against New World Leishmania species. J Photochem Photobiol B Biol 2012; 117: 157-163, doi: 10.1016/j.jphotobiol.2012.09.018.
https://doi.org/10.1016/j.jphotobiol.201...
) showed that PDT might have killed the parasites by a secondary route and not by directly acting on the parasite itself.

Ribeiro et al. (5151. Ribeiro JBP, Miranda-Vilela AL, Graziani D, Gomes MRA, Amorim AAS, Garcia RD, et al. Evaluation of the efficacy of systemic miltefosine associated with photodynamic therapy with liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL/6 mice. Photodiagnosis Photodyn Ther 2016; 13: 282-290, doi: 10.1016/j.pdpdt.2015.08.006.
https://doi.org/10.1016/j.pdpdt.2015.08....
) evaluated the effectiveness of the PDT technique when associating the topical use of AlClPC (liposomes containing chloroaluminium phthalocyanine) with the drug miltefosine, already used to treat CL (5252. Machado PRL, Penna G. Miltefosine and cutaneous leishmaniasis. Curr Opin Infect Dis 2012; 25: 141-144, doi: 10.1097/QCO.0b013e3283509cac.
https://doi.org/10.1097/QCO.0b013e328350...
-53. Dorlo TPC, Balasegaram M, Beijnen JH, de Vries PJ. Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis. J Antimicrob Chemother 2012; 67: 2576-2597, doi: 10.1093/jac/dks275.
https://doi.org/10.1093/jac/dks275...
5454. Almeida OLS, Santos JB. Advances in the treatment of cutaneous leishmaniasis in the new world in the last ten years: a systematic literature review. An Bras Dermatol 2011; 86: 497-506, doi: 10.1590/S0365-05962011000300012.
https://doi.org/10.1590/S0365-0596201100...
). After 20 days of treatment with miltefosine and PDT with AlClPC, they noted a reduction in the diameter of the infected paw accompanied by a considerable decrease in parasitic culture.

A nanoemulsion containing ZnPc was tested to optimize PDT studies in the treatment of CL (5555. de Siqueira LBO, Cardoso VS, Rodrigues IA, Vazquez-Villa AL, dos Santos EP, Guimarães BCLR, et al. Development and evaluation of zinc phthalocyanine nanoemulsions for use in photodynamic therapy for Leishmania spp. Nanotechnology 2017; 28: 065101, doi: 10.1088/1361-6528/28/6/065101.
https://doi.org/10.1088/1361-6528/28/6/0...
). First, free ZnPc was evaluated against promastigote forms of L. (L.) infantum and L. (L.) amazonensis; in contrast to previous findings (4747. Escobar P, Hernández IP, Rueda CM, Martínez F, Páez E. Photodynamic activity of aluminium (III) and zinc (II) phthalocyanines in Leishmania promastigotes. Biomedica 2006; 26: 49-56, doi: 10.7705/biomedica.v26i1.1499.
https://doi.org/10.7705/biomedica.v26i1....
), it was active in the presence and absence of light, although light caused greater inhibition of L. (L.) infantum. The results presented by nanoemulsion were even better after irradiation for L. (L.) amazonensis and L. (L.) infantum. As previously observed (4646. Dutta S, Ray D, Kolli BK, Chang KP. Photodynamic sensitization of Leishmania amazonensis in both extracellular and intracellular stages with aluminum phthalocyanine chloride for photolysis in vitro. Antimicrob Agents Chemother 2005; 49: 4474-4484, doi: 10.1128/AAC.49.11.4474-4484.2005.
https://doi.org/10.1128/AAC.49.11.4474-4...
,4848. Hernández IP, Montanari J, Valdivieso W, Morilla MJ, Romero EL, Escobar P. In vitro phototoxicity of ultradeformable liposomes containing chloroaluminum phthalocyanine against New World Leishmania species. J Photochem Photobiol B Biol 2012; 117: 157-163, doi: 10.1016/j.jphotobiol.2012.09.018.
https://doi.org/10.1016/j.jphotobiol.201...
), free PS was toxic to macrophages with or without irradiation. Like free PS, the nanoemulsion also showed cytotoxicity but with a certain selectivity for parasites compared to host cells. A reduction in the parasitic burden of macrophages was also found (5555. de Siqueira LBO, Cardoso VS, Rodrigues IA, Vazquez-Villa AL, dos Santos EP, Guimarães BCLR, et al. Development and evaluation of zinc phthalocyanine nanoemulsions for use in photodynamic therapy for Leishmania spp. Nanotechnology 2017; 28: 065101, doi: 10.1088/1361-6528/28/6/065101.
https://doi.org/10.1088/1361-6528/28/6/0...
).

Escobar et al. (5656. Escobar P, Vera AM, Neira LF, Velásquez AO, Carreão H. Photodynamic therapy using ultradeformable liposomes loaded with chlorine aluminum phthalocyanine against L. (V.) braziliensis experimental models. Exp Parasitol 2018; 194: 45-52, doi: 10.1016/j.exppara.2018.09.016.
https://doi.org/10.1016/j.exppara.2018.0...
) built on these studies by analyzing the topical use of UDL-ClAlPc in BALB/c mice infected with L. (V.) braziliensis and in vitro activity in L. (V.) braziliensis promastigotes and amastigotes and mammalian cells. In the in vitro assay, the UDL-ClAlPc internalized both infected and uninfected cells, and was active in both parasitic and host cells after PDT, with no selectivity. UDL-ClAlPc also induced ROS generation in infected macrophages after PDT. Since no activity was found after treatment with UDL-ClAlPc and only low levels of ROS were produced without PDT, the authors suggested that ROS production after PDT may have been responsible for killing the parasites. Damage to the DNA was found with or without PDT, even in the empty UDL, but the mechanism behind the damage was not studied. The study did not find any effect in the BALB/c infected mice treated with topical UDL-ClAlPc and PDT, which was explained as possibly due to photobleaching or low penetration in the skin (5656. Escobar P, Vera AM, Neira LF, Velásquez AO, Carreão H. Photodynamic therapy using ultradeformable liposomes loaded with chlorine aluminum phthalocyanine against L. (V.) braziliensis experimental models. Exp Parasitol 2018; 194: 45-52, doi: 10.1016/j.exppara.2018.09.016.
https://doi.org/10.1016/j.exppara.2018.0...
).

These results contrast with those found by Ribeiro et al. (5151. Ribeiro JBP, Miranda-Vilela AL, Graziani D, Gomes MRA, Amorim AAS, Garcia RD, et al. Evaluation of the efficacy of systemic miltefosine associated with photodynamic therapy with liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL/6 mice. Photodiagnosis Photodyn Ther 2016; 13: 282-290, doi: 10.1016/j.pdpdt.2015.08.006.
https://doi.org/10.1016/j.pdpdt.2015.08....
), who also used the PS within liposomes. The latter study (5151. Ribeiro JBP, Miranda-Vilela AL, Graziani D, Gomes MRA, Amorim AAS, Garcia RD, et al. Evaluation of the efficacy of systemic miltefosine associated with photodynamic therapy with liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL/6 mice. Photodiagnosis Photodyn Ther 2016; 13: 282-290, doi: 10.1016/j.pdpdt.2015.08.006.
https://doi.org/10.1016/j.pdpdt.2015.08....
) combined topical use of the liposome with miltefosine, which may have been responsible for reducing the lesions present in the animals. Another difference is in the type of formulation: the first study (5656. Escobar P, Vera AM, Neira LF, Velásquez AO, Carreão H. Photodynamic therapy using ultradeformable liposomes loaded with chlorine aluminum phthalocyanine against L. (V.) braziliensis experimental models. Exp Parasitol 2018; 194: 45-52, doi: 10.1016/j.exppara.2018.09.016.
https://doi.org/10.1016/j.exppara.2018.0...
) used a lipid film while the second one (5151. Ribeiro JBP, Miranda-Vilela AL, Graziani D, Gomes MRA, Amorim AAS, Garcia RD, et al. Evaluation of the efficacy of systemic miltefosine associated with photodynamic therapy with liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL/6 mice. Photodiagnosis Photodyn Ther 2016; 13: 282-290, doi: 10.1016/j.pdpdt.2015.08.006.
https://doi.org/10.1016/j.pdpdt.2015.08....
) created a gel formulation. These differences may explain the effectiveness of the liposome on mouse lesions.

According to the excellent results obtained previously in a study of the association of miltefosine with PDT using AlClPC as PS (5151. Ribeiro JBP, Miranda-Vilela AL, Graziani D, Gomes MRA, Amorim AAS, Garcia RD, et al. Evaluation of the efficacy of systemic miltefosine associated with photodynamic therapy with liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL/6 mice. Photodiagnosis Photodyn Ther 2016; 13: 282-290, doi: 10.1016/j.pdpdt.2015.08.006.
https://doi.org/10.1016/j.pdpdt.2015.08....
), Ribeiro et al. (5757. Ribeiro JBP, Miranda-Vilela AL, Amorim AAS, Garcia RD, Moreira JR, Gomes CM, et al. Study of the efficacy of N-methyl glucamine antimoniate (SbV) associated with photodynamic therapy using liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL6 mice. Photodiagnosis Photodyn Ther 2019; 26: 261-269, doi: 10.1016/j.pdpdt.2019.04.004.
https://doi.org/10.1016/j.pdpdt.2019.04....
) decided to evaluate the effectiveness of the association between AlClPC and PDT with the drug N-methyl glucamine (NMG) in mice infected with L. (L.) amazonensis. The standard recommendation of NMG for the CL treatment is 20 mg SbV.kg-1.day-1 (5858. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet 2018; 392: 951-970, doi: 10.1016/S0140-6736(18)31204-2.
https://doi.org/10.1016/S0140-6736(18)31...
), so the researchers decided to test 20 mg SbV/kg/day + PDT + AlPlPC (NMG20 + PDT) and 10 mg SbV.kg-1.day-1 + PDT + AlPlPC (NMG10 + PDT). The latter concentration was used to verify whether the PDT can decrease the NMG dosage, which may diminish adverse effects.

The treatment with NMG20 + PDT decreased the diameter of the animal's paw in 60 days after the end of treatment, a decrease similar to the negative control. It also showed negative results for amastigotes and parasitic cultures after 20 days of treatment and 60 days after the end of treatment. Cell viability was reduced after 10 and 20 days of treatment, even for NMG10 + PDT, although only NMG20 + PDT could maintain this reduction at 60 days after the end of the treatment. The results of NMG10 + PDT were similar to those of NMG20 alone (standard treatment), suggesting that it could be used in the future to minimize the adverse effects caused by the drug alone. It is important to note that this decrease is related to PDT + AlPlPC, indicating that AlClPC may be of interest as a PS (5757. Ribeiro JBP, Miranda-Vilela AL, Amorim AAS, Garcia RD, Moreira JR, Gomes CM, et al. Study of the efficacy of N-methyl glucamine antimoniate (SbV) associated with photodynamic therapy using liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL6 mice. Photodiagnosis Photodyn Ther 2019; 26: 261-269, doi: 10.1016/j.pdpdt.2019.04.004.
https://doi.org/10.1016/j.pdpdt.2019.04....
).

Phthalocyanines have proven to be an excellent PS for use in PDT against different species of Leishmania, although more in-depth studies should be carried out on the selectivity and toxicity in the host cells. However, studies involving liposomal formulations and the association with existing drugs for the disease have shown satisfactory results both in terms of reducing parasitic load and skin lesions, as well as reducing the usual concentration and thus achieving a possible improvement in adverse effects.

Phenothiazinium salts

Phenothiazine derivatives have been extensively studied, showing promising results against bacterial activity. Changes in structure, including the addition of methyl, nitro, and primary amine groups, the positioning of these groups, and the hydrophobicity of the molecule can improve the effects (5959. Wainwright M, Phoenix DA, Marland J, Wareing DRA, Bolton FJ. A study of photobactericidal activity in the phenothiazinium series. FEMS Immunol Med Microbiol 1997; 19: 75-80, doi: 10.1111/j.1574-695X.1997.tb01074.x.
https://doi.org/10.1111/j.1574-695X.1997...
-60. Wainwright M. Phenothiazinium photosensitisers: V. Photobactericidal activities of chromophore-methylated phenothiazinium salts. Dye Pigment 2007; 73: 7-12, doi: 10.1016/j.dyepig.2005.10.001.
https://doi.org/10.1016/j.dyepig.2005.10...
6161. Usacheva MN, Teichert MC, Biel MA. The role of the methylene blue and toluidine blue monomers and dimers in the photoinactivation of bacteria. J Photochem Photobiol B 2003; 71: 87-98, doi: 10.1016/j.jphotobiol.2003.06.002.
https://doi.org/10.1016/j.jphotobiol.200...
). Studies on the differences in chemical structure have shown that spatial constraints and the geometry of the phenothiazine derivatives are also important, for example, in aggregation (6262. Bacellar IOL, Pavani C, Sales EM, Itri R, Wainwright M, Baptista MS. Membrane damage efficiency of phenothiazinium photosensitizers. Photochem Photobiol 2014; 90: 801-813, doi: 10.1111/php.12264.
https://doi.org/10.1111/php.12264...
). In addition to antibacterial activity, phenothiazine derivatives have also been studied for the treatment with PDT of other diseases such as Kaposi's sarcoma, herpes, and diabetic foot (6363. Tardivo JP, Giglio A Del, Paschoal LH, Baptista MS. New photodynamic therapy protocol to treat aids-related Kaposi's sarcoma. Photomed Laser Surg 2006; 24: 528-531, doi: 10.1089/pho.2006.24.528.
https://doi.org/10.1089/pho.2006.24.528...
-64. Tardivo JP, Wainwright M, Baptista MS. Local clinical phototreatment of herpes infection in São Paulo. Photodiagnosis Photodyn Ther 2012; 9: 118-121, doi: 10.1016/j.pdpdt.2012.01.002.
https://doi.org/10.1016/j.pdpdt.2012.01....
6565. Tardivo JP, Adami F, Correa JA, Pinhal MAS, Baptista MS. A clinical trial testing the efficacy of PDT in preventing amputation in diabetic patients. Photodiagnosis Photodyn Ther 2014; 11: 342-350, doi: 10.1016/j.pdpdt.2014.04.007.
https://doi.org/10.1016/j.pdpdt.2014.04....
).

Methylene blue (MB) is the most studied photosensitizer of the phenothiazine class against Leishmania species. MB is an interesting photosensitizer due to its high singlet oxygen quantum yield of around 0.5 (6666. Redmond RW, Gamlin JN. A compilation of singlet oxygen yields from biologically relevant molecules. Photochem Photobiol 1999; 70: 391-475, doi: 10.1111/j.1751-1097.1999.tb08240.x.
https://doi.org/10.1111/j.1751-1097.1999...
) and its absorption band between 550 and 700 nm. MB is capable of forming dimers, although this characteristic depends on the concentration, the ionic strength, and the presence or not of charged interfaces. The absorption spectrum of monomers and dimers is different, with maximum absorption at 665 and 580 nm, respectively (6767. Junqueira HC, Severino D, Dias LG, Gugliotti MS, Baptista MS. Modulation of methylene blue photochemical properties based on adsorption at aqueous micelle interfaces. Phys Chem Chem Phys 2002; 4: 2320-2328, doi: 10.1039/b109753a.
https://doi.org/10.1039/b109753a...
,6868. Severino D, Junqueira HC, Gugliotti M, Gabrielli DS, Baptista MS. Influence of negatively charged interfaces on the ground and excited state properties of methylene blue. Photochem Photobiol 2003; 77: 459-468, doi: 10.1562/0031-8655(2003)077<0459:IONCIO>2.0.CO;2.
https://doi.org/10.1562/0031-8655(2003)0...
).

Song et al. (6969. Song D, Lindoso JAL, Oyafuso LK, Kanashiro EHY, Cardoso JL, Uchoa AF, et al. Photodynamic therapy using methylene blue to treat cutaneous leishmaniasis. Photomed Laser Surg 2011; 29: 711-715, doi: 10.1089/pho.2010.2915.
https://doi.org/10.1089/pho.2010.2915...
) performed in vitro and in vivo studies to assess the effectiveness of PDT and MB as a PS for treatment of CL. Although MB was effective without irradiation, the half inhibitory concentration (IC50) value in the parasitic cells was reduced after irradiation in a dose-dependent manner. After the in vitro assay, one patient with three lesions caused by Leishmania (L.) amazonensis underwent treatment with a low concentration of pentavalent antimonial and PDT + MB, with one lesion treated with SbV alone. The results showed that although the lesions were reduced and cured with SbV treatment alone, PDT accelerated this process.

Peloi et al. (7070. Peloi LS, Eduardo C, Biondo G, Kimura E, José M, Valdrinez M, et al. Photodynamic therapy for American cutaneous leishmaniasis: the efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis. Exp Parasitol 2011; 128: 353-356, doi: 10.1016/j.exppara.2011.04.009.
https://doi.org/10.1016/j.exppara.2011.0...
) performed an in vivo PDT study using 10 nM of MB in a lotion or aqueous formulation in hamsters infected with L. (L.) amazonensis. MB alone could not decrease the animal's footpad size, while animals treated with MB and light showed a significant reduction in footpad size, with no differences between the formulations applied. Some 40% of the infected animals had ulcerated lesions, and after treatment with MB in lotion and water plus light, 40 and 50% of the lesions were cured, respectively, after 12 weeks of treatment. The analysis of the parasitic load in the spleen of infected and treated hamsters found no presence of the parasite regardless of the formulation used. However, parasites were found in lymph nodes of treated hamsters in a much lower percentage compared to untreated hamsters.

To verify how PDT and MB can interfere in the interaction between macrophages and L. (V.) braziliensis, in vitro studies were performed (7171. de Oliveira S, da Ordem Trahamane EJ, Monteiro J, Santos GP, Crugeira P, Sampaio F, et al. Leishmanicidal effect of antiparasitic photodynamic therapy—ApPDT on infected macrophages. Lasers Med Sci 2017; 32: 1959-1964, doi: 10.1007/s10103-017-2292-9.
https://doi.org/10.1007/s10103-017-2292-...
). The parasitic load was decreased both in the group that received only MB and in the group that received MB + light, within the first 24 h of infection. After 48 h of infection, there was a significant 38% reduction in the group that received MB + light compared to the other groups. A 33% reduction in infectivity was observed within 24 h of treatment with MB and 58% when using MB + light. The infection rate of parasite macrophages was 71% lower in the group that received MB + light than the control. Also, when compared to the group that received only MB, the infection rate was 48% lower in the group that received MB + light after 24 h of treatment.

The studies by Song et al. (6969. Song D, Lindoso JAL, Oyafuso LK, Kanashiro EHY, Cardoso JL, Uchoa AF, et al. Photodynamic therapy using methylene blue to treat cutaneous leishmaniasis. Photomed Laser Surg 2011; 29: 711-715, doi: 10.1089/pho.2010.2915.
https://doi.org/10.1089/pho.2010.2915...
) and de Oliveira et al. (7171. de Oliveira S, da Ordem Trahamane EJ, Monteiro J, Santos GP, Crugeira P, Sampaio F, et al. Leishmanicidal effect of antiparasitic photodynamic therapy—ApPDT on infected macrophages. Lasers Med Sci 2017; 32: 1959-1964, doi: 10.1007/s10103-017-2292-9.
https://doi.org/10.1007/s10103-017-2292-...
) showed that, although MB has activity in parasitic cells, the use of PDT together with MB enhances its action, in addition to allowing the decrease in PS concentration. Furthermore, Song et al. (6969. Song D, Lindoso JAL, Oyafuso LK, Kanashiro EHY, Cardoso JL, Uchoa AF, et al. Photodynamic therapy using methylene blue to treat cutaneous leishmaniasis. Photomed Laser Surg 2011; 29: 711-715, doi: 10.1089/pho.2010.2915.
https://doi.org/10.1089/pho.2010.2915...
) and Peloi et al. (7070. Peloi LS, Eduardo C, Biondo G, Kimura E, José M, Valdrinez M, et al. Photodynamic therapy for American cutaneous leishmaniasis: the efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis. Exp Parasitol 2011; 128: 353-356, doi: 10.1016/j.exppara.2011.04.009.
https://doi.org/10.1016/j.exppara.2011.0...
) found that the use of PDT and MB healed the wounds caused by L. (L.) amazonensis. Therefore, the use of PDT associated with the phenothiazine compound is effective in both in vitro and in vivo assays, helping to decrease the parasitic rate and infection rate, as well as reducing the concentration of drugs already used in current therapy.

Pinto et al. (7272. Pinto JG, Martins JFS, Pereira AHC, Mittmann J, Raniero LJ, Ferreira-Strixino J. Evaluation of methylene blue as photosensitizer in promastigotes of Leishmania major and Leishmania braziliensis. Photodiagnosis Photodyn Ther. 2017; 18: 325-330, doi: 10.1016/j.pdpdt.2017.04.009.
https://doi.org/10.1016/j.pdpdt.2017.04....
) carried out a study in which they evaluated the internalization and cell location of the MB along with cell viability and morphology after the application of PDT in the species L. (L.) major and L. (V.) braziliensis. The internalization results showed that PS does not accumulate in organelles but rather remains in the cytosol of the parasitic cell. The most intriguing results were demonstrated in the tests of cell viability and mitochondrial activity. Although mitochondrial activity was altered in the control groups, the trypan blue viability assay showed that MB is not toxic in any species without light exposure. Furthermore, after the application of PDT, viability was significantly reduced, showing that the variation in mitochondrial activity does not necessarily mean a change in cell viability.

These results are in contrast to those reported by Song et al. (6969. Song D, Lindoso JAL, Oyafuso LK, Kanashiro EHY, Cardoso JL, Uchoa AF, et al. Photodynamic therapy using methylene blue to treat cutaneous leishmaniasis. Photomed Laser Surg 2011; 29: 711-715, doi: 10.1089/pho.2010.2915.
https://doi.org/10.1089/pho.2010.2915...
), who verified MB activity even in the dark after a mitochondria activity test using MTT. In their study, this mitochondrial activity did not always interfere with the cell viability itself since parasites were shown to be viable even with changes in the activity of the mitochondria. In addition to the above, PDT using MB as PS caused morphological changes in the promastigote forms of both species, suggesting interaction with the parasitic cell membrane (7272. Pinto JG, Martins JFS, Pereira AHC, Mittmann J, Raniero LJ, Ferreira-Strixino J. Evaluation of methylene blue as photosensitizer in promastigotes of Leishmania major and Leishmania braziliensis. Photodiagnosis Photodyn Ther. 2017; 18: 325-330, doi: 10.1016/j.pdpdt.2017.04.009.
https://doi.org/10.1016/j.pdpdt.2017.04....
).

Sbeghen et al. (7373. Sbeghen MR, Voltarelli EM, Campois TG, Kimura E, Aristides SMA, Hernandes L, et al. Topical and intradermal efficacy of photodynamic therapy with methylene blue and light-emitting diode in the treatment of cutaneous leishmaniasis caused by Leishmania braziliensis. J Lasers Med Sci 2015; 6: 106-111, doi: 10.15171/jlms.2015.03.
https://doi.org/10.15171/jlms.2015.03...
) conducted a study comparing the action of PDT + MB administered intradermally and topically in lesions caused by L. (V.) braziliensis on hamster footpads. The authors observed that the MB applied intradermally did not cure or decrease the lesion. However, topical treatment reduced and healed lesions in 30% of the animals after nine weeks. Similar to the findings of Peloi et al. (7070. Peloi LS, Eduardo C, Biondo G, Kimura E, José M, Valdrinez M, et al. Photodynamic therapy for American cutaneous leishmaniasis: the efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis. Exp Parasitol 2011; 128: 353-356, doi: 10.1016/j.exppara.2011.04.009.
https://doi.org/10.1016/j.exppara.2011.0...
), the parasitic burden observed in the lymph nodes and spleen was low for animals treated with topical MB. Also, the treatment restored the lesion area and decreased inflammation (7373. Sbeghen MR, Voltarelli EM, Campois TG, Kimura E, Aristides SMA, Hernandes L, et al. Topical and intradermal efficacy of photodynamic therapy with methylene blue and light-emitting diode in the treatment of cutaneous leishmaniasis caused by Leishmania braziliensis. J Lasers Med Sci 2015; 6: 106-111, doi: 10.15171/jlms.2015.03.
https://doi.org/10.15171/jlms.2015.03...
).

MB proved to be an interesting photosensitizer for the treatment of Leishmania along with the PDT technique. We found that the studies showed good results both in vitro and in vivo, leading to a decrease of parasite load, reduction of lesions in both animals and humans, as well as action on several Leishmania species.

Concluding remarks

PDT has proven to be a useful technique for the treatment of CL since it has a low cost, is non-invasive, and has low toxicity compared to conventional therapies. Although some adjustments are necessary, the PSs studied so far have shown promising results. In this review, a range of PSs and several methodologies were explored. These studies are important to assist in the search for increasingly efficient PSs against parasitic forms.

We can highlight some of the aspects observed to be important in the development of a new PS, including the lipophilicity and amphiphilicity of compounds, the charges, the electrostatic interaction, and the presence or absence of metal (Figure 3). All of these factors interfered in some way during the studies.

Figure 3
Key features to consider in increasing the effectiveness of photosensitizers (PS) against Leishmania spp.

As seen, porphyrins are active against parasitic forms, and a prodrug such as ALA can be used. Although it is not active on its own, ALA can be transformed into porphyrin (2626. Kaneko JJ. Porphyrins and the porphyrias. Clin Biochem Domest Anim 2008; 241-258, doi: 10.1016/B978-0-12-370491-7.00008-8.
https://doi.org/10.1016/B978-0-12-370491...
,3030. Kennedy JC, Pottier RH. New trends in photobiology. Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy. J Photochem Photobiol B 1992; 14: 275-292, doi: 10.1016/1011-1344(92)85108-7.
https://doi.org/10.1016/1011-1344(92)851...
,3131. Kosaka S, Akilov OE, O'Riordan K, Hasan T. A mechanistic study of δ-aminolevulinic acid-based photodynamic therapy for cutaneous leishmaniasis [6]. J Invest Dermatol 2007; 127: 1546-1549, doi: 10.1038/sj.jid.5700719.
https://doi.org/10.1038/sj.jid.5700719...
). Furthermore, cationic porphyrins interacted better with parasitic cells since the parasite has a negative charge on its membrane surface, improving the electrostatic interaction of cationic compounds (4040. Bristow CA, Hudson R, Paget TA, Boyle RW. Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis. Photodiagnosis Photodyn Ther 2006; 3: 162-167, doi: 10.1016/j.pdpdt.2006.04.004.
https://doi.org/10.1016/j.pdpdt.2006.04....
,4141. Zhang K, Beverley SM. Phospholipid and sphingolipid metabolism in Leishmania. Mol Biochem Parasitol 2010; 170: 55-64, doi: 10.1016/j.molbiopara.2009.12.004.
https://doi.org/10.1016/j.molbiopara.200...
).

The presence of metal in the structure is another point to be considered, since an improvement in the activity of PSs has been observed. In addition, care should be taken when examining hydrophobicity, as highly hydrophobic molecules may not interact optimally. Like the amphiphilic molecules, they present better activity compared to hydrophobic molecules (4242. Andrade CG, Figueiredo RCBQ, Ribeiro KRC, Souza LIO, Sarmento-Neto JF, Rebouças JS, et al. Photodynamic effect of zinc porphyrin on the promastigote and amastigote forms of: Leishmania braziliensis. Photochem Photobiol Sci 2018; 17: 482-490, doi: 10.1039/C7PP00458C.
https://doi.org/10.1039/C7PP00458C...
,4747. Escobar P, Hernández IP, Rueda CM, Martínez F, Páez E. Photodynamic activity of aluminium (III) and zinc (II) phthalocyanines in Leishmania promastigotes. Biomedica 2006; 26: 49-56, doi: 10.7705/biomedica.v26i1.1499.
https://doi.org/10.7705/biomedica.v26i1....
).

Finally, the use of techniques such as adding PSs to liposomes seems interesting. As we have seen in some studies, this strategy improves the efficiency of PSs and decreases toxicity (5151. Ribeiro JBP, Miranda-Vilela AL, Graziani D, Gomes MRA, Amorim AAS, Garcia RD, et al. Evaluation of the efficacy of systemic miltefosine associated with photodynamic therapy with liposomal chloroaluminium phthalocyanine in the treatment of cutaneous leishmaniasis caused by Leishmania (L.) amazonensis in C57BL/6 mice. Photodiagnosis Photodyn Ther 2016; 13: 282-290, doi: 10.1016/j.pdpdt.2015.08.006.
https://doi.org/10.1016/j.pdpdt.2015.08....
,5555. de Siqueira LBO, Cardoso VS, Rodrigues IA, Vazquez-Villa AL, dos Santos EP, Guimarães BCLR, et al. Development and evaluation of zinc phthalocyanine nanoemulsions for use in photodynamic therapy for Leishmania spp. Nanotechnology 2017; 28: 065101, doi: 10.1088/1361-6528/28/6/065101.
https://doi.org/10.1088/1361-6528/28/6/0...
,5656. Escobar P, Vera AM, Neira LF, Velásquez AO, Carreão H. Photodynamic therapy using ultradeformable liposomes loaded with chlorine aluminum phthalocyanine against L. (V.) braziliensis experimental models. Exp Parasitol 2018; 194: 45-52, doi: 10.1016/j.exppara.2018.09.016.
https://doi.org/10.1016/j.exppara.2018.0...
).

Therefore, the study of PSs, especially in planning and development studies, should pay special attention to the efficiency in producing singlet oxygen while also adopting a molecular perspective, observing all the structural aspects important for the interaction between the PS and the target (1717. Bacellar IOL, Tsubone TM, Pavani C, Baptista MS. Photodynamic efficiency: From molecular photochemistry to cell death. Int J Mol Sci 2015; 16: 20523-20559, doi: 10.3390/ijms160920523.
https://doi.org/10.3390/ijms160920523...
).

Acknowledgments

This research was funded by the São Paulo Research Foundation (FAPESP, grant #2018/24190-7), CEPID Redoxoma (grant #2013/07937-8), and CNPq (grant #303831/2019-7).

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Publication Dates

  • Publication in this collection
    29 Oct 2021
  • Date of issue
    2021

History

  • Received
    17 May 2021
  • Accepted
    26 Aug 2021
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