Endosome-targeting photodynamic therapy of cutaneous leishmaniasis due to Leishmania tropica
1Saad
Alkahtani, 2Ahmed Al-Qahtani, 3Dennis K. P. Ng, 4Chin Shen Chang, 5Kayoko Waki, 5Pankaj Tripathi, 5Bala Kolli, 5Kwang Poo Chang
of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia, Riyadh 11352; 2Department of Infection and Immunity, Research Center, King Faisal Specialist
Hospital and Research Centre, Riyadh, Saudi Arabia; 3Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong; 4LeishVac LLC, Kenilworth, IL 60043,
USA; 5Department of Microbiology/Immunology, Chicago Medical School/RFUMS, North Chicago, IL 60064, USA
1Department
The sensitivity of this and other L tropica
strains to oxidative photo-inactivation was first
assessed and determined by fluorescent
microscopy,
showing
that
cultured
promastigotes accumulated endosomally two
novel polyamine-phthalocyanins (Pcs 1-2)
(Figs. 5-6). By microscopy and MTT reduction
assay, the Pc-loaded Leishmania were
subsequently found susceptible to photoinactivation by singlet oxygen and other ROS
that were generated by a brief red-light
illumination at low intensity. Leishmania was
found 1,000 X more sensitive than their J774
host cells, as determined by the ED50 of Pcs 1-2
used (Fig. 7). This host-parasite differential
sensitivity predicts the potential utility of these
Pcs for chemo-/immuno-therapy against L
tropica-caused
cutaneous
leishmainasis.
Toward that end, we have begun to generate
transgenic L trpoica for expressing egfp (Fig. 8)
to enhance their sensitivity for quantitative
evaluation of infection via flurimetric assays in
vitro in macrophages and in animal models in
response to Pcs 1-2-mediated oxidative photoinactivation as before (Dutta et al. 2011 PLoS
ONE 6, e20786). The results from these studies
are expected to yield data, indicative of the
feasibility to develop Pcs 1-2 for future
chemotherapy
against
the
cutaneous
leishmaniasis. Transgenic approach is also
planned for producing porphyrinogenic L
tropica, as accomplished previously for other
Leishmania spp. (Sah et al. 2002 J Biol Chem
277, 14902). and used for photodynamic
vaccination (Kumari et al. 2009 Eur J Immunol
38, 1; Dutta et al. 2012 Euk Cell 11, 430). We
anticipate to use these porphyric mutants of L
tropica in combination with the external Pcloading for double photo-inactivation (Dutta et
al. 2012 Photochem Photobiol 88, 620), thereby
ensuring their safety and efficacy for future
use in immuno-therapy and -propylaxis against
cutaneous leishmaniasis. (Supported by KSA
NPSTI 12-MED2681-02)
8
10 8
Total number of J774 cells per culture
7
10 0
3
6
9
12
15
10 7
1000
0
3
6
9
12
Days post infection
Days postatinfection
The rate of
infection was quantitatively determined
the time
points indicated by phase contrast/fluorescent microscopic
counting of ~100 cells for % infection and the average # of
Leishmania/cell. The total # of Leishmania per culture
determined from these data (Left) and the total # of J774
macrophages/culture (Right). Note: The axenic amastigotes
are more infective than promastigotes.
15
Fig. 5. Polyamine Si-phthalocyanines: Novel
photosensitizers synthesized by Prof. Dennis Ng, Chinese
University of Hong Kong used for this study.
O
Pc 1
N
H
N
N
Si
N
N
N
N
N
H
Pc1
O
O
C40H36N10O2Si
Exact Mass: 716.28
Mol. Wt.: 716.87
N
H
N
H
N
N
N
N
Si
N
N
N
N
Note: Promastigotes (Left) lose flagellum and become amastigotelike (Right) at 33 C, pH 5.3 Giemsa Stain. Bar = 5 uM
N
H
N
H
O
Pc2
C46H50N12O2Si
Exact Mass: 830.39
Mol. Wt.: 831.05
Pc 2
10
1
1
0.01
0.1
1
Pc Concentration (µM)
N
N
Axenic amstigote
100
10
0.001
Fig. 2 Promastigote to axenic amastigote differentiation of L.
tropica EP41
Promastigote
Axenic amstigote
100
10 6
10
0.001
0.01
0.1
1
Pc Concentration (µM)
Parasite vs host differential sensitivity to Pc-mediated photoinactivation: [1] Leishmania promastigotes and J774 macrophages
grown in vitro were sensitized in the dark separately by incubation in
the presence of increasing Pc concentrations overnight; [2] After
washing to remove the Pcs, sensitized cells were exposed to red light
(~600 nm wavelength) for 20 minutes; [3] Illuminated cells were
incubated overnight in culture medium; [4] Viability of the cells were
assessed by MTT assays; [5] The ED50 of photo-inactivation was
determined to be in the range of nM for Leishmania and uM for
macrophages, indicative of their differential sensitivity by 1,000 fold
against Leishmania.
Fig. 8. Cutanotropic Leishmania tropica transfected with
p6.5/GFP under selection with 10 ug tunicamycin/ml (40X).
Fig. 3. Infection of J774 A1 macrophages with L. tropica EP 41
Fig. 6. Fluorescent images, showing uptake of Pc1 by Leishmania
promastigotes into their endosomes (20X) Cells incubated with 1
µM Pc1 in HBSS +0.01% BSA overnight in dark, washed and
digitally imaged by fluorescent microscopy.
Infection was initiated with axenic amastigotes grown for 1 week at
a host-parasite ratio 1:10. Samples were collected 3 days after
infection. Left panel, Phase contrast; Right panel, fluorescent
microscopy in the presence of acridine orange. Note: Leishmania in
vacuoles, as indicated by their fluorescent kinetoplast and nucleus.
Endosome uptake of Pcs 1-2: [1] Leishmania grown as promastigotes
were incubated in the presence of the photosensitizers, Pcs 1in the dark
overnight; [2] Pc-exposed cells were washed by centrifugation and
resuspended in HBSS-BSA. [3] Fluorescent microscopy of these Pcsensitized cells by using cy5 filter revealed the presence of fluorescent
vacuoles in the cytoplasm (Left), suggestive of Pc-uptake by
Leishmania into their endosomes. Right, Phase contrast photo of the
same material.
1. Leishmania tropica is a genetically heterogeneous
complex, consisting of many homozygous and
heterozygous genotypes. Clinical samples isolated in
culture are known to vary significantly in their
infectivity. It is crucial to use isolates with phenotypes
closely simulating the typical clinical disease, such as
their ability to produce lesion in mice (Fig. 1), to
undergo differentiation into the mammalian stage (Fig.
2) and to infect macrophages (Figs. 3-4). The isolate
EP41 is verified to have these desirable phenotypes and
are thus chosen to initiate the work. Additional isolates
will be used to further verify the findings as universally
applicable, regardless of their geographic origin.
Macrophage
Promastigote
AxAm
Pm
Discussion
1000
Macrophage
Viability (%)
10
We have begun our work by screening clinical
isolates for those, which are cultivable in vitro
and retain disease-causing capacity in animal
models (Figs. 1-5). Strain EP41 was identified
for our initial use on account of its lesioncausing capacity in mice (Fig. 1), for its ease of
differentiation into the mammalian infective
stage (Fig. 2) and its productive infection of
macrophages, such as the J774 cell line (Figs.
3-4).
Fig. 7. MTT viability assay for parasite vs host sensitivity to Pcs
1-2-mediated oxidative photo-inactivation.
Leishmania promastigotes/axenic amastigotes grown to stationary
phase, resuspended in HBSS-BSA to 108 cells/ml, and J774
macrophages were exposed separately in the dark overnight to
increasing Pcs 1-2 concentrations as shown. Sensitized cells were
exposed to red light for ~20 min and assessed for viability by MTT
assay after overnight incubation. Note: Pcs 1-2 were >1,000 fold
more photolytic to Leishmania than to the host macrophages.
Fig. 4 Differential infectivity between axenic amastigotes and
promastigotes of L. tropica EP41 to J774 macrophages
Viability (%)
Fig. 1 Footpad lesion of a BALB/c mouse 45 days after infection
with Leishmania tropica (Photo courtesy of Prof. Ahmet Ozbilgin)
Total number of parasites per culture
Abstract
Leishmania tropica is one of the dominant
species,
responsible
for
cutaneous
leishmaniasis – a common skin disease
epidemic in Saudi Arabia, and other countries
in the Middle East and other parts of Asia and
Africa. Photodynamic immuno-/chemo-therapy
represents a new approach to minimize the
development of drug-resistance and toxicity
that has long been noted with the current use
of few ineffective compounds for treatment of
this disease.
Transfection of cutaneous L tropica with green fluorescent
protein: [1] A strain of L tropica identical to those from S. Arabia
were transfected with p6.5/GFP by electroporation according to
established methodology; [2] Transfectants were selected with
tunicamycin at 5-20 ug/ml; [3] GFP fluorescence was observed in
some of the transfected cells by fluorescent microscopy (Right),
suggestive of GFP expression in the transfectants. Left panel, Phast
contrast photo of the same material.
10
2. Pcs 1-2 (Fig. 5) are novel photosensitizers of Silicaphthalocyanines, which are conjugated with polyamines
by our collaborator, Professor Ng of Chemistry
Department, the Chinese University of Hong Kong. The
unique feature of Pcs 1-2 is their design for cellular
uptake via polyamine transporters, which are active in
proliferative cells, like cancer cells and Leishmania.
This constitutes a significant element of our studies that
differs from previous published work, including our
own.
3. Endosomal uptake of Pcs 1-2 (Fig. 6) is critical to
ascertain effective photo-inactivation. Excitation of the
Pcs by illumination with light of the red range generates
singlet oxygen whose life span amounts to only
milliseconds. Such ROS generated extracellularly does
not lasts long enough to travel for entering cell and is
thus non-cytolytic. the finding of Pcs 1-2 in the
endosomes of Leishmania instead of their cellular
membrane is indicative of not only their effectivenss but
also their specificity.
4. Leishmania vs macrophage differential photoinactivation by Pcs 1-2 (Fig. 7) represents another
critical finding, showing the selectivity of Pcs 1-2mediated photo-inactivation at a very large margin
against Leishmania versus macrophage host cells. The
results foretell the potential application of Pcs 1-2 in the
clinical setting for photodynamic therapy with high
specificity against the target with minimal collateral
damages to the patients.
5. Cutaneous L tropica GFP transfectants (Fig. 8) will be
made available to serve 2 essential purposes: [1] L
tropica is a major causative agent of leishmaniasis wide
spread in Saudi Arabia and many other countries; and
[2] The use of such fluorescent transfectants will
facilitate further investigation by increasing the
sensitivities of Leishmania detection all assays.