ORIGINAL ARTICLE

Photodynamic effects of zinc phthalocyanineson intracellular amastigotes of Leishmania amazonensisand Leishmania braziliensis

Emanoel Pedro de Oliveira Silva & Josane Mittmann &

Vitória Tonini Porto Ferreira & Maria Angélica Gargione Cardoso &

Milton Beltrame Jr.

Received: 29 August 2013 /Accepted: 17 September 2014# Springer-Verlag London 2014

Abstract This study investigated the photoactivity of fourzinc phthalocyanines (PcZns) on a murine macrophage cellline infected with Leishmania amazonensis or Leishmaniabraziliensis. Infected and uninfected cells were incubated withPcZns at different concentrations (1–10 μM) for 3 h and thenexposed to an LED device in continuous wave mode at660 nm with a fluency of 50 J/cm2 (25 mV). Enzymaticactivity was determined by MTT assay 24 h after light treat-ment. The results demonstrated that all PcZns exhibited highphotoactivity, particularly when used at 10 μM. The photody-namic effects were different for uninfected cells versusparasite-infected cells and among the four PcZns. Uninfectedcells were more sensitive to photoactivity than infected cells.Although PcZns photodynamic therapy provided promisingresults, further studies are necessary to better understand itsmechanism of action in the treatment of leishmaniasis.

Keywords Leishmania amazonensis . Leishmaniabraziliensis . Murinemacrophages (J774.A1) . Photodynamictherapy . Zinc phthalocyanines

Introduction

Cutaneous leishmaniasis (CL) is an infectious disease causedby genus Leishmania protozoa. This parasite is transmitted tohumans through the bite of female Lutzomyia sandfly species(although 30 other species are also known to transmit leish-maniasis) and occurs in 88 countries [1]. The life cycle ofprotozoa involves two distinct forms: the promastigote, whichis found in the vector and is involved in transmission, and theamastigote, the intracellular form found in mammalian cells[2]. Because of its high rate of detection and ability to causebodily deformities, leishmaniasis is considered to be one ofthe six most important infectious diseases [1]. Clinical mani-festations vary with the Leishmania sp. and include chroniculcers (skin), facial disfigurement (cutaneous), and, in severecases, symptoms involving the reticuloendothelial system(systemic) [3]. The World Health Organization (WHO) esti-mated that 350 million people are at risk of contracting leish-maniasis, with approximately 58,000 new cases of CL and220,000 cases of visceral leishmaniasis (VL) reported per year[1]. In addition, 90 % of all CL cases on record occurred inonly six countries: Iran, Saudi Arabia, Syria, Afghanistan,Peru, and Brazil [4]. From 1990 to 2010, an annual averageof 31,680 autochthonous cases of CL was recorded in Brazil.Additionally, in 2003, autochthony was confirmed in all Bra-zilian states [5–7]. According to the Brazilian Center of Epi-demiological Surveillance, between 1998 and 2012, 8338cases of leishmaniasis were recorded in São Paulo state, withan additional of 237 cases in 2012 [8].

Although antiparasitic drugs are commercially available,the number of cases of leishmaniasis has not decreasedbecause Leishmania sp. has been acquiring resistance tocommonly prescribed medications [9]. Conventionaltreatment includes the administration of pentavalent an-timonials for at least 20 days; however, these drugs

E. P. d. O. Silva :V. T. P. Ferreira :M. Beltrame Jr. (*)Laboratório de Síntese Orgânica, Instituto de Pesquisa eDesenvolvimento, Universidade do Vale do Paraíba, 2911 ShishimaHifumi Avenue, 12244-000 São José dos Campos, SP, Brazile-mail: beltrame@univap.br

J. MittmannLaboratório de Parasitologia e Biotecnologia, Instituto de Pesquisa eDesenvolvimento, Universidade do Vale do Paraíba, 2911 ShishimaHifumi Avenue, 12244-000 São José dos Campos, SP, Brazil

M. A. G. CardosoLaboratório de Imunologia, Instituto de Pesquisa eDesenvolvimento, Universidade do Vale do Paraíba, 2911 ShishimaHifumi Avenue, 12244-000 São José dos Campos, SP, Brazil

Lasers Med SciDOI 10.1007/s10103-014-1665-6

should not be administered to pregnant women or pa-tients with renal or hepatic failure, cardiac arrhythmia,and Chagas disease [4, 10].

Many studies have investigated the use of alternativetherapies for the treatment of leishmaniasis, includingimmunotherapy and photodynamic therapy (PDT),which has demonstrated effectiveness against leishman-iasis [11]. Peloi et al. [12] and Gardner et al. [13]obtained promising results using PDT in combinationwith methylene blue or acenaphthoporphyrins, respec-tively, for treating leishmaniasis.

PDT is used for various treatments in several medicalfields, including treatment of cancer, skin diseases, and mi-crobial infections [5, 14]. PDT is based on the administrationof a photosensitizing agent followed by excitation with a lightsource. These processes produce reactive oxygen species(ROS) that are responsible for cell death [15, 16]. In addition,if the energy densities and optical power are high enough, thenthe PDT may also cause thermal damage by increasing thetemperature in the exposed tissue [17].

Phthalocyanines (Pcs), which are classified as second-generation photosensitizers, have advantageous photophysicalproperties, and extensive studies have demonstrated their po-tential for PDT [18]. Due to their strong absorption spectrum inthe red light range (670–780 nm), low dark toxicity, highefficiency in generating ROS, and excellent selectivity fortumor targeting, phthalocyanines have been used for industrial,technological, and medical applications [19, 20].

Zinc phthalocyanines (PcZns) have been intensively stud-ied for their d10 configuration of the central Zn+2 ion, resultingin an optical spectrum that is not influenced by additionalbands. They also have strong absorption in the red range andhigh quantum yield in their singlet and triplet states. There-fore, PcZns are optimal for PDT, with significant results inin vitro and in vivo studies [20, 21]. Therefore, based on thesepromising findings regarding the use of PDT to treat leish-maniasis, this study evaluated the photodynamic activity oftetrasubstituted zinc phthalocyanines in an in vitro model ofinfection in murine macrophages (J774.A1) by Leishmaniaamazonensis and Leishmania braziliensis.

Materials and methods

General The molecular structures of photosensitizes areshown in Fig. 1. The PcZn and zinc tetrasulfonate phthalocy-anine (PcZn4SO3H) used in this study were purchased fromSigma-Aldrich (Sigma-Aldrich Chemie Gmbh, Steinheim,Germany). The zinc tetranitrophthalocyanine (PcZn4NO2)and zinc tetraaminephthalocyanine (PcZn4NH2) used in thisstudy were prepared and characterized as previously described[22]. 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazoliumbromide (MTT) was purchased from Sigma-Aldrich. Absorp-tion measurements were carried out on the sample with aspectrophotometer UV/VIS (Cary 50 BIO, Varian Inc.Scientific Instruments, Australia) and quartz cuvettes type

Fig. 1 Molecular structures oftetrasubstituted zincphthalocyanines. Zincphthalocyanine (PcZn), zinctetranitrophthalocyanine(PcZn4NO2), zinc tetraminophthalocyanine (PcZn4NH2), andzinc tetrasulfonatephthalocyanine (PcZn4SO3H)

Lasers Med Sci

111-QS 10 mm (Hellma Analytics, Müllheim, Germany).Irradiation treatment was carried out with an LED dispositive(Biopd i / I r r ad -Led 5 , Equ ipamentos Médicos eOdontológicos, São Carlos, Brazil). The MTT analyses wereperformed in an enzyme-linked immunosorbent assay(ELISA) Spectracount Reader (Packard Instrument CompanyInc., Netherlands).

Cell culture J774.A1 murine macrophages (American TypeCulture Collection [ATCC], no. TIB 67) were cultured in 25-cm2 flasks (TPP, Switzerland) with RPMI medium (Gibco,Invitrogen Corp., Grand Island, NY, USA), supplementedwith 10 % fetal bovine serum and 1 % antibiotic andantimycotic (Gibco). Cells were maintained in a humidifiedatmosphere containing 5 % CO2 at 37 °C.

Parasites L. amazonensis (M2269) and L. braziliensis(M2904) promastigotes were cultured in LIT medium supple-mented with 10% heat-inactivated fetal bovine serum, 2.5 μg/mL hemin, 2 % urine and 1 % penicillin (10,000 UI/mL), and10,000 μg/mL streptomycin at 26 °C.

PDT The study was performed twice in duplicate exper-iments using four experimental groups: control (cellswith no photosensitizer and no light), LED (cells withno photosensitizer and irradiated with LED light), dark(cells incubated with PcZns and no LED irradiation),and PDT (cells incubated with PcZns and irradiatedwith LED light). J774.A1 cells were counted, and 1×104 cells/mL/well were seeded in 96-well plates andcultured for 24 h. Infection assays were performed usinga macrophage to paras i te ra t io of 1 :10 wi thL. amazonensis or L. braziliensis stationary-phasepromastigotes for 24 h. This condition provided aninfection rate of 98 % with 5–7 amastigotes per cell[23]. Infected and uninfected cells in the dark and PDTcell groups were incubated with photosensitizers dilutedin phosphate-buffered saline (PBS) at 1, 5, or 10 μM,then incubated in the dark at 37 °C with 5 % CO2 for3 h [24]. After this period, the supernatant wasdiscarded, and the wells were washed twice with PBSto remove unabsorbed photosensitizers. Afterwards,200 μL of PBS was added in each well to the platescontaining cultures (LED and PDT groups). Next, theplates were irradiated with an LED device composed of54 red LEDs, each one with 70 mW uniformly distrib-uted across the device [25]. This distribution allowed ahomogenous illumination of the entire surface of the 96-well plate with a light intensity of 25 mW/cm2 at660 nm and a fluency of 50 J/cm2 in continuous wavemode for 33 min. The LED device had a coolingsystem to maintain the temperature, preventing thermaldamage. After irradiation, PBS was removed, and RPMI

medium was added to each well. The plates were thenincubated under the growth conditions described abovefor 20 h.

Determination of mitochondrial dehydrogenase activity Theenzymatic activity of mitochondrial dehydrogenase was basedon the reduction of MTT. Viable cells produce purpleformazan crystals as a reaction product, indicating the levelof cell respiration. After each period of incubation, cells wereincubated with 0.5 mg/mL MTT for 2 h according to themethods described by Simioni et al. [26]. Results were readusing an ELISA plate reader with a 570-nm filter.

Statistical analysis Graphs of the average shunting line stan-dard were generated, and statistical analyses were performedusing GraphPad software, version 5.0 (GraphPad Software,Inc., La Jolla, CA, USA). The differences between control andtreatment groups were measured using analysis of variance(ANOVA), and differences with p values of less than 0.01were considered statistically significant. The data were plottedas a percentage of the control.

Results

All PcZns studied were essentially nontoxic in the dark, butexhibited substantial photodynamic activity against J774.A1cells uninfected or infected with L. amazonensis orL. braziliensis after LED irradiation (Figs. 2, 3, 4, and 5).Cultures treated with light only exhibited no significantchanges in cell viability, as indicated by levels of mitochon-drial dehydrogenase activity. Figure 2 shows the means andstandards deviations of enzymatic activity in uninfectedJ774.A1 macrophages (Fig. 2a) and macrophages infectedwith L. amazonensis (Fig. 2b) or L. braziliensis (Fig. 2c)20 h after treatment with PcZns. Significant variations inenzymatic activity in uninfected and infected macrophageswere observed (p<0.01 using two-way ANOVA) in relationto the control. The use of 10 μM photosensitizers was suffi-cient to reduce to the enzymatic activity of uninfectedJ774.A1 cells to 37.7 % (Fig. 2a), that of L. amazonensis-infected cells to 35.9 %, and that of L. braziliensis-infectedcells to 77.9 % that of the control.

The means and standard deviation of mitochondrial dehy-drogenase activity after treatment of uninfected and infectedcells with PcZn4NO2 are shown in Fig. 3. The most dramaticresults were obtained with 10 μM photosensitizers for bothuninfected and infected J774.A1 cells. The enzymatic activityin uninfected J774.A1 cells (Fig. 3a) was reduced to 45.8 %,while those of J774.A1 cells infected with L. amazonensis orL. braziliensis were reduced to 52 % (Fig. 3b) and 44.3 %(Fig. 3c), respectively. Differences in the enzymatic activities

Lasers Med Sci

of uninfected and infected macrophages were significant ac-cording to two-way ANOVA (p<0.01) in relation to thecontrol.

PcZn4NH2 was able to induce a photodynamic effect at allconcentrations tested in both uninfected and infected J774.A1cells (Fig. 4). A dramatic reduction in enzymatic activity afterPDTwas obtained with 10 μM photosensitizer for both unin-fected and infected J774.A1 cells. The viability of uninfectedJ774.A1 cells (Fig. 4a) was reduced to 1.7 %, while those of

J774.A1 cells infected with L. amazonensis or L. braziliensiswere 30.4 % (Fig. 4b) and 5.4 % (Fig. 4c), respectively. Thereduction in enzymatic activity of mitochondrial dehydroge-nase in uninfected and infected macrophages was significantaccording to two-way ANOVA (p<0.01) in relation to thecontrol.

Cells treated with PcZn4SO3H photosensitizer exhibitedthe greatest differences (Fig. 5). We observed a strong and

Fig. 2 Enzymatic activity determined by MTTassay 20 h after PDT andzinc phthalocyanine (PcZn) treatment. a Control, uninfected J774.A1macrophages, b J774.A1 macrophages infected with Leishmaniaamazonensis, and c J774.A1 macrophages infected with Leishmaniabraziliensis. Analysis of variance (ANOVA) showed that the differencesamong the concentrations were significant in relation to the control group(***p<0.01) and between the dark and PDT groups (*p<0.05). Data areexpressed as means±SDs (n=3)

Fig. 3 Enzymatic activity determined by MTTassay 20 h after PDT andzinc tetranitrophthalocyanine (PcZn4NO2) treatment. aControl, uninfect-ed J774.A1 macrophages, b J774.A1 macrophages infected with Leish-mania amazonensis, and c J774.A1 macrophages infected with Leish-mania braziliensis. Analysis of variance (ANOVA) showed that the dif-ferences among the concentrations were significant in relation to thecontrol group (***p<0.01) and between the dark and PDT groups(*p<0.05). Data are expressed as means±SDs (n=3)

Lasers Med Sci

statistically significant (p<0.01) reduction of enzymatic mito-chondrial dehydrogenase activity in uninfected J774.A1 cells,indicating that only 0.2 % of cells were viable (Fig. 5a). Thesame photodynamic effect was observed in infected J774.A1cells. The enzymatic activity of J774.A1 infected withL. amazonensis was reduced to 2.9 % (Fig. 5b), while thatof cells infected with L. braziliensis was reduced to 1.6 %(Fig. 5c). The results from ANOVA showed that the

differences among the treatments were statistically significant(p<0.01).

J774.A1 cells infected with L. amazonensis were less pho-tosensitive when treated with PcZn4NO2, PcZn4NH2, andPcZn4SO3H than those infected with L. braziliensis; however,this effect was not observed when the cells were treated withthe compound PcZn (Fig. 2c). J774.A1 cells infected with theparasites were more resistant to photodynamic effects of the

Fig. 4 Enzymatic activity determined by MTTassay 20 h after PDT andzinc tetraminophthalocyanine (PcZn4NH2) treatment. a Control, unin-fected J774.A1 macrophages, b J774.A1 macrophages infected withLeishmania amazonensis, and c J774.A1 macrophages infected withLeishmania braziliensis. Analysis of variance (ANOVA) showed that thedifferences among the concentrations were significant in relation to thecontrol group (***p<0.01) and between the dark and PDT groups(*p<0.05). Data are expressed as means±SDs (n=3)

Fig. 5 Enzymatic activity determined by MTTassay 20 h after PDT andzinc tetrasulfonate phthalocyanine (PcZn4SO3H) treatment. a Control,uninfected J774.A1 macrophages, b J774.A1 macrophages infected withLeishmania amazonensis, and c J774.A1 macrophages infected withLeishmania braziliensis. Analysis of variance (ANOVA) showed that thedifferences among the concentrations were significant in relation to thecontrol group (***p<0.01) and between the dark and PDT groups(*p<0.05). Data are expressed as means±SDs (n=3)

Lasers Med Sci

tested PcZns when compared to uninfected cells. Aconcentration-dependent response was observed in all PcZnstested in both L. amazonensis- and L. braziliensis-infectedJ774.A1 cells; the light treatment showed that photoactivitywas enhanced when the concentration of photosensitizerincreased.

J774.A1 cells exhibited concentration-dependent re-sponses to PcZn4NO2 and PcZn4NH2 with light treatment,as photoactivity increased with increasing concentrations ofphotosensitizers; however, this effect was not observed whencells were treated with PcZn or PcZn4SO3H. The most effec-tive concentration for infected J774.A1 cells was 10 μM,except for J774.A1 cells infected with L. braziliensis, forwhich the enzymatic activity was 77.9 %.

Discussion

Current treatments for leishmaniasis include injection with apentavalent antimony derivate, amphotericin B, pentamidine,or paromycin [12]. However, these treatments require paren-teral administration and vary in efficacy, toxicity, and cost[27]. PDT treatment of leishmaniasis has been shown to havepromising results and may be a cost-effective therapeuticmodality. PDT does not require expensive, technologicallyadvanced equipment and is convenient to use, even in devel-oping countries [28].

In this in vitro study, PDT using tetrasubstituted zincphthalocyanines in J774.A1 macrophages infected withL. amazonensis or L. braziliensis was strongly dependent onthe cell model studied as well as the type and concentration ofphotosensitizer used. In this study, we only examined 1, 5, and10 μM concentrations of photosensitizers. While the highestconcentration of PcZns seemed to be more effective, aconcentration-response curve could not be fully established.Concentrations below 1 μM demonstrated no photodynamicactivity (data not shown). No significant difference in theenzymatic activity from mitochondrial dehydrogenase wasobserved between the control group (no treatment) and thedark and LED groups. These results were consistent with PDTprinciples, in which application of the dye or the light sourcealone has no effect on cell viability, but exhibits photodynamicactivity when irradiated [29].

PDT treatment showed no selectivity, reducing the enzy-matic activity in both infected and uninfected J774.A1 cells(health cells model). However, photodynamic activity mightbe controlled by the location at which the light is applied,restricting the PDT effect to the site of administration, such asa leishmaniasis lesion on the skin [3].

Differences in drug sensitivities between the two intracel-lular Leishmania strains and uninfected cells were observed.These results are believed to be related to molecular differ-ences between L. amazonensis and L. braziliensis. Nearly 15

species of Leishmania are known to cause disease in humans,and each species has specific biochemical and molecularcharacteristics that define its taxonomy and virulence [27,30]. These different characteristics are thought to be reflectedin the variable photosensitivity observed in J774.A1 cellsinfected with L. amazonensis and L. braziliensis. Similarresults were observed when aluminum phthalocyaninetetrasulfonate (10 μM) and irradiation (10 J/cm2) wasused against promastigotes of Leishmania major andL. braziliensis [5].

On the other hand, these photosensitive differences mightalso be responsible for the physicochemical differences be-tween the phthalocyanines. Most phthalocyanines are prone toself-aggregation and formation of dimers in aqueous solution,which are reported to be inactive or much more inefficientthan monomers as photosensitizers [27, 31]. However, themechanisms mediating the susceptibility of infected cells arestill unknown.

In this work, we compared four PcZns with different sub-stituents at 1–10μM, combinedwith irradiation at 660 nm anda fluency of 50 J/cm2 (25 mW/cm2) for 33 min. Differences inphotodynamic activity between PcZns could be attributed tothe presence of polar substituents (−NH2 and −SO3H) in thephthalocyanine structure.

The PcZns used in this study have the followed organicgroups as substituents: nitro (−NO2) for PcZn4NO2, amine(−NH2) for PcZn4NH2, and sulfonic acid (−SO3H) forPcZn4SO3H. Each PcZn-based molecule has characteristicsthat affect both chemical and physical properties of the mol-ecule and was compared with PcZn as a standard because thisparental molecule was unsubstituted.

Increased molecular polarity decreases self-aggregation inaqueous solution, and these changes could be responsible forenhanced photodynamic activity. In our study, thePcZn4SO3H compound exhibited the most photoactivity (inboth infected and uninfected cells). Because PcZn4SO3H hasthe highest polarity and the best water solubility comparedwith the other PcZns evaluated, the cellular uptake of thisphotosensitizer could have been improved, thereby contribut-ing to its increased photodynamic efficiency. Similar resultswere obtained by Akilov et al. [23] using phenothiaziniumanalogs; these results were attributed to the polarity of 5-ethylamino-9-diethylamino-benzo[α]phenosenazium chlo-ride, which had a higher photodynamic activity than its non-polar analogs against L. major. These results were furthersupported by more recent studies [20, 32].

The mechanism of action of PDT invokes the excited stateof a photosensitizer, activating local molecular oxygen intoROS, which ultimately induces cell death through direct andindirect cytotoxicity, thus reducing enzymatic activity [33,34]. The defense mechanism of intracellular parasites againstROS is considered to be involved in the efficient production ofreducing agents [15]. However, the mechanical functions of

Lasers Med Sci

PDT in intracellular Leishmania are still unknown. Subcellu-lar zinc octabromide phthalocyanine fluorescence in L929cells has been shown to be located in the perinuclear region,endosomes, and lysosomes [24]. PDTwith 10 μMpyridyloxyPcZn derivatives and 2 J/cm2 against intracellularL. amazonensis has been shown to reduce mitochondrialactivity by about 10 % [35]. Therefore, it is possible that celldeath in Leishmania is related to apoptosis-l ikephotosensitizing action on mitochondria [5, 13]. Further stud-ies are required to better understand if the mechanism of celldeath in Leishmania resembles apoptosis, necrosis, or both, asreported for other intracellular parasites [36].

Conclusion

In summary, our results showed that tetrasubstituted PcZnshad high photodynamic activity against macrophages infectedwith L. amazonensis or L. braziliensis. The photodynamicactivities of these compounds were greatly enhanced by theincreased polarity of its tetrasubstituents. PcZn4SO3H exhib-ited the highest in vitro photodynamic activity; however,further studies are needed to investigate its in vivo topicalactivity and to optimize the irradiation fluencies for imple-mentation of appropriate treatments for CL. The knowledgeobtained in this work can provide information to promote ourunderstanding of the structure-activity relationships of PcZns,which will improve the design of new drugs and PDTs againstleishmaniasis.

Acknowledgments The authors wish to thank Juliana Ferreira (Lab.Terapia Fotodinâmica—IP&D/UNIVAP) for the LED equipment used inthis study and Alene Alder-Rangel for assistance with English languagerevisions.

References

1. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano P, Jannin J,den Boer M (2012) Leishmaniasis worldwide and global estimates ofits incidence. PLoS ONE 7(5):e35671. doi:10.1371/journal.pone.0035671

2. Fernández MM, Malchiodi EL, Algranati ID (2011) Differentialeffects of paromomycin on ribosomes of Leishmania mexicana andmammalian cells. J Antimicrob Chemother 55(1):86–93. doi:10.1128/AAC.00506-10

3. Gardlo K, Horska Z, Enk CD, Rauch L, Megahed M, Ruzicka T,Fritsch C (2003) Treatment of cutaneous leishmaniasis by photody-namic therapy. J Am Acad Dermatol 48:893–896. doi:10.1067/mjd.2003.218

4. Gontijo B, Carvalho MLR (2003) Leishmaniose TegumentarAmericana. Rev Soc Bras Med Trop 36(1):71–80

5. Pinto JG, Soares CP, Mittmann J (2011) Assessment of Leishmaniamajor and Leishmania braziliensis promastigote viability after pho-todynamic treatment with aluminum phthalocyanine tetrasulfonate(AlPcS4). J Venom Anim Toxins Incl Trop Dis 17(3):300–307. doi:10.1590/S1678-91992011000300010

6. Ministério da Saúde (2011) Casos de Leishmaniose TegumentarAmericana. http://portal.saude.gov.br/portal/arquivos/pdf/lta_casos08_09_11.pdf. Accessed 28 August 2013

7. Ministério da Saúde (2011) Casos de Leishmaniose visceral. http://portal.saude.gov.br/portal/arquivos/pdf/lv_casos_05_09_11.pdf.Accessed 28 August 2013

8. Centro de Vigilância Epidemiológica (2012) São Paulo: Secretaria daSaúde. http://www.cve.saude.sp.gov.br/htm/zoo/lta_gve_notres.htm.Accessed 28 August 2013

9. Moreira W, Leprohon P, Ouellette M (2011) Tolerance to drug-induced cell death favours the acquisition of multidrug resistance inLeishmania. Cell Death Dis 2:e201. doi:10.1038/cddis.2011.83

10. Seifert K, Munday J, Syeda T, Croft SL (2011) In vitro interactionsbetween sitamaquine and amphotericin B, sodium stibogluconate,miltefosine, paromomycin and pentamidine against Leishmaniadonovani. J Antimicrob Chemother 66(4):850–854. doi:10.1093/jac/dkq542

11. Akilov OE, Yousaf W, Lukjan SX, Verma S, Hasan T (2009)Optimization of topical photodynamic therapy with 3,7-bis(di-n-butylamino)phenothiazin-5-ium bromide for cutaneousLeishmaniasis. Lasers Surg Med 41:358–365. doi:10.1002/lsm.20775

12. Peloi LS, Biondo CEG, Kimura E, Politi MJ, Lonardoni MVC,Aristides SMA, Dorea RCC, Hioka N, Silveira TGV (2011)Photodynamic therapy for American cutaneous leishmaniasis: theefficacy of methylene blue in hamsters experimentally infected withLeishmania (Leishmania) amazonensis. Exp Parasitol 128(4):353–356. doi:10.1016/j.exppara.2011.04.009

13. Gardner DM, Taylor VM, Cedeño DL, Padhee S, Robledo SM, JonesMA, Lash TD, Vélez ID (2010) Association of acenaphthoporphyrinswith liposomes for the photodynamic treatment of leishmaniasis.Photochem Photobiol 86(3):645–652. doi:10.1111/j.1751-1097.2010.00705.x

14. Machado AHA, Moraes KCM, Soares CP, Beltrame Junior M, daSilva NS (2010) Cellular changes after photodynamic therapy onHEp-2 cells using the new ZnPcBr8 phthalocyanine. Photomed LaserSurg 28:S143–S149. doi:10.1089/pho.2009.2561

15. Batista MS, Wainwright M (2011) Photodynamic antimicrobial che-motherapy (PACT) for the treatment of malaria, leishmaniasis andtrypanosomiasis. Braz J Med Biol Res 44:1–10. doi:10.1590/S0100-879X2010007500141

16. Çamur M, Ahsen V, Durmus M (2011) The first comparison ofphotophysical and photochemical properties of non-ionic, ionic andzwitterionic gallium (III) and indium (III) phthalocyanines. JPhotochem Photobiol A 219:217–227. doi:10.1016/j.jphotochem.2011.02.014

17. Simpson ER, Wilson BC, Corriveau C, Murphy J (1987) Thermaldamage and haematoporphyrin-derivative-sensitized photochemicaldamage in laser irradiation of rabbit retina. Laser Med Sci 2(1):33–40. doi:10.1007/BF02594129

18. Silva EPO, Barja PR, Cardoso LE, Beltrame M Jr (2012)Percutaneous permeation measurement of topical phthalocyanineby photoacoustic technique. J Appl Phys 112:104702. doi:10.1063/1.4761974

19. Jiang XJ, Huang JD, Zhu YJ, Tang FX, Ng DKP, Sun JC (2006)Preparation and in vitro photodynamic activities of novel axiallysubstituted silicon (IV) phthalocyanines and their bovine serum al-bumin conjugates. Bioorg Med Chem Lett 16:2450–2453. doi:10.1016/j.bmcl.2006.01.075

20. Durmus M, Yaman H, Göl C, Ahsen V, Nyokong T (2011) Water-soluble quaternized mercaptopyridine-substituted zinc-phthalocya-nines: synthesis, photophysical, photochemical and bovine serumalbumin binding properties. Dyes Pigments 91:153–163. doi:10.1016/j.dyepig.2011.02.007

21. Bolfarini GC, Siqueira-Moura MP, Demets GJ, Morais PC, TedescoAC (2012) In vitro evaluation of combined hyperthermia and

Lasers Med Sci

photodynamic effects using magnetoliposomes loaded withcucurbituril zinc phthalocyanine complex on melanoma. JPhotochem Photobiol B 115:1–4. doi:10.1016/j.jphotobiol.2012.05.009

22. Karaoğlana GK, GümrükçüaG, Kocab A, Gülc A, Avcıataa U (2011)Synthesis and characterization of novel soluble phthalocyanines withfused conjugated unsaturated groups. Dyes Pigments 90(1):11–20.doi:10.1016/j.dyepig.2010.10.002

23. Akilov OE, Kosaka S, O’riordan K, Hasan T (2007) Parasiticidaleffect of d-aminolevulinic acid-based photodynamic therapy for cu-taneous leishmaniasis is indirect and mediated through the killing ofthe host cells. Exp Dermatol 16:651–660. doi:10.1111/j.1600-0625.2007.00578.x

24. Machado AHA, Braga FMP, Soares CP, PelissonMMM,BeltrameMJr, da Silva NS (2007) Photodynamic therapy with a newphotosensitizing agent. Photomed Laser Surg 25:220–228. doi:10.1089/pho.2006.2035

25. Carvalho DPL, Pinto JG, Sorge CPC, Benedito FRR, Khouri S,Strixino JF (2014) Study of photodynamic therapy in the control ofisolated microorganisms from infected wounds - an in vitro study.Laser Med Sci 29:113–120. doi:10.1007/s10103-013-1283-8

26. Simioni AR, Primo FL, Tedesco AC (2012) Silicon (IV)phthalocyanine-loaded-nanoparticles for application in photodynam-ic process. J Laser Appl 24:012004. doi:10.2351/1.3669442

27. Escobar P, Hernández IP, Rueda CM, Martínez F, Páez E (2006)Photodynamic activity of aluminium (III) and zinc (II) phthalocya-nines in Leishmania promastigotes. Biomedica 26:49–56

28. Akilov OE, Kosaka S, O’Riordan K, Hasan T (2007) Photodynamictherapy for cutaneous leishmaniasis: the effectiveness of topicalphenothiaziniums in parasite eradication and Th1 immune responsestimulation. Photochem Photobiol Sci 6:1067–1075. doi:10.1039/B703521G

29. Costa AC, Chibebe J Jr, Pereira CA, Machado AK, Beltrame M Jr,Junqueira JC, Jorge AO (2010) Susceptibility of planktonic cultures

of Streptococcus mutans to photodynamic therapy with a light-emitting diode. Braz Oral Res 24(4):413–418. doi:10.1590/S1806-83242010000400007

30. Souza VL, Veras PS, Welby-Borges M, Silva TM, Leite BR, FerraroRB, Meyer-Fernandes JR, Barral A, Costa JM, de Freitas LA (2011)Immune and inflammatory responses to Leishmania amazonensisisolated from different clinical forms of human leishmaniasis inCBA mice. Mem Inst Oswaldo Cruz 106(1):23–31. doi:10.1590/S0074-02762011000100004

31. Neves SMT, Sguilla FS, Tedesco AC (2006) Photophysical studies ofzinc phthalocyanine incorporated into liposomes in the presence ofadditives. Braz J Med Biol Res 37:273–284. doi:10.1590/S0100-879X2004000200016

32. Kimani SG, Shmigol TA, Hammond S, Phillips JB, Bruce JI,MacRobert AJ, Malakhov MV, Golding JP (2013) Fully protectedglycosylated Zinc (II) phthalocyanine shows high uptake and photo-dynamic cytotoxicity in MCF-7 cancer cells. Photochem Photobiol89(1):139–149. doi:10.1111/j.1751-1097.2012.01204.x

33. Hooker JD, Nguyen VH, Taylor VM, Cedeño DL, Lash TD, JonesMA, Robledo SM, Vélez ID (2012) New application for expandedporphyrins: sapphyrin and heterosapphyrins as inhibitors of leish-mania parasites. Photochem Photobiol 88:194–200. doi:10.1111/j.1751-1097.2011.01034.x

34. Choudhary S, Nouri K, Elsaie M (2009) Photodynamic therapy indermatology: a review. Lasers Med Sci 24(6):971–980. doi:10.1007/s10103-009-0716-x

35. Dutta S, Ongarora BG, Li H, Vicente MGH, Kolli BK, Chang KP(2011) Intracellular targeting specificity of novel phthalocyaninesassessed in a host-parasite model for developing potential photody-namic medicine. PLoS ONE 6(6):e20786. doi:10.1371/journal.pone.0020786

36. Cuervo P, Fernandes N, Jesus JB (2011) A proteomics view ofprogrammed cell deathmechanisms during host-parasite interactions.J Proteomics 75(1):246–256. doi:10.1016/j.jprot.2011.07.027

Lasers Med Sci