Amino-phthalocyanine-mediated photodynamic inactivation of Leishmania

21 Photodynamic inactivation (PDT) of Leishmania spp. requires cellular uptake of 22 photosensitizers, e. g. endocytosis of silicon (IV)-phthalocyanines (PC) axially substituted with 23 bulky ligands. We report here that, when substituted with amino-containing ligands, the PCs 24 (PC1 and PC2) were endocytosed and displayed improved potency against Leishmania tropica 25 promastigotes and axenic amastigotes in vitro. The uptake of these PCs by both Leishmania 26 stages followed saturation kinetics, as expected. Sensitive assays were developed for assessing 27 PDT of Leishmania by rendering them fluorescent in two ways: transfection of promastigotes to 28 express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl 29 ester (CFSE). PC-sensitized Leishmania were seen microscopically to lose their motility, 30 structural integrity and GFP/CFSE fluorescence after exposure to red light (wavelength = ~650 31 nm) at a fluence of 1-2 J cm. Quantitative fluorescence assays based on the loss of GFP/CFSE 32 from live Leishmania showed that PC1 and PC2 dose-dependently sensitized both stages for 33 photo-inactivation, consistent with the results of a MTT cell viability assay. Leishmania are 34 >100 times more sensitive than their host cells or macrophages to PC1and PC-2-mediated 35 photo-inactivation, judging from the estimated EC50 values of these cells. Axial substitution of 36 the PC with amino groups instead of other ligands appears to increase its leishmanophotolytic 37 activity by up to 40 fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of 38 leishmaniasis and for oxidative photo-inactivation of Leishmania for use as vaccines or vaccine 39 carriers. 40 41 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom Introduction 42 Photodynamic therapy (PDT) uses photosensitizers (PS) that are light-excitable to 43 produce cytotoxic reactive oxygen species (ROS) in the presence of atmospheric oxygen for 44 clinical treatment of tumors, skin cancer and other cutaneous diseases (1). PS can be applied 45 exogenously, such as the case with phthalocyanines. Alternatively, PS can be induced 46 endogenously with delta-aminolevulinate (ALA) to up-regulate heme biosynthesis of the target 47 cells for over-producing photosensitive intermediates of this pathway, i. e. porphyrins (2). PDT 48 has the potential to solve the significant problems of emerging drug-resistance in infectious and 49 malignant diseases, since it is not known to elicit such resistance (3, 4). Light and PS alone are 50 non-cytotoxic and thus do not select for a resistance phenotype. In combination, a burst of 51 powerful cytotoxic ROS is rapidly generated, which simultaneously attack multiple cellular 52 molecules, making it unlikely to induce the development of resistance. This assumption is 53 supported experimentally by observations with porphyrinogenic Leishmania (see below). 54 The potential of PDT has been explored for treating infectious diseases caused by a 55 variety of different pathogens (5, 6), including trypanosomatid protozoa in the genus of 56 Leishmaina causing cutaneous leishmaniasis (CL). PDT with clinically acceptable 57 photosensitizers showed promising results by using day light for illumination in on-going trials 58 of CL patients (7) and for treating protracted infection in drug-resistant cases (8). Anti59 Leishmania PDT has been assessed experimentally in vivo in different animal models of CL and 60 in vitro in infected macrophages. More recent work includes the use of different photosensitizers, 61 e. g. hypericin, phthalocyanines, phenothiazines, methylene blue and zinc oxide, as such or after 62 PEGylation or in liposome/nanoparticle encapsulated forms, to generate ROS by illumination 63 using different light sources, e. g. LED, laser and sun light (9-17). The intrinsic susceptibility of 64 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom both stages of Leishmania, i. e. promastigotes and amastigotes, to PDT has been examined in 65 that context, but has rarely been explored at the cellular and molecular level. Such mechanistic 66 investigation is essential in attempt to improve the discriminatory efficacy of PDT and to 67 broaden the scope of its applications. 68 Previously, we have made Leishmania sensitive to PDT by targeting PS for intracellular 69 accumulation. For cytosolic accumulation of PS, Leishmania were first engineered transgenically 70 to express the 2 and 3 enzymes in the heme biosynthesis pathway missing in these 71 trypanosomatids (18-19). Such transfectants were then exposed to ALA, product of the 1 72 enzyme in this pathway, resulting in cytosolic accumulation of the uroporphyrin I (URO) in the 73 absence of downstream enzymes. These porphyric Leishmania are rapidly inactivated after a 74 brief exposure to light, which excites their cytosolic URO to generate singlet oxygen (O2) (20) – 75 an extremely short-lived (half-life = 1-3 microseconds), but highly reactive ROS. Mechanisms 76 for detoxification of O2 are not known to exist among non-photosynthetic organisms, like 77 Leishmania. Indeed, no development of PDT resistance was noted after as many as six 78 consecutive cycles of ALA-induced porphyrinogenesis followed by light-exposure for cytolysis. 79 After each PDT cycle, very few cells survived and were invariably aporphyric, attributable to 80 efflux of URO. However, the survivors remained as sensitive to the PDT as the parental 81 transfectants when recovered after each PDT cycle or cloning after the last cycle of the six 82 treatments (20). Photodynamic vaccination of hamsters based on this principle produced cell83 mediated immunity that is adaptively transferable to naïve animals against visceral leishmaniasis 84 (21). The porphyrinogenic mutants have the potential to serve as a universal platform for vaccine 85 delivery, as shown by the findings that ALA-induced porphyrinogenesis followed by photo86 inactivation of Leishmania in situ reversed immunosuppression of macrophages caused by the 87 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom infection (22). For endosomal accumulation of PS in Leishmania, silicon(IV)-phthalocyanines 88 (PC) attached axially with bulky ligands (PC14 and PC15) were identified as effective by 89 screening a panel of ~17 novel PCs (23). Endocytosis of PC14 and PC15 by Leishmania 90 sensitizes them to photo-inactivation. These PCs are more discriminatory than aluminum(III)-PC 91 against Leishmania versus their host cells or macrophages (24). When preoxidatively photo92 inactivated with PC15, Leishmania were shown to deliver transgenically expressed OVA 93 (ovalbumin) more effectively than conventional means to APCs (antigen-presenting cells) for 94 antigen presentation to activate OVA epitope-specific T cells (23). Significantly, oxidatively 95 photo-inactivated Leishmania after double-sensitization with URO and PC produced no lesion in 96 mice (25), indicative of their safety for applications. 97 In the present study, we screened additional PCs, leading to the identification of amino98 PC conjugates (PC1 and PC2), which are significantly more effective and discriminating than the 99 PCs previously identified for PDT of Leishmania. This efficiency was demonstrated in both 100 stages of L. tropica qualitatively by fluorescent microscopy and fluorimetry for the uptake of 101 PC1 and PC2, and quantitatively by fluorescence/metabolic cell viability assays after photo102 inactivation. The effectiveness and discriminatory potential of PC1 and PC2 are predicted from 103 the disparity between Leishmania and J774 macrophages in their EC50 (half maximal effective 104 concentration) values for photo-inactivation. 105 Materials and Methods 106 Novel phthalocyanines (see Fig. S1 for chemical structures): We examined 4 phthalocyanines 107 (PC): The amino-phthalocyanines PC 1 and PC 2, triethylene glycol substituted Zn(II)108 phthalocyanine, PC 4 (26, 27) and PC3, which was prepared from silicon(IV) phthalocyanine109 dichloride treated with triethylene glycol, (see legend to Fig. S1). All compounds were dissolved 110 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom in dimethyl sulfoxide (DMSO) for preparing 1 mM stock solutions, from which working 111 solutions (0.01 to 10 uM) were prepared by serial 10-fold dilutions. 112 Cells: Leishmania used included 2 virulent strains of L tropica EP41 (see Fig. S2) CBU10. 113 Promastigotes were grown to stationary phase at 25 C in HEPES-buffered Medium 199 or 114 Schneider’s Insect Medium with 10% heat inactivated fetal bovine serum (HIFBS) and as axenic 115 amastigotes at 33 C in Schneider’s Insect Medium with 10% HIFBS, pH 5.3 (23). Axenic 116 amastigotes are slightly larger than the amastigotes in infected macrophages in size and are more 117 infective than promastigotes in vitro and in vivo (Fig. S2 C-D). Macrophages of the J774 cell 118 line were cultured at 35 C without 5% (v/v) CO2 atmosphere in RPMI-1640 medium HEPES119 buffered to pH 7.4 plus 10% (v/v) HIFBS (22-24).Photosensitization of cells: Leishmania were 120 suspended to 5 X 10-10 cells ml in HBSS-BSA for exposure to serial dilutions of PCs 1-4 in 121 the dark overnight. Controls included cells exposed to diluent alone. In separate experiments, 122 promastigotes were exposed to FITC-dextran (MW ~4,000, Sigma) for two days followed by 3X 123 washing with HBSS-BSA by centrifugation and then exposure to 1 uM PC1 for 1 day in the 124 dark. J774 macrophages were PC-treated as a monolayer in Hank’s Balanced Salt Solution 125 (HBSS) HEPES-buffered to pH 7.4 plus 0.01% bovine serum albumin (HBSS-BSA) or culture 126 medium under otherwise the same conditions. All cultures were kept in the dark for various time 127 periods for up to 24 hrs. 128 Exposure of photo-sensitized cells to red light: Control and PC-sensitized Leishmania were 129 washed and suspended in HBSS-BSA. The cell suspensions in 24 well culture plates and 130 monolayers of J774 macrophages in 25 cm TC flasks were exposed from the bottom to red light 131 (wavelengths > 650 nm) via a red filter (Smith-Victor Co., Barlett, IL, USA; part # 650021) over 132 a light box for 20-30 min at a fluence of 1-2 J cm (22, 23). 133 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom Cell viability assay: 134 MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay was 135 done according to manufacturer’s protocol (Sigma, Molecular Probe kits) as before (22, 23). 136 Freshly prepared MTT stock solution (5 mg ml) was added to promastigote and axenic 137 amastigote cell suspensions at 0.5-1 X 10 ml. Formazan formed was solubilized for reading in 138 a Biotek Synergy HT plate reader (Texas Instruments) and analyzed by using GEN 5 version 139 5.11.1 software. 140 Quantitative fluorescence assay: 141 Used were L. tropica EP41 and CBU10 transfected by electroporation with egfp-p6.5 (28) to 142 express green fluorescent protein (GFP) as before (24). Stable transfectants were obtained and 143 grown in the presence of 10 ug tunicamycin ml. 144 Also used were Leishmania exposed for 10 min at 37 C to 5-10 uM Carboxyfluorescein 145 succinimidyl ester (CFSE) (Invitrogen) at 10 cells ml in HBSS-BSA. CFSE is converted by 146 intracellular non-specific esterase into cell-impervious fluorescent products, rendering live 147 Leishmania fluorescent (29). 148 GFP transfectants or CFSE-loaded Leishmania in HBSS-BSA were dispensed in triplicate of 100 149 ul aliqouts at 5 X 10 cells 100 ul well to 96 well microtiter plates for detergent lysis with 150 0.5% (V/V) Nanodet P-40 to release cellular GFP/CFSE. The lysates were read for fluorescence 151 at a sensitivity of 60 in a BIO-TEK Synergy HT plate reader (excitation wavelength of 485/20 152 nm and emission wavelength of 528/20 nm). Data collected were analyzed by using the same 153 software mentioned above. 154 Leishmania uptake of PCs assessed by fluorimetry: Promastigotes were exposed in the dark to 155 0.1 uM PC1 and PC2 at 5 X 10 cells ml under non-growing conditions in HBSS-BSA. 156 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom Controls were set up without cells under the same conditions. Samples were dispensed at 1-ml 157 aliquots in the dark at 25 C and harvested at different intervals for up to 24 hrs. Cells were 158 washed once and sedimented as pellets then stored at -20 C. At completion of the experiments, 159 PC was extracted from each frozen pellet with 2.5 ml DMSO. The fluorescent intensities of the 160 extracts were read (Excitation wave length at 608 nm, Emission wavelength at 682 nm) in an LS161 50B spectrofluorimeter (Perkin-Elmer) using FL WINLAB software. Concentrations of the PC in 162 the samples were expressed in nmoles 10 cells by interpolation of their fluorescent intensities 163 versus the readings from a linear standard curve of PC concentrations from 0 to 20 nM. 164 Assays for dark toxicity of PCs: GFP transfectants at 5x10 ml in culture media were divided 165 into 12 ml aliquots for exposure to a mixture of PC1 and PC2 in 10X serial dilutions from 0 to 1 166 uM. Cultures were handled in two ways to minimize light exposure: Normally used conditions: 167 These were the conditions referred to as “in the dark” in all experiments described. 12-ml 168 aliquots of cell suspension were each exposed to 0 to 1 uM PCs in separate culture vessels 169 wrapped in aluminum foil. Culture vessels were unwrapped for daily sample collection under the 170 incipient or ambient light with room light turned off. More stringent conditions: Samples from 171 different experiments to be collected daily were aliqouted (3 ml) and wrapped together in 172 aluminum foil for keeping them in total darkness for the duration of the incubation. Samples 173 under both conditions were processed daily for collecting cell pellets, which were kept frozen 174 until completion of the experiments when they were subjected to GFP fluorescence assays as 175 described. Data were presented as % of the controls from untreated cells collected at each time 176 point. 177 Fluorescence microscopy: Nikon Eclipse 80i and TE2000-S microscopes with CCD cameras 178 were used with Metamorphosis (version 6.1) or NIS Elements version AR 4.20.01 software for 179 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom image capture and analysis (18, 22-24). Cells were examined under living conditions by placing 180 concentrated cell suspensions in 3 ul aliquots each on a glass slide covered with an 18 mm glass 181 coverslip. The wet mounts were first scanned under phase contrast to localize cells. Cells 182 exposed to PCs were then imaged for subcellular localization of PC fluorescence using Cy5 filter 183 set. GFP/ CFSE fluorescent Leishmania were examined using FITC filter set. Images captured 184 under different settings were merged by using the software programs mentioned. The filter sets 185 (Chroma Technology Co., Brattleboro, VT) used were: [1] HQ620/60 (620-nm exciter), Q660LP 186 (660-nm dichroic), and HQ700/80 (700-nm emitter) for phthalocyanines. [2] HQ480/40 (480-nm 187 exciter), Q505LP (505-nm dichroic), and HQ535/50 (535-nm emitter) for GFP and CFSE. 188 Data analysis/presentation: All experiments were repeated at least twice and in most cases 189 three times using mainly EP41, but also CBU10 for confirmation. Results obtained were 190 comparable among repeat experiments. Data presented represent mean± standard errors of the 191 values obtained in triplicate for each of individual samples from representative experiments. 192 Statistical analyses were performed for pairwise data comparison by two-tail student t tests in 193 graphpad prism version 5. p values of < 0.05 are considered as significant. 194 195 Results 196 Leishmania uptake of amino-phthalocyanine conjugates (PC1 and PC2): Of the 4 197 phthalocyanines (PCs1-4) examined (Fig. S1), PC1 and PC2 were detected by fluorescent 198 microscopy in Leishmania, which are axially substituted each with two symmetrical ligands, 199 consisting of monoand di-amino groups, respectively. No uptake of PC3 and PC4 by 200 Leishmania was observed, irrespective of the concentrations and periods of incubation used (not 201 shown). 202 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom PC1 and PC2 were taken up by both Leishmania stages, as noted in the images captured 203 by using Cy5 filter set (Fig. 1). The uptake of both PCs was time-dependent. Cells examined 204 immediately after exposure to 0.1-1 uM PC1 or PC2 showed no intracellular fluorescence. 205 Fluorescence began to emerge in cells exposed to the PCs for >1 hour and increased gradually in 206 intensity upon further incubation. After >4 hours, PC fluorescence was readily discernable in 207 discrete cellular entities of promastigotes (Fig. 1, Promasitogte). PC fluorescence appeared in 208 vacuoles in the proximity of the flagellar pocket (short arrow), in multi-vesicular tubular 209 structures and vacuoles (long arrow) distant from the flagellar pocket (Fig 1, Promastigotes A-C, 210 A’C’ merged images). The axenic amastigotes also appeared to have PC fluorescence in the 211 similar intracellular structures, (Fig. 1, Amastigote, D-F arrows), although these small cells have 212 no external flagella for orientation. PC fluorescence varied with individual amastigotes (Fig. 1, 213 Amastigote D-F), but was present in all when the fluorescence intensity was digitally optimized 214 (Amastigote D’-F’). Fluorescence intensity appeared higher in both stages after exposure to PC2 215 than to PC1. PC1 was seen to co-localize with FITC-dextran in promastigotes (Fig. S3). 216 The uptake of PC1 and PC2 followed a saturation kinetics, as determined by fluorometric 217 quantitation of cell-associated PC (Fig. 2). Since the cells were washed before PC extraction, cell 218 surface associated PC was excluded from the readings obtained. Spontaneous precipitation of 219 PCs and cell proliferation did not contribute to the readings, since both were found minimal 220 within the timeframe under the conditions of incubation. Thus, the rise of cell-associated PC 221 with time was reflective of its uptake, reaching a plateau in ~7 hrs. The kinetics of the uptake 222 was comparable among different experiments, although the readings varied significantly from 223 one experiment to another, as reflected in the large standard errors (Fig. 2). We attribute this to 224 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom the inherent variations of different lots of cells used and/or different efficiencies of PC extraction 225 from different sample sets. 226 Microscopy of Leishmania photolysis mediated by PC1 and PC2 (Fig. 3): When sensitized 227 with PC1 and PC2 followed by exposure to red light, promastigotes were found to lose their 228 motility (not shown) and structural integrity (Fig. 3A 3, 5). In contrast, they remained motile and 229 intact in the groups of no treatment or treated with PC1 and PC2 alone without red light exposure 230 (Fig. 3A, 1, 2 and 4). This finding was further indicated by the loss of cellular fluorescence when 231 using GFP/CFSE fluorescent cells (Fig. S3). After red light exposure of PC1or PC2-loaded 232 GFP transfectants, the GFP fluorescence was reduced, especially with PC 2 (Fig. 3B and 3D, 3 233 and 5), but not in the control groups of no treatment and PC treatment alone (Fig. 3B and 3D, 1, 234 2 and 4). The images taken under Cy5 filter showed that PC1 and PC2 fluorescence was absent 235 in untreated cells (Fig. 3C and 3D, 1), as expected and differed little among PC1and PC2236 treated cells with or without red light exposure (Fig. 3C, 2 to 5). In merged images, PC-GFP co237 localization was evident in cells treated with PC1 or PC2 alone (Fig. 3D, 2, 4), but not at all or to 238 a lesser extent after exposure to red light (Fig. 3D, 3, 5). 239 Similar results were obtained by using other fluorescent cells of both EP41 and CBU10: 240 CFSE-loaded promastigotes and axenic amastigotes, and GFP transfected axenic amastigotes 241 (not shown, but see quantitative data presented in Fig. 4). 242 In the presence of PC3 and PC4, Leishmania showed no cellular fluorescence, and 243 retained their motility and cellular integrity with or without red light exposure (not shown). 244 PC dose-dependent sensitization of Leishmania for photo-inactivation (Fig. 4): MTT 245 reduction and loss of cellular GFP/CFSE fluorescence assays (see Materials and Methods) 246 showed that both promastigotes (Fig. 4. Panels A-C) and axenic amastigotes (Panels D-F) lost 247 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom their viability progressively with exposure to increasing concentrations of the PCs from 0 to 1 248 uM (Fig. 4 Panels A-F Bottom C, PC1 and PC2). The loss of fluorescence was significantly 249 higher among red light-exposed samples (All blank bars) than those not exposed to red light (All 250 dark bars) (Fig. 4, *** to *, p values = <0.001 to 0.05). Promastigotes (Panels A-C, blank vs dark 251 bars) were seen to respond more consistently to both PC1 and PC 2 than axenic amastigotes 252 (Panels D-F, blank vs dark bars). The differences between red light and no-light for each PC 253 concentration used are significantly greater for promastigotes (Fig. 4. Panels A-C, blank vs dark 254 bars, ***, p values = <0.001 ) than axenic amastigotes (Panels D-F, blank vs dark bars, *** to *, 255 p values = <0.001 to 0.05 ), indicating that the former are more susceptible to PC1and PC2256 mediated photo-inactivation. This conclusion is also evident by comparing each set of the values 257 under the light condition at each PC concentration for both stages, especially at the highest 258 concentration of PCs (Fig. 4 Panels A-C vs D-F, Blank bars at 1 uM PC, ***, p values = 259 <0.001). At the highest PC concentration of 1 uM used, PC2 appeared to be more effective than 260 PC1 against promastigotes (Fig. 4 Panels A-C, PC1 vs PC2 Blank bars at 1 uM, *** to **, p 261 values = <0.001 to 0.01). This difference was less pronounced for axenic amastigotes (Panels D262 F, PC1 and PC2 Blank bars at 1 uM), especially PC1-treated GFP transfectants in response to red 263 light (Fig. 4, Panel E, Dark vs light bars at PC1 0.01-1 uM). 264 Growth of promastigotes in the presence of PC1 and PC2 (Fig. 5): Room light exposure was 265 minimized under “normally used” and “more stringent” conditions as defined (see Materials and 266 Methods). Daily collected samples included small aliquots from all for brief microscopic 267 observations, which showed no gross differences in motility, integrity and density of the 268 promastigotes exposed to all PC concentrations (0-1 uM) under both conditions throughout the 269 incubation period of 4 days. GFP fluorescence assays of the samples showed differences in PC270 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom mediated growth inhibition between the two conditions. Under “normally used” conditions, GFP 271 fluorescence was reduced in samples exposed to all PC concentrations used by up to 25% of the 272 PC-untreated controls on day 4 (Fig. 5A). The loss of fluorescence increased progressively with 273 time at PC concentrations from 0.1 to 1 uM, but was not evident until day 2 at 0.01 uM. These 274 data are consistent with those for photo-inactivation under “dark” conditions presented in Fig. 4. 275 Under dark conditions controlled more stringently, there was also loss of GFP fluorescence at all 276 PC concentrations used, but to a much smaller extent, especially notable at 0.01-0.1 uM (Fig. 277 5B). 278 Differential sensitivity of Leishmania versus J774 macrophages to PC1and PC2-mediated 279 photo-inactivation (Fig. 6): Leishmania are far more sensitive than their host cells or J774 280 macrophages to PC-mediated photo-inactivation. The J774 cells actively endocytosed PC1 and 281 PC2 into their endosomes, which accumulated in the peri-nuclear region (Fig. S4). The PC282 loaded macrophages remained unchanged morphologically (Fig. S4, 3 row) and metabolically 283 at all PC concentrations used up to 10 uM, irrespective of exposure to red light (Fig. 6 A and B, 284 open and solid squares). The EC50 of both PCs for these cells is thus >10 uM. Under the same 285 conditions, both Leishmania stages lost their viability to photo-inactivation progressively with 286 increasing concentrations of PCs up to 1 uM (Cf. Fig. 4 A, D), Their loss of cell viability was 287 complete at 10 uM with or without red light exposure (Fig. 6 A and B, Circles and triangles at 10 288 uM). The EC50 values of PC1 and PC2 for reducing Leishmania viability were estimated from 289 the graphs presented in Fig. 6. Under the dark conditions, the EC50 values of both PCs appeared 290 to fall between 1-10 uM, comparable for both Leishmania stages (Fig. 6A and B Solid triangles 291 and circles). The EC50 values are significantly lowered by red light exposure, varying with PCs 292 and Leishmania stages. The estimated EC50 values of PC1 are ~0.3 uM and ~0.1 uM, and those 293 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom of PC2 are 0.2 uM and 0.07 uM for axenic amastigotes and promastigotes, respectively. Thus, 294 PC2 is 1.5 times more effective than PC1 against both Leishmania stages, while axenic 295 amastigotes are ~3 times more resistant than promastigotes to both PCs. All EC50 values were 296 estimated from data obtained by the MTT assay – the only method usable to assess the viability 297 of both Leishmania and macrophages. These EC50 values of PC1 and PC2 for Leishmania are 298 consistent with the data presented in Fig. 4 (A, D) under the light conditions, but appeared higher 299 than those from the fluorescence assays, judging from the data presented in Fig. 4 (C-E). 300 301 302 Discussion 303 The major contribution of the work presented is the discovery of new PC’s, which are 304 more effective than their congeners found previously against Leshmania. Evidence is provided 305 from in vitro observations at the cellular level based on the previously established criteria for 306 effective photodynamic inactivation of Leishmania. 307 The uptake of PS by Leishmania is a prerequisite for its PDT effectiveness (18, 20, 23). 308 PC1 and PC2 are clearly taken up by both stages of L tropica, as evidenced by fluorescent 309 microscopy (Fig. 1). The emergence of PC-positive vesicles near the flagellar pockets, lysosome310 like multi-vesicular bodies and in distal vacuoles are all structural recapitulation of endocytic 311 pathway, as shown previously by using different PS’s of similar properties, including PC14 and 312 PC15 (23) and re-confirmed here by co-localization with FITC-dextran as an endosome marker 313 (Fig. S3, Co-localization). The uptake of all these PCs by Leishmania is thus akin to endocytosis 314 of soluble molecules at the lining membrane of their flagellar pocket where endosomes are 315 formed for shuttling endocytosed cargo to lysosomes. The uptake of these PCs follows saturation 316 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom kinetics, consistent with endocytic activity (Fig. 2). The absence of PC fluorescence in the 317 cytosol is indicative of very little, if any uptake of PC1 and PC2 via transporters of the plasma 318 membrane for soluble molecules, such as polyamines (30), consistent with the previous finding 319 that spermidine did not compete with PC1 and PC2 for their uptake by mammalian cells (26). 320 PC1 and PC2 taken up by L tropica sensitize them effectively to red light for photo321 inactivation. This is underscored by the facts that PC3 and PC4 are not taken up and have no 322 photodynamic activity, consistent with previous observations using similarly ineffective PS’s 323 (18, 20, 23). Qualitative and quantitative data provided convincing evidence for PC1and PC2324 mediated photo-inactivation of Leishmania. This observation is indicated clearly by seeing red 325 light-induced cellular changes of PC-loaded Leishmania, i. e. flagellar immobilization, cell 326 disintegration and loss of cellular GFP/CFSE fluorescence (Fig. 3). This loss of cell viability as 327 indicated is conclusively demonstrated quantitatively by three different assays, showing that it is 328 PC dose-dependent for both stages after red light exposure (Fig. 4). The use of three different 329 methods lends credence to the results obtained. Some discordance of the data obtained from the 3 330 different methods is not unexpected, considering that they are based on different mechanisms, i. 331 e. MTT reduction activity and detergent release of fluorescence from GFP/CFSE labeled cells. 332 While MTT assay is a gold standard extensively used to assess cell viability (20, 23), it is less 333 sensitive than the fluorescence assays developed (data not shown). The two fluorescence assays 334 have relative merits for assessing anti-Leishmania PDT. GFP is sensitive as a protein to 335 oxidative photo-denaturation for loss of fluorescence (20), but its use is limited by the necessity 336 of producing transfectants for each species. In contrast, CFSE is universally applicable to any 337 Leishmania by one-step incubation, although the fluorescence is less detergent soluble, thereby 338 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom reducing its sensitivity. Together, the qualitative and quantitative data presented leaves no doubt 339 that PC1 and PC2 are effective PS’s for anti-Leishmania PDT. 340 The efficacy of PC1and PC2-mediated photo-inactivation of Leishmania is actually 341 greater than what is reflected in the differences seen between dark and light conditions used (Fig. 342 4). Unavoidable ambient light apparently contributes to photo-inactivation of PC-loaded 343 Leishmania under the routine dark conditions used, since the growth inhibition under these 344 conditions was reduced when dark conditions were controlled more stringently (Fig. 5). While 345 dark toxicity of PC1 and PC2 to Leishmania cannot be totally ruled out, it appears to be rather 346 marginal in comparison to their anti-Leishmania photo-inactivation. 347 For photo-inactivation of Leishmania, the amino-PCs used here are 10-40 times more 348 effective, depending on Leishmania stages, than both the endosome-targeting pyridyloxy PCs 349 and mitochondrion-targeting anilinium PCs previously examined (23), as indicated by comparing 350 the EC50s of PC1 and PC2 (Fig. 6) versus PC14 and PC3.5 (23, Fig. 2). Even more striking are 351 the host-parasite differential sensitivities to these PCs for photo-inactivation, as reflected in their 352 EC50s that is in favor of anti-Leishmania PDT greatly by >100 times for PC1 and PC2 (Fig. 6), 353 but only ~3 times for PC14 and negative against Leishmania by 10-20 folds for PC3.5 (23). The 354 differences of these PCs in mediating Leishmania photo-inactivation are due neither to the use of 355 different Leishmania spp. (data not shown) nor to their photodynamic properties per se, e. g. 356 quantum yields (26), but apparently to their differences in the axial substitutions with different 357 ligands. Axial substitution of the Si-PC with 2 symmetrical monoor di-amino groups (Fig. S1) 358 instead of pyridyloxy groups (23) appears to significantly improve its endocytic bioavailability 359 (cationicity and solubility) to Leishmania cells for photosensitization in aqueous milieu. While 360 the precise mechanisms remain unknown, empirical observations showed that PC1 and PC2 361 on July 0, 2017 by gest httpaac.asm .rg/ D ow nladed fom stayed soluble longer in the culture media than the other PCs. Also unknown is the unexpected 362 finding of the huge host-parasite disparity seen in their susceptibility to PC1and PC2-mediated 363 photo-inactivation under the experimental conditions used. At greater fluence of illumination, 364 PC1 and PC2 have been shown to inactivate mammalian cells other than macrophages (26). It 365 awaits further study to determine if the latter are more PDT-resistant because these phagocytes366undergo respiratory burst and thus may be equipped with a higher capacity of anti-oxidant367mechanisms than other mammalian cells.368In summary, the results obtained identify PC1 and PC2 as excellent PS’s for oxidative369photo-inactivation of Leishmania for use as potential vehicles in drug/vaccine delivery,370considering that they are up to 40 times more effective than those used previously (23). In371addition, the finding that Leishmania are >100 times more susceptible than macrophages to372sensitization by PC1 and PC2 for photo-inactivation suggests their potential utility for anti-373Leishmania PDT, pending further evaluation in in vitro and in vivo models of experimental374leishmaniasis.375376Acknowledgements: This Project was funded by the National Plan for Science, Technology and377Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdome of378Saudi Arabia, Award Number (12-MED2681-02). We thank Dr. Ken Neet for reviewing this379manuscript.380381onJuly0,2017bygesthttpaac.asm.rg/Downladedfom References3823831. Oleinick NL, and Evans HH. 1998. The photobiology of photodynamic therapy:384Cellular targets and mechanisms. Radiat Res 150, S146-1564.3852. Zhao B and He YY 2010. Recent advances in the prevention and treatment of skin386cancer using photodynamic therapy. Expert Rev Anticancer Ther 10: 1797-1809.3873. Lønning, P E 2010. 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Genetic and biochemical analysis of protozoal polyamine475transporters. Methods Mol Biol. 720:309-326.476477 onJuly0,2017bygesthttpaac.asm.rg/Downladedfom Figure legends478Fig. 1. Fluorescent microscopy of Leishmania tropica, showing uptake of amino-phthalocyanine479 conjugate PC2 by both stages.480Panels A-C/A’-C’: Promastigotes; Panels D-F/D’-F’: Axenic amastigotes.481 A/A’, D/D’: Phase contrast; B/B’, E/E’: Cy5 fluorescence for phthalocyanine PC2; C/C’, F/F’:482 Phase/Cy5 merged images.483Cells were loaded in the dark with 1 uM PC2 for ~16 hours and washed once before imaging.484See Materials/Methods for experimental details.485Short arrow, PC-positive endosomes near flagellar pockets; Long arrow, PC-positive486filamentous multi-vesicular bodies and endosomes distant from the flagellar pocket.487488Fig. 2. Saturation kinetics of amino-phthalocyanine uptake by Leishmania tropica.489Horizontal axis: Time periods in days for sample collections after incubation of promastigotes490with a mixture of 1 uM PC1 and 1 uM PC2 in the dark for ~24 hours.491Vertical Axis: Cellular concentrations of PCs in nmoles/10 cells, as determined by fluorimetry492of the extraction from PC-loaded cells taken at different times after incubation.493See Materials/Methods for experimental details for PC extraction with DMSO, fluorimetric494readings of the extracts for fluorescence intensities and determination of PC concentrations by495extrapolation against the values from a standard curve of graded PC concentrations.496Note: The fluorescence readings of cell-associated PCs follow saturation kinetics with times of497 incubation within each experimental set, but vary considerably with different experiments,498accounting for the large standard errors seen.499500onJuly0,2017bygesthttpaac.asm.rg/Downladedfom Fig. 3. Phase contrast and fluorescence microscopy of GFP-transfected Leishmania tropica501 promastigotes, showing PC-mediated photo-inactivation.502[A] to [D]: the settings used to capture the images of GFP-transfected promastigotes. [A]503PHASE CONTRAST for assessing cellular integrity; [B] FITC/GFP: FITC filter set for GFP504fluorescence; [C] Cy5/PC: Cy5 filter set for PC fluorescence; and [D] FITC/Cy5: Merged505 images of [B] and [C].506[1] to [5]: Treatments of cells. [1] None: No treatment; [2] PC1: Exposure to 1 uM PC1 for 1507day in the dark; [3] PC1+Light: Sample [2] further exposed to red light for 30 min followed by5081-day incubation; [4] PC2: Exposure to 1 uM PC2; for 1 day in the dark; [5] PC2+Light:509Sample [4] further exposed to red light for 30 min followed by 1-day incubation. See Materials510and Methods for experimental details.511Note: Cellular disintegration ([A]-[3] and [A]-[5]) and loss of GFP fluorescence ([B][D]-[3] and512[B][D]-[5]) of the promastigotes only after photo-inactivation.513 514Fig. 4. PC dose-dependent photo-inactivation of both Leishmania tropica stages, as determined515by MTT reduction and fluorescence assays516Panels A-C, Promastigotes; Panels D-F, Axenic amastigotes. Panels A, D: Relative MTT517reduction activities; Panels B, E: Relative intensities of GFP fluorescence detergent-released518from GFP-transfectants; Panels C, F: Relative intensities of FITC fluorescence detergent-519released for CFSE-loaded cells.520Horizontal axis: C, Controls without treatments; PC1 and PC2, Cells exposed to 0.01, 0.1 and5211 uM PC1 or PC2 overnight, respectively; Black and blank bars, Samples with and without red522light treatment.523onJuly0,2017bygesthttpaac.asm.rg/Downladedfom Vertical axis: % of control for MTT reduction activities and fluorescence intensities by524 normalizing the values of the experimental groups against those of no-treatment controls.525 The p-values (*, 0.01-0.05; **, <0.01-0.001; ***, <0.001) were calculated by paired Student-t526 tests for the data between dark (black bar) and light (blank bar), and for paired data connected by527 brackets.528Fig.5. Effect of amino-phthalocyanines on the growth of Leishmania tropica promastigotes529under dark conditions of different stringencies.530Panels A and B: Cells grown under “normally used” and “more stringent” dark conditions,531respectively, as described in Materials and Methods.532Horizontal axis: Growth period in days after cultivation of GFP-promastigotes in the presence533of 0-1 uM PCs; Vertical axis: GFP fluorescence intensities in % of no-treatment controls,534reflective of cell growth in graded PC concentrations. See Materials and Methods for the culture535conditions used, daily collections of samples for storage as frozen pellets and assay for cell536growth based on fluorescence of detergent-released GFP.537Note: Cell growth is more affected under the “normally used” dark conditions (Panel A) than538“more stringent” dark conditions (Panel B).539540541Fig. 6. Disparity of Leishmania tropica and macrophages in their susceptibility to PC-mediated542photo-inactivation.543Panels A and B: Viability of J774 macrophages and both stages of L tropica under light (open544symbols) and dark (closed symbols) conditions after exposure to graded concentrations (0.001-54510 uM) of PC1 and PC2, respectively.546onJuly0,2017bygesthttpaac.asm.rg/Downladedfom Horizontal axis: PC concentrations used from 0.001 to 10 uM;547Vertical axis: Cell viability in % of un-treated controls based on MTT reduction assays.548AxAm Light and Dark (Open and closed triangles), PC-treated axenic amastigotes with and549 without exposure to red light, respectively;550Pm Light and Dark (Open and closed circles), PC-treated promastigotes with and without551 exposure to red light, respectively;552Mac Light and Dark (Open and closed squares), PC-treated J774 macrophages with and553without exposure to red light, respectively.554See Materials and Methods for incubation conditions, MTT assay and red light exposure for 20555min.556 onJuly0,2017bygesthttpaac.asm.rg/Downladedfom