Combinational Sensitization of Leishmania with Uroporphyrin andAluminum Phthalocyanine Synergistically Enhances their PhotodynamicInactivation in vitro and in vivo†

Sujoy Dutta*, Kayoko Waki and Kwang Poo Chang

Department of Microbiology ⁄ Immunology, Chicago Medical School ⁄ RFUMS, North Chicago, IL

Received 18 October 2011, accepted 27 December 2011, DOI: 10.1111 ⁄ j.1751-1097.2012.01076.x

ABSTRACT

Leishmania were previously shown to undergo photolysis when

their transgenic mutants were induced endogenously to accumu-

late cytoplasmic uroporphyrin or when loaded exogenously with

aluminum phthalocyanine chloride. A combinational use of both

is reported here, which renders Leishmania far more susceptible

to photolysis. Fluorescence microscopy of cells loaded with the

two photosensitizers localized them to different subcellular sites.

Pre-exposure of Leishmania to both synergistically sensitized

them for photolysis as extracellular promastigotes and intracel-

lular amastigotes in infected macrophages in vitro when illumi-

nated at specific wavelengths to excite the respective photosen-

sitizers for production of reactive oxygen species. Both

Leishmania stages lost their viability completely when doubly

photosensitized optimally and illuminated at low intensity, the

host cells being left unscathed. Inoculation of mice with

photoinactivated Leishmania produced no lesions, which invari-

ably developed in the control groups during a period of

observations for 8 weeks. Pretreatment of Leishmania with both

photosensitizers rendered these cells susceptible to clearance

from the ear dermis by white light illumination. The results

suggest that double photosensitization for synergistic activity

enhances the efficacy and safety of photodynamic therapy in

general and for Leishmania in particular.

Abbreviations: ALA, delta-aminolevulinate; AlPhCl, aluminum

phthalocyanine chloride; DT, uroporphyrinogenic Leishmania;

PS, photosensitizer; ROS, reactive oxygen species; ST, control

transfectants; URO, uroporphyrin I.

INTRODUCTION

Photodynamic therapy (PDT) uses photosensitizers (PS) to

generate cytolytic reactive oxygen species (ROS) in thepresence of atmospheric oxygen for clinical treatment of skintumors and other cutaneous diseases (1). PS can be deliveredexogenously, such is the case with phthalocyanines or pro-

duced endogenously, for example, with delta-aminolevulinate(ALA) for over-production of porphyrins in the target cells (2).

Illumination of PS-loaded diseased cells ⁄ tissues results in theirdestruction via the generation of powerful cytolytic ROS,

which attack multiple cellular targets, making it unlikely toselect for resistance—a universal problem in radio andchemotherapy of all diseases (3). PDT is potentially usable

to treat all infectious and noninfectious diseases, provided thatthe PS can be targeted specifically to the causative agents ordiseased cells, thereby minimizing collateral damage to the

surrounding healthy cells ⁄ tissues (4,5).We have begun to explore a novel strategy of targeting PS

by exploiting the unusual mechanism of Leishmania parasit-ism. In mammalian hosts, these trypanosomatid protozoa find

their way to parasitize specifically the phagolysosomes ofmacrophages and other antigen-presenting cells (APC), i.e. thedendritic cells (6,7). Leishmania spp. are genetically deficient in

heme biosynthesis, making it possible to produce transgenicmutants, which are inducible with ALA for accumulation ofuroporphyrin I (URO) as a PS (8–10). Selective photolysis of

these uroporphyric mutants (DT) in the APC phagolysosomeswas found to ensue by illumination of infected APCs with dimlight. This outcome not only indicates the susceptibility of

Leishmania to PDT but also suggests the potential use of suchphotoinactivated mutants to release drugs ⁄ vaccines in thephagolysosomes for activation ⁄ presentation in chemo ⁄ immu-notherapy and prophylaxis (8–11). Similarly, preloading of

Leishmania with externally supplied AlPhCl or other phathlo-cyanines has the potential to achieve the same aims (12,13).

In the present study, we explore a combinational approach

by loading the Leishmania DT mutants both endogenouslywith URO via the use of ALA and exogenously with AlPhCl.This approach is expected to enhance photodynamic inactiva-

tion of these mutants, thereby improving their utility after suchmodification as an efficient and safe vehicle for drug ⁄ vaccinedelivery in future application. Herein, we report a synergisticactivity of the URO ⁄AlPhCl combination in photosensitiza-

tion of Leishmania for photolysis to completion under both thein vitro and in vivo conditions used.

MATERIALS AND METHODS

Cells and mice. Uroporphyrinogenic mutants of Leishmania amazon-ensis (RAT ⁄BA ⁄ 74 ⁄LV78) clone 12-1, doubly transfected withpX-alad and p6.5-pbgd (DT; 8–10), were routinely grown as prom-astigotes at 25�C in medium 199 (Sigma) buffered with 25 mMM HEPESto pH 7.4 and supplemented with 10% heat inactivated fetal bovineserum (HIFBS). Mutants were placed under the selective pressures of

†This paper is part of the Symposium-in-Print on ‘‘Antimicrobial PhotodynamicTherapy and Photoinactivation.’’

*Corresponding author email: sujoy.dutta@rosalindfranklin.edu (Sujoy Dutta)� 2012 Wiley Periodicals, Inc.Photochemistry and Photobiology� 2012 The American Society of Photobiology 0031-8655/12

Photochemistry and Photobiology, 2012, 88: 620–625

620

tunicamycin and G418 to express ALAD and PBGD, the second andthird enzymes in the heme biosynthetic pathway (14). Before exposureof these DT mutants to ALA for inducing cytoplasmic accumulationof uroporphyrin 1 (URO), they were briefly grown in drug-freemedium to stationary phase to avoid the potential cytotoxicity of thedrugs carried over to the host cells.

Mouse macrophages of the J774 line were grown at 35�C in RPMI1640 supplemented with 10% HIFBS. Male BALB ⁄ c · C57BL ⁄ 6 mice(ca 25 gm, 8–12 weeks old) were used for the in vivo studies.

Leishmania infection of macrophages. J774 cells were infected withLeishmania by mixing a suspension of these cells and that of the DTs inRPMI 1640 + 20% HIFBS at a host-parasite ratio of 1:10, i.e. 106

host cells and 107 DTs per mL. The mixture was plated at 4 mL per25 cm2 TC flask or 1 mL per well in a 12 well plate. The host cell-parasite mixtures were incubated for 16–24 h at 35�C for adhesion ofthe J774 cells to the substratum and their infection by the DTs.Infected cultures were subsequently maintained under the sameconditions with daily medium renewal. To estimate the infectionquantitatively, cells were flattened under a glass coverslip andexamined under phase contrast with a 100· oil immersion lens fortallying infected and noninfected cells, and the total number ofintracellular amastigotes in up to 100 infected cells. The values soobtained were used to estimate the total parasite load per culture, i.e.[total number of macrophages per flask] · [% infected cells] · [averagenumber of Leishmania ⁄ host cell; 13].

Leishmania infection of mice. All mice were housed in the animalfacility of Rosalind Franklin University and handled per IACUC-approved protocol (no. #05-02). Mice were anaesthetized underisoflurane vapor and each inoculated subcutaneously with 106–107

stationary phase promastigotes in 20–100 lL of phosphate bufferedsaline. Mice were grouped at 4–6 ⁄ cage according to the sites ofinoculation, i.e. tailbase, footpad and ear dermis. Mice were monitoredtwice weekly for development of lesions, which were measured, ifpresent, with a Vernier caliper. At the conclusion of experiments, i.e.8 weeks, lesions or tissues at the injection sites were excised sterilely andemulsified in a dounce homogenizer. The homogenates were cleared oftissue debris by passing through a nylon mesh. Parasite loads in thehomogenates were assessed first bymicroscopic counting of amastigotesin a hemocytometer and then by their growth for 2 weeks after limitingdilutions of the homogenates in the complete medium.

Phototsensitization of Leishmania with URO and AlPhCl. In vitro:For cytoplasmic URO accumulation, DT mutants at 50 · 106 cellsmL)1 were exposed to 1 mMM ALA in Hank’s Balanced Salt Solution(pH 7.4) +0.01% BSA (HBSS + BSA) for ca 48 h at 25�C in the dark(8–10). Control DT mutants were simultaneously prepared under thesame conditions without ALA (Sigma). Aluminum phthalocyaninechloride (AlPhCl; Sigma) was added to both uroporphyric andnonuroporphyric DT preparations in 10· serial dilutions of 0.01–1 lg mL)1 to produce URO + AlPhCl double and URO or AlPhClsingle photosensitization. Double photosensitization of uroporphyricDTs by exposing them to 0.01 and 0.1 lg AlPhCl mL)1 was referred toheretofore as protocols 1 and 2, respectively. Uroporphyric DTswithout further exposure to AlPhCl served as the URO singlephotosensitization. All cells were treated overnight in the dark andwashed by centrifugation at 3 500 · g for 5 min at 4�C thrice in HBSSto remove extracellular ALA and ⁄ or AlPhCl. As the exposure of thesecells to PS in the dark per se produced no adverse effect, they wereincubated overnight to optimize the loading efficiency. Nonphotosen-sitized cells without any treatment or exposed to light alone wereprepared similarly in HBSS + BSA. All cells were finally resuspendedin HBSS + BSA at a cell density of 108 cells mL)1 before use.

Photosensitivity of the doubly sensitized DTs was further studied asintracellular amastigotes in macrophages. J774 macrophages wereinfected with URO + AlPhCl-loaded DTs (as described elsewhere inthe text). The infected cells were further exposed to 1 mMM ALA for24 h to boost the URO loading of intracellular DTs. Infected cultureswere washed and incubated in ALA-free complete media for 1–2 days.All photosensitized cultures were incubated in the dark and handledwhen necessary under minimal light exposure until completion of theexperiment.

In vivo: Mice (4–6 per group) were inoculated with photosensitizedand control DTs as described elsewhere in the text. One day afterinoculation, ALA was injected at the same site to foster uroporphyri-nogenesis of the intracellular DTs.

Illumination of photosensitized Leishmania. Suspensions of photo-sensitized and control cells were placed at 108 cells mL)1 per well in12 well tissue culture plates and illuminated in two different ways: (1)sequentially with a long wave UV lamp (366 nm) from the top (with thelid of the culture plates removed) for 20 min (9,10) and red light(>650 nm; 2.5 mW cm)2) from the bottom for 5 min (fluence =0.75 J cm)2; 13) to photodynamically excite URO and AlPhCl, respec-tively. A fluorescent light box covered with a red filter (12) was used asthe source of the red light; and (2) cells were also illuminated with whitelight at a distance of 5 cm from the bottom of the well using an opticcable, consisting of 75 end-emitting fibers, connected to a 250 W metalhalide lamp as the light source (Eclipse II; Supervision; 10 J cm)2) (13).This light source was also used to spot-illuminate the site that wasinoculated with photosensitized and controlDTs inmice. The fiber opticcable was placed 5 cm away from the skin to spot-illuminate theinjection sites each for 20 min to deliver a fluence of 50 J cm)2.Under allconditions of illumination used, samples were not subjected to heatingabove the ambient temperature, as determined by reading a thermom-eter placed in the same location for the same duration.

Cell viability assay. The viability of cells treated in vitro was assessedin two different ways: (1) microscopic assessment for the loss of cellintegrity based on their permeability to propidium iodide. Cells werestained with propidium iodide (50 lg mL)1) for 5 min on ice andwashed thrice by centrifugation. Aliquots were mounted on glassslides, covered with cover slips and observed first under phase contrastand then epifluorescence using specific filter sets for propidium iodide(9). Greater than 500 cells were examined in randomly chosen fields.DTs were considered as dead when permeable to propidium iodide, asindicated by the fluorescence of their nuclei. These dead cells werecounted to estimate their proportion against the total number of cellsexamined. (2) aliquots of treated samples were each inoculated at a celldensity of 106 cells mL)1 in complete culture media for growth aspromastigotes. Cultures were incubated for up to 5 days and viabilityassessed by MTT assay (9,12).

The viability of treated Leishmania in mice was assessed by theirability to produce a lesion during an 8-week period. Parasite loadswere estimated at the end point, as already described in ‘‘Leishmaniainfection of mice.’’

Estimation of cell-associated URO and AlPhCl. Both URO andAlPhCl were extracted after optimal cell-loading conditions from thepellets of known cell numbers for fluorimetric evaluation of theiramounts per cell population. URO was extracted with a mixture ofperchloric acid ⁄methanol and quantitatively assessed as described(8,10). AlPhCl was extracted by vigorous vortexing of the cell pelletswith a mixture of chloroform and methanol (ratio 2:1 vol ⁄ vol)(200 lL). Samples were cleared by centrifugation at 13 000 g for15 min. AlPhCl in the clear supernatants was estimated by comparisonto a standard curve of chemically pure AlPhCl in serial dilutions versustheir fluorescence intensities (640 nm excitation per 660 nm emissionwavelength).

Fluorescence microscopy of URO- and AlPhCl-loaded cells. Aliquotsof photosensitized and control cells were observed under livingconditions in a Nikon Eclipse 80i microscope fitted with appropriatefilter sets: D405 ⁄ 10 (405 nm exciter), Q485DCXR (485 nm dichroic)and RG610LP (610 nm emitter; Chroma Tech Co., Brattleboro, VT)for URO; and HQ620 ⁄ 60 (620 nm exciter), Q660LP (660 nm dichroic)and HQ700 ⁄ 80 (700 nm emitter) for AlPhCl. Images were capturedwith a Cool Snap ES camera in conjunction with Metamorphosisimage acquisition software (version 6.2r6; Molecular Devices, Down-ington, PA).

Statistical analysis. All experiments were repeated at least twicewith triplicate samples for each treatment. Student’s t-test and one-wayANOVA were used to calculate statistical significance of the datawhere appropriate.

RESULTS

AlPhCl and URO photosensitize Leishmania at different

subcellular sites

Loading of live Leishmania with externally supplied AlPhClproduced a distinct pattern of cellular fluorescence, which

differed from what was produced by ALA-induced accumulation

Photochemistry and Photobiology, 2012, 88 621

of URO in the DTs (Fig. 1A versus B). Incubation of thesecells with AlPhCl alone resulted in cellular fluorescence that

outlined the cell body, flagellar reservoir, nucleus and otherunidentified intracellular cellular structures, but not theflagella (Fig. 1A phase contrast versus fluorescence). The

cellular structures were clearly, albeit incompletely, delineatedby fluorescence of intermittent intensity. Treated cells retainedthis pattern of cellular fluorescence when exposed to increasing

concentrations of AlPhCl up to 1 lg mL)1. AlPhCl is hydro-phobic, suggesting that it is associated with cellular mem-branes, accounting for the fluorescent image observed. Incontrast, the endogenously produced URO gave a diffused

pattern of cellular fluorescence, consistent with the previousfindings (9,10) in that it is present in the cytosol of these cells,their flagella, and in their endocytic vacuoles (Fig. 1B). URO is

distributed unequally in these cellular compartments and isapparently absent in some small vesicles, accounting for thepatchy pattern of cytoplasmic fluorescence. Thus, these two

PSs localize at two distinctly different subcellular compart-ments.

Sensitization of Leishmania with AlPhCl and URO for their

photolysis is more effective when used in combination than

individually in vitro

Double photosensitization of Leishmania DTs with AlPhCland URO makes it possible to photolyze them in both stages

to completion by illumination under the conditions used. Nocytotoxicity was noted after loading of these cells with either orboth of the two PSs in the dark. DT promastigotes so treated

remained microscopically as intact and motile as untreatedcontrols, barring photoinactivation by prolonged exposure tomicroscope illumination. These cells were doubly photosensi-

tized under the conditions which were previously optimized forsingle presensitization of promastigotes with the respectivePSs, i.e. AlPhCl (13) or URO (9,10). The viability of these

sensitized cells was initially assessed 2 h post illuminationbased on their permeability to propidium iodide for nuclearfluorescence (Fig. 2A [I], [II]). Sequential exposure of the

singly sensitized cells to longwave UV and then red light underthe described conditions did not result in their complete loss ofviability, but reduced it to ca 17% and 3% of the control with

Figure 1. Cellularlocalization of aluminum phthalocyanine chloride(AlPhCl) and uroporphyrin I (URO) in Leishmania amazonensis DTpromastigotes. (A) DT promastigotes exposed in the dark to AlPhCl at1 lg mL)1 for 16 h; and (B) DT promastigotes exposed in the dark toALA (1 mMM) for 48 h. Left panel, phase contrast; right panel,fluorescence microscopy. Note: membrane association of AlPhClversus cytoplasmic accumulation of uroporphyrin I. Bar scale =10 lm.

Figure 2. Synergistic sensitization of Leishmania amazonensis DTpromastigotes with AlPhCl and URO for their photolysis. (A andB): Viability of uroporphyric Leishmania decreased by additionalphotosensitization with increasing concentrations of AlPhCl followedby illumination at wavelengths specific to the two photosensitizers.(a) Nonphotosensitized control DT promastigotes; (b) DTs photo-sensitized with AlPhCl by incubation with 0.01 (protocol 1) and0.1 (protocol 2)lg mL)1 in [I] and [II], respectively; (c) DTs exposed to1 mMM ALA for cytoplasmic URO accumulation; (d) DTs photosen-sitized with both PSs under the conditions as described. The valuesgiven at the bottom denote the cellular amounts of AlPhCl and UROat pmoles ⁄ 108 cells calculated as described in the Materials andMethods section. All samples were illuminated sequentially with longwave UV (maximum 366 nm) and red light (>650 nm) for photo-dynamic excitation of URO and AlPhCl, respectively. The percent cellviability was determined by: (A) propidium iodide permeability assays2 h after illumination; and (B) MTT assay after culturing aliquotsof the treated cells under promastigote culture conditions for 24 h.P-values were calculated by paired Student’s t-tests.

622 Sujoy Dutta et al.

the higher concentration (0.1 lg mL)1 overnight) of AlPhClused for loading (Fig. 2A [II; b] versus [a]) and the UROaccumulated by exposure to ALA (Fig. 2A [c] versus [a]),respectively. This differential outcome is not unexpected,

considering the difference in the loading efficiencies calculatedfor the two PSs, i.e. 500 versus 28 pmoles per 108 cells for UROand AlPhCl, respectively. Notably, presensitization of cells

with AlPhCl alone with increasing concentrations from 0.01 to0.1 lg mL)1 did not proportionally increase their photolysis;namely, a ca 10-fold increase in the cellular loads of AlPhCl

from ca 2.4 to 28 pmoles per 108 cells gave only a five-folddecrease of their viability from ca 87% to 17% of the control(Fig 2A [I; b] versus [II; b]). Significantly, a complete loss of

cell viability after illumination, not attainable by any of thesingle presensitization conditions used, was achieved by doublepresensitization according to protocol 2, i.e. a combination ofURO and the higher concentration (0.1 lg mL)1) of AlPhCl

used for loading (Fig. 2A [II; d]). The profiles of cell viabilityunder the various conditions described were generally consis-tent with the patterns of cell growth after inoculation of

treated cells into complete culture media (Fig. 2B [I], [II]). Cellviability assessed by MTT assay after incubation for 1 day alsoshowed no survivors after protocol 2 double photosensitiza-

tion (Fig. 2B [II; d] protocol 2 versus the rest). This negativeoutcome was further verified after prolonged incubation ofthese cultures for up to 5 days. Similar results of synergisticsensitization for photolysis were also obtained when the

sequential illumination regimen of longwave UV and red lightwas replaced with white light at a fluence of 10 J cm)2 (notshown).

Double presensitization of Leishmania DTs with AlPhCl +URO also synergized their photolysis in macrophages byillumination of the infected cultures (Fig. 3A solid circle versus

rest of the groups for all the other experimental conditions).DT promastigotes subjected to protocol 2 double photosen-

sitization (Fig 2 [II; d]) were found to infect J774 macrophagesas well as the control groups, all giving a total number ofintracellular parasites of ca 107 per culture 3 days afterinfection (Fig. 3A, Day 3). After incubation for 2 days, the

infected macrophages were pulsed for 24 h with another doseof ALA (1 mM;M; Fig. 3, arrow) to further boost the UROloading of the uroporphyric parasites (Fig. 3A). These cultures

were incubated for 1 day in ALA-free media to clear the smallamounts of porphyrins in the host cells, resulting from theirALA-induced porphyrinogenesis. After illumination on day 4

with white light at 10 J cm)2 (Fig. 3, arrow head), the parasiteloads of infected cultures were assessed every 3–4 days up today 12. The results showed that illumination cleared the

parasite loads progressively to completion from the cultureswith the doubly presensitized DTs (Fig. 3A, solid circle), butnot from those with singly presensitized DTs (Fig. 3A, opencircle and triangle). Parasite loads remained essentially

unchanged or marginally decreased in all the other controlgroups, including singly and doubly presensitized DTs withoutexposure to light (Fig. 3A, open square and solid triangle).

These observations were verified by the results from thegrowth of Leishmania in suitable media. Promastigotes grewfrom all samples, except those which were infected with

protocol 2 doubly prephotosensitized DTs. Throughout the12 day period of observation, the host cells were not signif-icantly affected by any of the treatments used (Fig. 3B). Thus,double photosensitization of Leishmania synergized their

photolysis not only as extracellular promastigotes but also asintracellular amastigotes in infected macrophages.

Doubly photosensitized Leishmania were susceptible to white

light-mediated lysis in vivo

During a period of observations for 8 weeks, DTs doublyprephotosensitized according to protocol 2 produced no visible

lesion in mice after inoculation into their tailbase followed byan ALA boost (Fig. 4A, long arrow) and spot-illumination(short arrow) at the site of injection (Fig. 4A, open triangle).

Lesions invariably developed during the same period ofobservation among all the control groups included. The onsetof their lesion development varied with the treatments, startingon week 2 with the untreated samples or light alone (Fig. 4A,

solid square) and +URO-Light (open square), on week 3 withthose of +AlPhCl ) URO + Light (open circle), on week 4with those of +URO + Light (solid square) and on week 6

with those of the protocol 1 AlPhCl + URO + Light (solidtriangle). In all control groups for the duration of this study,lesions increased in size progressively, reaching 6–7 mm in

diameter, except those produced by DTs doubly presensitizedby protocol 1, in which case a much smaller lesion of ca 2 mmin diameter was observed (Fig. 4A, solid triangle). Thedifferences in the lesion size were already notable on week 6

among the major groups, i.e. +URO ) Light > + URO +Light > + AlPhCl(1) + URO + Light > + AlPhCl(2) +URO + Light (Fig. 4B, arrow head pointed to lesions). The

results obtained demonstrated the synergistic activity ofAlPhCl + URO to enhance the photosensitivity of Leishmaniain vivo, as indicated by suppressing their ability to produce

lesions in mice.After the same 8 week period, no parasite loads were

detectable after injection of mice with DTs, which were doubly

Figure 3. Selective and complete photolysis of intracellular DTs doublyphotosensitized with URO and AlPhCl. Leishmania DT promastigotesprephotosensitized under optimal conditions with URO and ⁄ orAlPhCl were used to infected J774 macrophages. About 2 days afterinfection, all cultures were again pulse-exposed to 1 mMM ALA (arrow)for 24 h. One day after removal of ALA, infected cultures wereexposed to white light (10 J cm)2; arrow head) for 1 h. Infectedcultures not exposed to ALA ()URO) or light ()Light) served ascontrols. (A) The parasite loads and (B) the total number of themacrophages per culture were quantitatively estimated at the timepoints under various conditions (±URO ± AlPhCl ± light), asindicated; +AlPhCl(2) denotes conditions according to protocol 2 inFig. 2. P-values were calculated by one-way ANOVA.

Photochemistry and Photobiology, 2012, 88 623

presensitized and spot-illuminated in situ under the sameregimen, but only at a dose of 106 DTs per site in the ear

dermis (Fig. 5 [II] Ear). When inoculated with the doublypresensitized DTs at 107 per site, subsequent spot-illuminationof the injection sites decreased the parasite loads in the order

of ca 1 log in all sites examined, i.e. tailbase, footpad and ear(Fig. 5 [I] blank versus black bars). The reduction in theparasite loads to this extent is apparently sufficient to accountfor the difference that was observed in the development of a

small lesion or no lesion by the doubly sensitized DTs(Fig. 4A–B). When the inoculum was reduced by 10-fold to106 doubly presensitized DTs per site, subsequent spot-

illumination dropped the parasite loads dramatically at anorder of ‡2 logs in the tailbase and footpad, and to anundetectable level in the ear (Fig. 5 [II] solid versus blank

bars). Cultivation of the ear tissue homogenates yielded nopromastigote growth, confirming the absence of viable DTs at

this inoculation site under the conditions described. Thesynergistic activity of URO + AlPhCl thus sensitized DTsin situ for photoinactivation not only to prevent them fromproducing lesions but also their elimination, even though this

occurred at the specific site of ear dermis inoculated with asmaller cell number.

DISCUSSION

In the present study, we demonstrate for the first time thatloading of Leishmania with two different PSs (Fig. 1) syner-

gistically sensitize them for photolysis to completion underboth in vitro and in vivo conditions used (Figs. 2–5). Herein,synergy is defined in the biological context; namely, the two

PSs function together to produce specific photolysis ofLeishmania that is not obtainable by PS alone. This definitionis analogous to the synergistic effects that were described for

the finding that tumoricidal activities were enhanced using twolaser dyes with different intracellular targeting sites, i.e.rhodamine-123 and merocyanine-540 (15). In our case, Leish-

mania DTs were doubly loaded with URO that was induced toaccumulate optimally via their exposure to 1 mMM ALA (8–10)and with AlPhCl, which was also optimized previously to theloading concentrations of £0.1 lg mL)1 that is nontoxic to

the host cells (13). The synergistic effect was clearly shown bythe absence of any viable Leishmania after double photosen-sitization, but not single photosensitization under the same

illumination conditions used, i.e. longwave UV + red light orwhite light. The simplest explanation for the observed syner-gism may lie in the differences in the photodynamic and other

properties between URO and AlPhCl. Indeed, these two PSssensitize Leishmania at different subcellular sites (Fig. 1). Inaddition, it is known that they are photoexcitable at different

wavelengths to produce different primary species of ROS.Thus, illumination of the doubly presensitized cells mayhypothetically generate more ROS both in quantity andquality, which attack more targets in these cells than those

singly presensitized. Moreover, the application of two differentPSs is less likely than single photosensitization to leave cells inthe population as a whole unsensitized. Indeed, loading of

Leishmania DTs via either ALA-induced uroporphyrinogene-sis or exposure to externally supplied AlPhCl at the concen-trations used is stochastic, always leaving a small percent of

nonsensitized and thus photolytically insensitive cells in thepopulation (9,13). Double photosensitization of Leishmaniamust have eliminated such cells altogether or reduced them toa negligible number to account for the complete photolysis

observed.The in vivo results obtained in mice warrant further

discussion beyond their provision of evidence for the syner-

gism of double photosensitization. The doubly presensitizedLeishmania as promastigotes and amastigotes in infectedmacrophages are evidently cleared completely in vitro by

sequential illumination with longwave UV and red light orwhite light, as shown by microscopy and cultivation(Figs. 2–3). This is also the case in vivo, but only for those

inoculated into the mouse ear dermis illuminated with whitelight (Fig. 5). Photolytic clearance of the parasites from thissite, but not from the footpad and tailbase, is perhapsattributable to the translucency of ear dermis to illumination.

However, as the parasite loads produced by a larger inoculum

Figure 4. Photodynamic inactivation of doubly photosensitzed Leish-mania in vivo. (A) Mice in groups of 4–6 were each inoculated (107

parasites ⁄ tailbase) with control DTs and those photoinactivated underconditions, as indicated in Fig. 4A (±AlPhCl ± URO ± Light).Each site was injected with 100 lL of 100 mMM ALA 1 day afterinoculation and spot-illuminated with white light (50 J cm)2) 36 hthereafter. Lesions were measured weekly and the values of theirdiameter plotted with time for a period of 8 weeks, as shown. P-valueswere calculated by one-way ANOVA. (B) Photographs of the lesionson week 6 produced by Leishmania treated under different conditionsas indicated.

624 Sujoy Dutta et al.

were reduced, but not cleared by illumination at this site, it stillpresents a barrier to white light, despite its spectrum includesboth URO and AlPhCl-excitable wavelengths. The results

obtained provide information of relevance to further investi-gation in photodynamic vaccination. Of interest to determineis whether photolysis of uroporphyric DTs after additionalphotosensitization with AlPhCl used here or other effective

phthalocyanines (12) would trigger an immune clearance ofparasites that may survive in the ear dermis or escape fromthat site to other places, e.g. lymph nodes after the 8 week

experimental protocol used in our study. Previously, vaccina-tion of hamsters with uroporphyric DTs photodynamicallylysed in vivo was shown to protect them from challenges with

Indian L. donovani by eliciting immunity that is adoptivelytransferrable with splenic lymphocytes to naı̈ve animals (11). Itwill be of interest to evaluate this by doubly sensitized

Leishmania in the mouse model, for which experimentalconditions are expected to differ from those used for the L.donovani-hamster model.

In summary, results presented demonstrate that sensitiza-

tion of Leishmania with two different PSs acts synergisticallyto produce an outcome of complete photolysis in contrast tosingle photosensitization both in vitro and in vivo. This

approach will help us in future experimental designs forassessing the efficacy and safety of photoinactivated Leish-mania as a potential carrier for drug ⁄ vaccine delivery to the

phagolysosomes of APC for eliciting effective photodynamicimmune therapy and prophylaxis (8–13).

Acknowledgements—This work is supported by NIH grants AI-083951

and AI-068835 to KPC. Thanks are due to Dr. John Keller for

reviewing this manuscript.

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Figure 5. Clearance of doubly photosensitzed Leishmania in ear dermisby spot-illumination. Mice in another group of four were eachinoculated with 107 (panel [I]) or 106 (panel [II]) of doubly andoptimally presensitized DTs (+AlPhCl[2] + URO) at differentsites ⁄ group as indicated. Each site received another 10 lL of 1 MM

ALA 2 days later and processed with (solid bar) and without (blankbar) spot-illumination (±white Light at 50 J cm)2) after another3 days. All groups of mice were humanely euthanized at week 8 todetermine parasite loads (see the Materials and Methods section).P-values were calculated by paired Student’s t-tests. #P < 0.05 and*P < 0.01.

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