Fig 1.
Effects of PHMB on L. major morphology and behavior.
(b and d) TEM images of L. major promastigotes (right) showing morphological changes such as shrinkage, extensive cytoplasmic vacuolization (V), marked loss of cytosolic contents and condensed nucleus (N). Images b and d were taken after treatment with 2 μM PHMB for 48 and 24 h, respectively. (a and c) The untreated controls (left) show normal elongated morphology of promastigotes with intact and clear distinct kinetoplast (kDNA), N, mitochondrion (M), lipid vacuoles (LV) and glycosome (G). (e-h) Light microscopy images showing morphological and behavioral changes (no more clamp formation as indicated by arrowheads) after treatment with 2 μM PHMB for 24 h. Scale bars = (a) 1.1 μm, (b) 0.6 μm, (c and d) 0.25 μm, (e and f) 7.5 μm and (g and h) 25 μm.
Table 1.
Antileishmanial and cytotoxic effects of PHMB and PHMB/DNA polyplexes.
The tables show antileishmanial efficacy, cytotoxicity and selectivity of the compounds. The IC50 values and/or selectivity index (SI) of the compounds as compared to standard antileishmanial drugs are shown. The table values are the average IC50 values of 3–6 independent experiments for each compound. IC50 = minimum concentration of the substances required to kill 50% of the population; SI = mean IC50 against BMDM /mean IC50 against L. major amastigotes; ND = not determined.
Fig 2.
Mechanism(s) of action of PHMB on L. major promastigotes.
PHMB disrupted membrane integrity and condensed DNA in L. major promastigotes. Representative FACS histogram (a) propidium iodide (PI) and (b) YO-PRO-1dye staining of promastigotes after treatment with 2 μM PHMB for indicated time points, showing time-dependent effect of PHMB on the membrane integrity. Heat-killed promastigotes and amphotericin B at 2 μM concentration were used as positive controls. (c) Fluorescent microscopy analysis showing condensed and damaged DNA materials of L. major promastigotes after treatment with PHMB at 2 μM for 48 h as compared to the mock control (distilled water). Scale bars = 5 μm.
Fig 3.
Cell localization of PHMB-FITC in promastigotes and BMDM.
Confocal images showing uptake of PHMB-FITC by (a) promastagotes and (b) BMDM. Note apparent nuclear exclusion in BMDM cells but not parasites, scale bars = 10 μm.
Fig 4.
PHMB/DNA interactions and physicochemical characterization of polyplexes.
Formation of PHMB/DNA polyplexes confirmed by 1% agarose gel, TEM and color change. (a) PHMB/gDNA polyplexes, (b) TEM picture showing PHMB/CpG ODN polyplexes formation at 2:1 ratio, (c) PHMB/CpG ODN polyplexes, (d) temporary turbidity change during PHMB/CpG ODN complexation, (e) PHMB/CpG-R polyplexes and (f) the same gel (e) with fluorescence measurement of CpG-R. With the exceptions of negative control and PHMB alone, all wells contained equal amounts (weight) of DNA with various concentrations of PHMB. M represents 2-Log DNA ladder (0.1–10.0 kb, New England Biolabs) and N represents water used as a negative control. All indicated PHMB/DNA ratios are in relative weight (w/w). Indicates two month old PHMB/CpG ODN polyplexes. Scale bar = 300 nm.
Fig 5.
Physicochemical characterization of PHMB/CpG ODN nanopolyplexes.
The figures show quantitative measurements of the strength of interaction between CpG ODN and PHMB by ITC with the corresponding thermodynamic parameters: binding affinity (Ka), enthalpy changes (ΔH) and entropy changes (ΔS). (a) Raw data generated by ITC, (b) and (c) analyzed ITC data using two data analysis and plotting tools: Origin and SigmaPlot, respectively.
Table 2.
Dynasore rescues killing of L. major amastigotes inside macrophages.
Dynasore inhibited killing of the intracellular L. major amastigotes and promastigotes by PHMB or PHMB/CpG ODN polyplexes. Values are the average of two independent experiments and the values are given as mean ± SE. The concentration values given are for PHMB within polyplexes.
Table 3.
Physicochemical characterization of PHMB/CpG ODN nanopolyplexes.
The table shows particle size and zeta potential determination by using DLS and ELS with their PDI. The table summarizes the results of three independent experiments. Size distribution and zeta potential measurements were performed using Millipore water and ISA water (0.15M KCl), respectively.
Fig 6.
Cellular uptake of PHMB and CpG ODN, and their polyplxes by BMDM.
The overlay histograms show time dependent uptake of (a) PHMB-FITC and (b) PHMB-FITC/CpG ODN polyplexes into macrophages. Whereas (c) shows uptake of CpG-R by BMDM as polyplex form compared to its free form. The histograms are representative of three independent experiments.
Fig 7.
Cellular uptake of PHMB-FITC and PHMB-FITC/CpG ODN polyplexes and effects of selective endocytosis inhibitors.
The bar graphs show the effects of different inhibitors and temperature on cellular uptake of (a) PHMB-FITC, (b) PHMB-FITC/CpG ODN polyplexes, (c) dextran/FITC used as positive control to exclude the effect of FITC that might interfere with the cellular uptake mechanisms of PHMB and (d) alexa-448-labeled transferrin used as positive control for clathrin-dependent endocytosis. Normalized MFI values of three independent flow cytometry experiments are shown and the values are given as mean ± SE.
Fig 8.
Effects of CpG ODN and PHMB/CpG ODN polyplexes on cytokine production by BMDM.
The bar graphs show the levels of (a-b) IL-6, (c-d) IL-10 and (e-f) IL-12 production after CpG ODN or PHMB/CpG ODN polyplexes were added to BMDM that had been pre-activated with CpG ODN (15 μg/ml) for 30 min and further incubated for 48 h. Controls were cells without any pre-stimulation and the 0 μM doses were cells pre-stimulated by CpG ODN but without any further stimulation by CpG ODN or the complex. The concentrations shown for the polyplexes represent the dose of PHMB and the concentrations of CpG ODN are half of the indicated doses. The error bars show standard errors from three independent experiments.