In vitro assays, parasites and infections

(i)Assays on promastigotes

Promastigotes of Leishmania major LV39 (MRHO/Sv/59/P), Leishmania mexicana (MNYC/BZ/62/M379), Leishmania donovani infantum LV9 (MHOM/ET/67/HU3), Leishmania donovani donovani sudanese 1S2D (MHOM/SD/62/1S-CL2D), Leishmania donovani archibaldi 2211 (MHOM/SD/91/D1783; LEM 2211), Leishmania mexicana amazonensis LV78 (MPRP/BR/72/M1845), Leishmania (Viannia) panamensis (MHOM/87/CO/UA140), Leishmania (Viannia) guyanensis (MHOM/SR/87/TRUUS4) and Leishmania tarantolae (TarIIWT) in log phase of growth (5 days of culture) were exposed to OlPC in duplicate alongside non-treated controls. Stock solution of OlPC (100 mM) was prepared in endotoxin-free PBS (Wisent) and consecutively diluted (2 × serial dilutions; 125 ul/well in 96 well plates) to obtain final concentrations ranging from 0.1 μM to 51.2 μM in presence of 125 μl of Leishmania culture. Plates were incubated at 25°C for 7 days (stationary phase) in presence of SDM medium pH 7.3 supplemented with 10% heat inactivated FBS and hemine, then Leishmania concentrations were determined by optical density (600 nm)9. Parasite concentrations in treated cultures were expressed as percentages of concentration in non-treated cultures.

(ii)Cell culture

Murine macrophages cells line B10R10 and J774A.1 (ATCC) were maintained in culture in DMEM (B10R) or RPMI (J774A.1) supplemented with final concentration of 10% decomplemented fetal bovine serum (FBS - Gibco) and 1× penicillin/streptomycin/glutamine (PSG - Wisent). Cells were passed every two days with starting concentration for B10R: 0.3 × 106/8 ml/100 mm2 dish (BD falcon); and J774A.1: 0.7 × 106/8 ml/100 mm2 dish (BD falcon). Cell detachment was done using rubber cell scrapper (B10R) or re-suspended with culture medium (J774A.1).

Determination of % of infected cells by light microscopy

One coverslip (Fisher) was added/well in 24 wells plate (BD Falcon). B10R (0.1 × 106 cells/1 ml of DMEM 10% FBS/well in 24 wells plate) and J774A.1 (0.2 or 0.1 × 106 cells/1 ml of RPMI 5% FBS/well in 12 wells plate) were plated on the day before the experiment. The next day medium was replaced by 300 μl of fresh medium containing Leishmania major (NIH S (MHOM/SN/74/Seidman) clone A2) or L. mexicana(MNYC/BZ/62/M379) in 1:20 (B10R) or 1:10 (J774A.1) ratios, assuming that cells had divided once since plating (i.e. considering double amount of cells compared to the starting numbers). After 6 h of infection, cells were washed with PBS and coverslips were put in presence of OlPC-liposome concentrations. Cells were incubated for an additional 18 hrs. After 24 h of culture, cells were washed twice with PBS and coverslips were collected and stained with Diff Quick (Behring #84132-1A). Subsequently dried coverslip were mounted on slides with Permount media (Fisher) and % of infected cells were determined using 3–5 count of 100 cells by light microscopy (Leica).

Animals

Balb/c female mice of 6 to 9 weeks of age (weights of about 20 g) were purchased from Charles River (Montreal, Canada) and housed at McGill University. All experiments were performed in accordance to the guidelines of the Canadian Council on Animal Care. Animal protocols were approved by the Animal Care committee of McGill University (UACC); protocol number: AUP#6088.

In vivo infections

Mice (10 mice per/group) were shaved at tail base and injected subcutaneously (s.c.) with 50 μl of sterile saline containing 10 × 106 of Leishmania major Friedlin-luciferase (stably transfected with luciferase reporter gene)11 or with Leishmania mexicana using a 1 ml syringe and 27 G1/2 needle. Mice were then checked regularly to monitor the appearance and growth of the lesions until they reached ~5 mm2 in surface (average 8–12 weeks post-infection). Following lesion development, mice were selected according to lesion size to ensure similar levels of infection and were divided into groups (6–9 mice showing lesion/group) for treatment. Control groups were treated with PBS while treatment groups were treated with OlPC-liposomes. Each group was treated daily for 10 days (2 × 5 days) with the permanent make-up (PMU) delivery system, by intraperitoneal injection, or by simply dropping the liposomal drug on the lesion (see below).

Substance and Substance delivery

Oleylphosphocholine (OlPC; Dafra Pharma Research and Development, Belgium) was formulated in liposome and stored as previously described4. The OlPC -liposomes were used directly from the stock (18 mg/ml, final concentration) for tattooing or freshly diluted in medium for the in vitro experiments.

Tattooing-mediated drug delivery was performed using a commercial professional permanent make-up apparatus (Nouveau contour Digital 1000 machine, Micro-Pigmentation Inc., Missisauga, Canada) and needles (Nouveau contour Needle #5 Magnum (5-needles head)). The substances (PBS vehicle or anti-leishmanial therapy) were administered on anesthetized mice (isoflurane mask) to facilitate the procedure. Each tattooing session was done by performing 12 times repeated two second lasting administrations with the 5 needles oscillating at 100 Hz (i.e. 100 punctures per second), for a total of (5 × 12 × 2 × 100) 12000 punctures; excess drug was not wiped off after the procedure. We estimate that ~2–5 μl of OlPC-liposomes (36–90 μg) is injected into the skin during every tattooing session3. The device was adjusted to allow exposure of 1.5 mm of the needle tip beyond the barrel guide. Gentle pressure was used to facilitate penetration of the needle into the skin resulting in the deposition of the substances into the dermis5. Topical treatments were done by applying 50 μl of liposomal drug (i.e. 900 μg of OlPC) on the lesion for 30 seconds on anesthetized mice (isoflurane mask) before waking up the animal. Intraperitoneal treatments were performed at the dose of 30 mg/kg for 10 days (2 × 5 days) without anesthesia. Following each drug administration mice were closely monitored for evidence of inflammation, pain and distress. Animal weights were also measured as signs of toxicity.

In vivo treatment evaluation

Treatment efficacy was evaluated using three different parameters: (i) lesion areas; (ii) skin histolopathology; and (iii) tissue parasite burden as measured by luciferase activity and dilution assays.

(i) Lesion areas

For each experiment pictures of the lesions were taken on Day 0, 7, 14, 21 and 28 to document the evolution of the sizes. Lesion areas were individually calculated from the pictures using the Adobe photoshop® software and were expressed in mm2. Specifically, the ruler tool was first used to determine the pixel number in 1 cm on the ruler present on a given picture. These values were used to set the measurement scale (Pixel number = L1 valor, Logical length = 1, Logical unit = cm). Subsequently, the lesion borders were delimited using the lasso tool and the measurement was recorded. A mouse with no apparent lesion was given a lesion area of the smallest area possible to draw ~ 0.0007 ± 0.00042 mm2.

(ii) Histopathology

After Day 28, the infected animals were humanely euthanized in a CO 2 chamber and the skin around the lesion was taken and washed in PBS, fixed in 10% neutral formalin, processed and embedded in paraffin. Blocks were cut at 4 μm, mounted on slides and stained with H&E and Giemsa.

(iii) Quantification of tissue parasite burdens

Limiting dilution assay was done as previously described12,13, with some modifications. Briefly, on Day 28 mice were euthanized and individual lesions were disinfected, excised and homogenized in 1 ml of sterile PBS. Tubes were spun down (500 RPM for 30 seconds) and 100 μl of the lysate was diluted again in 10 ml of PBS (1:100). A hundred μl of extracted parasites (in 1 ml or 100 times diluted) were serially diluted in a 96 well plate in duplicate and put in culture. Eight days later the number of viable parasites was determined from the highest countable dilution using an inverted microscopy. This highest dilution was multiplied by a factor 10 (1000 μl/100 μl) or 1000 (1000 μl/100 μl × 100) to obtain the parasite burden. Lesions infected with luciferase-labeled parasites were processed as above, then homogenized material from each lesion was spun down (13,000 rpm/10 min) and the pellet were frozen at −80°C. Frozen pellets were lysed with 30 μl of Cell Culture Lysis Reagent (Promega) diluted 1:5 in ddH 2 O, vortexed, incubated on ice 15 min and spun down at 13000 rpm/10 min at 4°C. Five μl of lysate were then mixed with 90 μl of Luciferase Assay Reagent (Promega) and luciferase activity were determined using a Mini Lumat LB 9506 luminometer (EG&G, Berthold, Germany). Protein concentrations were measured using Bradford reagent (Bio-Rad). Results were reported as relative light units (RLU)/μg of protein.

Statistical analysis

IC50 on promastigotes were calculated by non-linear regression using GraphPad Prism5® software. For the statistical analysis in biological assays, means ± standard errors of the mean (SEM) were calculated by the GraphPad Prism5® software. Multiple comparisons between groups were made with a one-way analysis of variance (ANOVA) followed by Dunnet and Tukey tests. P values of less than 0.05 were considered significant (*).