Photochemotherapeutic strategy against Acanthamoeba infections

Article abstract of DOI:10.1128/AAC.05126-14

Acanthamoeba is a protist pathogen that can cause serious human infections, including blinding keratitis and a granulomatous amoebic encephalitis that almost always results in death. The current treatment for these infections includes a mixture of drugs,

Full text of DOI:10.1128/AAC.05126-14

AAC Accepted Manuscript Posted Online 9 March 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.05126-14
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Photochemotherapeutic strategy against Acanthamoeba infections
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1Yousuf Aqeel, 1Ruqaiyyah Siddiqui, 2Ayaz Anwar, 2Muhammad Raza Shah, 1Shahrukh Khoja,
1Naveed Ahmed Khan
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1Department of Biological and Biomedical Sciences, Aga Khan University; Karachi, and
2International Center for Chemical and Biological Sciences, H.E.J. Research Institute of
Chemistry, University of Karachi, Karachi, Pakistan.
Running Head: Photochemotherapy against Acanthamoeba
Corresponding author: Dr. Naveed Ahmed Khan, Department of Biological and Biomedical
Sciences, Aga Khan University, Stadium Road, Karachi, Pakistan.
Tel: 92-(0)21-3486-4540. Fax: 92-(0)21-3493-4294. E-mail: naveed5438@gmail.com
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Abstract
Acanthamoeba is a protist pathogen that can cause serious human infections including a
blinding keratitis and a granulomatous amoebic encephalitis that almost always results in death.
Current treatment includes a mixture of drugs and even then infection recurrence can occur.
Photochemotherapy has shown promise in the treatment of Acanthamoeba infections, however
selective targeting of pathogenic Acanthamoeba has remained a major concern. The mannose-
binding protein is an important adhesin expressed on the surface membranes of pathogenic
Acanthamoeba. To specifically target Acanthamoeba, the overall aim of this study was to
synthesize photosensitising compound (porphyrin)-conjugated with mannose and test its efficacy
in vitro. The synthesis of mannose-conjugated porphyrin was achieved by mixing benzaldehyde
and pyrrole yielding tetra-phenyl porphyrin. Tetra-phenyl porphyrin was then converted into
mono-nitro phenyl porphyrin by selectively nitrating the para position of phenyl rings as
confirmed by NMR spectroscopy. The mono-nitro phenyl porphyrin was reduced to mono-amino
phenyl porphyrin in the presence of tin dichloride and confirmed by peak at 629 m/z. Finally,
mono-amino porphyrin was conjugated with mannose resulting in the formation of imine bond.
Mannose-conjugated porphyrin was confirmed through spectroscopic analysis and showed that it
absorbed light of wavelength ranging from 425-475nm. To determine antiacanthamoebic effects
of the derived product, amoebae were incubated with mannose-conjugated porphyrin for 1 h, and
washed 3X to remove extracellular compound. Next, amoebae were exposed to light of the
appropriate wavelength for 1 h. The results revealed that mannose-conjugated porphyrin
produced potent trophicidal effects and blocked excystation. In contrast, A. castellanii incubated
with mannose alone and porphyrin alone did not exhibit anti-amoebic effect. Consistently, pre-
treatment with mannose-conjugated porphyrin reduced A. castellanii-mediated host cell
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cytotoxicity from 97% to 4.9%. In contrast, treatment with porphyrin, mannose or solvent alone
had no protective effects on host cells. These data suggest that mannose-conjugated porphyrin
has application for the targeted photodynamic therapy of Acanthamoeba infections and may
serve as a model in the rationale development of therapeutic interventions against other
eukaryotic infections.
Keywords: Acanthamoeba; mannose-conjugated porphyrin; photochemotherapy; Targeted
therapy; In vitro assays.
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Introduction
Pathogenic Acanthamoeba are well known to produce serious infections, including a fatal
granulomatous amebic encephalitis (GAE), and a painful, sight-threatening keratitis (reviewed in
1, 2). The most distressing aspect is that the mortality associated with GAE due to pathogenic
Acanthamoeba has remained significant (more than 90%) despite our advances in antimicrobial
chemotherapy and supportive care (1, 2). Similarly, current diagnosis of Acanthamoeba keratitis
is challenging (1, 2), and the available treatments are lengthy and not fully effective against all
strains (3), in part due to the ability of amoebae to transform into a resistant cyst form (4),
resulting in infection recurrence. A complete understanding of the pathogenesis and
pathophysiology will undoubtedly lead to the development of diagnostic advances and
therapeutic interventions (5, 6). Recent studies have identified a mannose-binding protein (MBP)
as an important adhesin expressed on the surface membranes of Acanthamoeba, as free
exogenous mannose blocks the parasites’ adhesion to primary human brain microvascular
endothelial cells and corneal epithelial cells (7, 8). Notably, oral immunization with recombinant
mannose-binding protein ameliorates Acanthamoeba keratitis in the Chinese hamster model (9).
Acanthamoeba mbp contains 6 exons and 5 introns that spans 3.6kbp. The 2.5kbp cDNA codes
for an 833amino acids precursor protein with a signal sequence (residues 1 – 21 aa), an N-
terminal extracellular domain (residues 22 – 733 aa) with five N- and three O-glycosylation sites,
a transmembrane domain (residues 734 – 755 aa), and a C-terminal intracellular domain
(residues 756 – 833 aa) (7).
Photochemotherapy is a promising technology that can target pathogens and thus, has the
potential for therapeutic interventions. The basis of this technology is that visible light (or light
of appropriate wavelength) activates the photosensitising compound, resulting in production of
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singlet oxygen and other reactive oxygen species (ROS) to induce cell death in the target
pathogen/tissue (10, 11). However, it is possible that the photosensitising compound may bind to
host cells and subsequent irradiation could lead to their destruction. In this context, selective
targeting of pathogenic Acanthamoeba has remained a major concern (12). To specifically target
Acanthamoeba, the overall aim of this study was to synthesize the photosensitising compound
(porphyrin)-conjugated with mannose and test its efficacy in vitro.
Materials and Methods
Acanthamoeba castellanii cultures
All chemicals were purchased from Sigma Laboratories (Poole, Dorset, England), unless
otherwise stated. A clinical isolate of A. castellanii belonging to the T4 genotype was obtained
from American Type Culture Collection (ATCC 50492). Amoebae were routinely grown in 10
mL PYG medium [proteose peptone 0.75% (w/v), yeast extract 0.75% (w/v) and glucose 1.5%
(w/v)] in T-75 cm2 tissue culture flask at 37°C as previously described (12). Media were
refreshed 15-20 h prior to experiments. Amoebae adherent to the flask represented the
trophozoite form and were collected by placing the flask on ice for 20 min with gentle agitation
and used in all subsequent experiments.
Human brain microvascular endothelial cell (HBMEC) cultures
Primary brain microvascular endothelial cells were of human origin and were isolated
and cultured as previously described (8, 13). The endothelial cells were purified by fluorescent
activated cell sorting, and their purity tested using endothelial markers such as expression of F-
VIII, carbonic anhydrase IV and uptake of acetylated low density lipoprotein (DiI-AcLDL) (8,
13), resulting in >99% pure endothelial cultures. The human BMEC were routinely grown in rat
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tail collagen-coated tissue culture dishes in RPMI-1640 containing 10% heat inactivated fetal
bovine serum, 10% Nu-serum, 2 mM glutamine, 1 mM Na-pyruvate, 100 U penicillin/mL, 100
µg streptomycin/mL, non-essential amino acids, and vitamins and incubated at 37°C in a 5%
CO2 incubator. For cytotoxicity assays, human BMEC were grown in 24-well plates by
inoculating 106 cells/mL/well. At this cell density, confluent monolayers were formed within 24
h and used in experiments.
Synthesis of mannose-conjugated porphyrin
Mannose-conjugated porphyrin was synthesized as follows: Initially 40 mL of propionic
acid were heated at 90°C. A solution was prepared by mixing 1.9 mL (18.85 mM) benzaldehyde
in propionic acid (2.5 mL). Benzaldehyde was added very slowly in hot propionic acid. After
continuous heating of the solution at reflux temperature, pyrrole 1.3 mL (18.85 mM) dissolved in
propionic acid (2.5 mL) was added in above mixture in a drop-wise manner for 30 min. The
reaction was refluxed and monitored by thin layer chromatography (TLC) (14). After completion
(about 1.5 h), the reaction mixture was cooled, and crude tetra-phenyl porphyrin in ethyl acetate
(3 x 50 mL) was extracted from 2.5 M (50 mL) NaOH aqueous solution. Solvent was evaporated
and solid product was passed from silica column (ethyl acetate:hexane, 1:9) to get the pure
product in 7% yield as described previously (14). Next, 160 mg (0.26 mM) of tetra-phenyl
porphyrin was nitrated using 90 mg (1.3 mM) sodium nitrite (NaNO2) in 10 mL trifluoroacetic
acid (TFA), and the reaction mixture was stirred for an hour. By controlling the amount of TFA
and reaction time, mono-nitrated phenyl porphyrin was obtained as a major product along with
di-nitro phenyl porphyrin. The reaction was quenched with excess of water and was extracted in
dichloromethane (DCM, 3 x 25 mL). Next, it was evaporated in vacuum, and mono-nitrated
phenyl porphyrin was isolated by using column chromatography (DCM:hexane, 3:1 to 1:1 ratio)
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to get 30% yield. After this, 100 mg (0.15 mM) of mono-nitro phenyl porphyrin together with
100 mg (0.44mM) tin dichloride dihydrate was added in 0.5 mL concentrated HCl and heated at
60°C for 2.5 h. The progress of reaction was monitored by TLC. After completion, ammonium
hydroxide was added to neutralize the reaction mixture and was extracted wit DCM (3 x 25 mL)
and purified with column chromatography (DCM:hexane, 1:3 to 3:1 ratio) to afford mono-amino
phenyl porphyrin in 53% yield. Finally, 15 mg (0.079 mM) of mannose along with 50 mg (0.079
mM) of mono-amino phenyl porphyrin was dissolved in ethanol (8 mL). Three drops of glacial
acetic acid were added and reaction was refluxed overnight. Precipitates formed were filtered
and washed with DCM and methanol separately and purity confirmed through spectroscopic
analysis. The residue was dissolved in dimethyl sulfoxide (DMSO) and was subjected to NMR to
determine product purity. The synthesized product absorbed photons of 450 nm, which is within
the range of blue light.
Amoebicidal assays
To determine cidal effects of mannose-conjugated porphyrin, amoebicidal assays were
performed as described previously but with slight modifications (12). Briefly, A. castellanii were
incubated with 10µM (4 µL volume) and 50µM (20 µL volume) concentrations of mannose-
conjugated porphyrin, as well as porphyrin and mannose alone in RPMI (106 amoebae/mL/well)
in 24-well plates for 1 h. Amoebae were collected by centrifugation at 1000 x g for 5 min. The
supernatant was aspirated and pellet resuspended in 0.5 mL of RPMI-1640. This process was
repeated 3X to remove any remaining residual mannose-conjugated prophyrin. Finally, amoebae
pellet was resuspended in PYG. Next, the lids were removed and plates were exposed to blue
light for 1 h in a biosafety hood at room temperature using blue round light-emitting diode
(LED), that is lead-free and at 20mA emits minimum wavelength of 465nm and maximum
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wavelength of 480nm, while typical dominant wavelength of 470nm at pulse width of 0.1msec
by placing the 5-mm LED approximately 15 cm above the 24-well plate (Cree Inc., Durham,
NC, USA). Following this, plates were incubated for 24 h at 37°C. Next day, amoebae counts
were determined using a haemocytometer and images obtained using a phase contrast inverted
microscope. For solvent controls, amoebae were incubated in same amounts of DMSO (4 µL and
20 µL volume), chloroform:Methanol, 50:50 (1.34 µL and 6.7 µL volume), and RPMI alone. For
positive controls amoebae were incubated with antiacanthamoebic drug, chlorhexidine (20µM in
RPMI as a solvent), which exhibited 100% kill.
Host cell cytotoxicity assays
To determine antiamoebic effects of mannose-conjugated porphyrin in vitro, assays were
performed using human BMEC. A. castellanii were pre-treated with mannose-conjugated
porphyrin and exposed to light as described above. Next, amoebae were collected by
centrifugation at 1000 x g for 5min. The supernatant were aspirated and pellet resuspended in 0.5
mL of RPMI-1640. This process was repeated 3X to remove any remaining residual mannose-
conjugated prophyrin. Finally, amoebae were inoculated on human BMEC monolayers, grown in
24-well plates. Plates were incubated at 37°C in a 5% CO2 incubator. Next day, cell-free
conditioned medium was collected and host cell cytotoxicity was determined by measuring
lactate dehydrogenase (LDH) release using Cytotoxicity Detection kit (Roche, Indianapolis, IN).
The principle of this assay is that cell supernatant containing LDH catalyzes the conversion of
lactate (solution from kit) to pyruvate, generating NADH and H+. In the second step, the catalyst
(diaphorase, solution from kit) transfers H and H+ from NADH and H+ to the tetrazolium salt p-
iodo-nitrotetrazolium violet (INT), which is reduced to formazon (dye), and absorbance is read at
492 nm. The human BMEC incubated alone were used as negative controls, whereas monolayers
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lysed with 1% Triton X-100 for 30 min at 37°C were used as 100% cell death (high value).
Absorbance of samples was converted to percentage cytotoxicity as follows: (sample value –
negative value) / (high value – negative value) × 100 = % cytotoxicity.
In addition to amoebae pre-treatment, antiamoebic effects of mannose-conjugated
porphyrin were determined by co-incubating A. castellanii + human BMEC + mannose-
conjugated porphyrin and exposed to light as described above. Next, plates were incubated at
37°C in a 5% CO2 incubator for 24 h and cell-free conditioned medium was collected and host
cell cytotoxicity was determined by measuring LDH release.
Excystation assays
For excystation assays, A. castellanii cysts were prepared using non-nutrient agar plates
(3% Oxoid Agar technical using distilled water) as previously described (12). A. castellanii
trophozoites (2 x 106 amoebae) were inoculated on non-nutrient agar plates and incubated at
37°C for up to 14 days to allow trophozoite differentiation into cysts. Following this incubation,
no trophozoite was seen when observed under a phase contrast inverted microscope. Next, agar
plates were flooded with 15 mL of PBS and placed on a shaker for 30 min at room temperature.
After this incubation, cysts were gently scraped off the agar surface using a rubber scrapper.
Cysts were collected in a 50 mL tube, followed by centrifugation at 1500 x g for 10 min. The
supernatant was discarded and the pellet resuspended in 10 mL PBS. This process was repeated
3X to wash A. castellanii cysts and counted using a hemocytometer. To determine the effects of
mannose-conjugated porphyrin on A. castellanii cysts, excystation assays were performed as
described previously (15). Briefly, A. castellanii cysts (5 x 105 cysts/mL/well) were incubated
with 10 µM and 50 µM mannose-conjugated porphyrin, as well as porphyrin and mannose alone
for 1 h. Amoebae were collected, washed 3X with PBS and resuspended in RPMI in 24-well
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plates. Next, lids were removed and plates were exposed to blue light for 1 h as described above.
Finally, cysts were collected and resuspended in growth medium, PYG (0.5 mL PYG
medium/well) in 24 well plates. Plates were incubated at 37°C for 72 h and the emergence of
actively growing trophozoite was observed microscopically. The trophozoite counts were
determined using a hemocytometer.
Results
Synthesis of mannose-conjugated porphyrin was confirmed using NMR spectroscopy
The synthesis of mannose-conjugated porphyrin was achieved in four linear steps (Fig.
1). Initially tetra-phenyl porphyrin was obtained by mixing benzaldehyde and pyrrole in the
presence of propionic acid. Tetra-phenyl porphyrin was then converted into mono-nitro phenyl
porphyrin by selectively nitrating the para position of phenyl rings. As a result, a mixture of
mono and di-nitro phenyl porphyrins were obtained, which were separated by column
chromatography. By controlling the equivalents of nitrating mixture, mono nitro product was
obtained in higher yield and checked using NMR spectroscopy (Fig. 2). Next, mono-amino
phenyl porphyrin was obtained by reducing mono-nitro phenyl porphyrin in the presence of tin
dichloride in hydrochloric acid. The reaction yielded mono amino porphyrin that resulted in
slight shifting of signals in NMR spectrum (Fig. 3A), however the mass spectra of the product
revealed the disappearance of mono-nitro phenyl porphyrin mass peak and formation of desired
mono-amino porphyrin was confirmed by peak at 629 m/z (Fig. 3B). Finally, mannose-
conjugated porphyrin was obtained by conjugating mono-amino porphyrin with mannose
resulting in the formation of imine bond. Mannose-conjugated porphyrin was obtained as solid
product which was then washed with dichloromethane and methanol several times to remove
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excess starting materials and confirmed through spectroscopic analysis. The purity of mannose-
conjugated porphyrin was 75% as determined by NMR analysis (Fig. 4A). The
spectrophotometric analysis of the obtained product showed that it absorbed light of wavelength
ranging from 425-475nm (Fig. 4B).
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Mannose-conjugated porphyrin inhibited viability of A. castellanii
To determine the effects of mannose-conjugated porphyrin on A. castellanii trophozoites,
amoebae were incubated with 10 µM and 50 µM of mannose-conjugated porphyrin and
amoebicidal effects were determined. The results revealed that 50 µM of mannose-conjugated
porphyrin exhibited significant amoebicidal effects (P<0.01 using 2 sample T-test; two-tailed
distribution) (Fig. 5). Additionally, amoebicidal effects were confirmed by observing under the
microscope (Fig. 6). In contrast, A. castellanii incubated with 50 µM mannose alone and
porphyrin alone did not exhibit any killing effects. Also, solvent controls such as RPMI, DMSO
and 50% chloroform:methanol had no effect on the viability of A. castellanii (Fig. 5 and 6).
Mannose-conjugated porphyrin inhibited A. castellanii mediated host cell cytotoxicity
Pre-treatment of A. castellanii with mannose-conjugated porphyrin, followed by host cell
cytotoxicity assays were performed. Without mannose-conjugated porphyrin, A. castellanii
produced 95%±8.5 BMEC cell deaths within 24 h. Pre-treatment of A. castellanii with mannose-
conjugated porphyrin reduced host cell cytotoxicity significantly to 4.9%±2.5 (P<0.01, using 2
sample T-test; two-tailed distribution) compared to amoeba alone (Fig. 7A). In contrast,
incubation of A. castellanii with porphyrin, mannose or solvent alone had no protective effects
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on host cells (Fig. 7A). Similarly, solvent alone had no effect on BMEC cell cytotoxicity (Fig.
7A).
In co-incubation assays, A. castellanii + human BMEC + 50 µM mannose-conjugated
porphyrin also reduced host cell cytotoxicity but to a lesser extent of 58%±4 (Fig. 7B) (P<0.01,
using 2 sample T-test; two-tailed distribution). In the absence of mannose-conjugated porphyrin,
A. castellanii produced 97%±5 host cell cytotoxicity. Similarly, other compounds and solvents
alone did not show any effect on A. castellanii-mediated host cell cytotoxicity (Fig. 7B).
Mannose-conjugated porphyrin blocked excystation in A. castellanii
Cysts were pre-treated with and without mannose-conjugated porphyrin and inoculated in
fresh growth medium. The findings revealed that A. castellanii cysts incubated with PYG alone
emerged as viable trophozoites (1.2 x 106 amoebae) (Fig. 8). However, 50 µM mannose-
conjugated porphyrin significantly inhibited A. castellanii excystation within 72 h (P<0.01, using
2 sample T-test; two-tailed distribution), compared with cysts inoculated in PYG alone. In
contrast, viable trophozoites emerged in wells containing A. castellanii cysts treated with
mannose alone, porphyrin alone and solvents alone such as DMSO and 50% chloroform:
methanol within 72 h (Fig. 8).
Discussion
The successful treatment of Acanthamoeba infections is challenging. For example,
current treatment of Acanthamoeba keratitis is problematic and involves hourly topical
application of a mixture of drugs including chlorhexidine, polyhexamethylene biguanide,
propamidine isethionate, neomycin, lasting up to a year and even then recurrence can occur (3).
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One intriguing report was made by Ficker et al. (16), which showed the development of
propamidine resistance during the course of therapy for Acanthamoeba keratitis, which lead to
recurrence of the infection. Of concern, several studies have shown the resistance of
Acanthamoeba to antimicrobial chemotherapy (2, 4, 5). This is of particular concern in the
absence of available alternative chemotherapeutic agents. Our recent studies have shown the
potential of photochemotherapy, namely the application of photodynamic compounds followed
by exposure to a suitable source of UV-Vis radiation (17), however selective targeting of
Acanthamoeba has remained a major concern. By synthesizing photo-activated compound,
porphyrin and its conjugation with mannose to selectively target Acanthamoeba, the potential of
photochemotherapy against Acanthamoeba was investigated in the present study. The results
showed that mannose-conjugated porphyrin exhibited potent amoebicidal effects and blocked
excystation, in vitro. The basis of this technology involves the activation of photosensitizing
molecule by exposure to visible light (light of certain wave length). This in turn releases the
singlet oxygen or reactive oxygen species which causes cell death (10, 11, 16). However, a major
challenge in the use of photochemotherapeutic compound in treating infections caused by
eukaryotic pathogens is the specific delivery of photo-activated agent to the pathogen. Previous
studies have shown that pathogenic Acanthamoeba specifically binds to mannose but not other
sugars and have identified MBP as an important adhesin, expressed on the surface membranes of
Acanthamoeba (7-9). In the present study, mannose sugar was attached to a porphyrin ring to
synthesize mannose-conjugated porphyrin, for the targeted delivery of this photo-activated
compound to Acanthamoeba, which proved highly effective in killing parasite and exhibited both
trophicidal effects as well as abolished excystation against A. castellanii, in vitro.
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Notably, pre-treatment and co-treatment of A. castellanii with mannose-conjugated
porphyrin showed varied host cell cytotoxicity, 4% versus 58% host cell cytotoxicity
respectively. In contrast, no treatment resulted in more than 95% host cell cytotoxicity. A likely
explanation of higher cell cytotoxicity in co-treatment assays is due to the production of reactive
oxygen species produced by photosensitizing compound in the presence of human cells that may
have contributed to increased host cell cytotoxicity. It is also interesting to note that mannose-
conjugated porphyrin blocked excystation. At 50µM of mannose-conjugated porphyrin, complete
inhibition of excystation was observed. This is unexpected as previous studies show that cysts do
not exhibit adhesion to the host cells (18), and it was presumed that MBP is not expressed on the
external cell wall membranes, however the expression of MBP in Acanthamoeba cysts and if so,
then its precise localization has not been investigated. The inhibition of excystation as observed
in this study could be due to lipid soluble material or prolonged exposure to the photo-activated
compound (19, 20), affecting trophozoite emergence from the dormant cyst stage, however the
underlying molecular mechanisms of accumulation of photodynamic compound at the cyst wall,
its possible traversal across the cell wall, and/or through the operculum membranes to target
residing trophozoite inside the shell remains to be investigated, as well as its efficacy in vivo and
these studies will be the subject of further studies. Future studies are also needed to conjugate
porphyrin with Acanthamoeba-specific antibody and testing its potential for enhanced targeted
therapy against Acanthamoeba. Additionally, there is a need to test the possibility of using
various photosensitive molecules with high intensity light but shorter duration (pulsed interval
treatments) that may prove to be effective, and more feasible for clinical application. If
successful, this technology will be particularly useful in treating Acanthamoeba keratitis cases
due to ease of visible light usage and the fact that the current mode of eye inspection is exposure
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of visible light, in addition to disseminated/tissue infections. For latter, several lines of evidence
suggest that photodynamic therapy has proven ability against a range of malignant and other
diseased cells and tissues (21-26). With targeted killing of pathogenic Acanthamoeba, as
proposed in this study, photodynamic therapy can be recognized as a treatment strategy which is
both minimally invasive and minimally toxic and should be investigated further in vivo.
Acknowledgements: This work was funded by the Aga Khan University, Karachi, as well as the
Higher Education Commission, Pakistan.
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Conflict of interest: The author(s) declare no conflict of interest.
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Figure legends
Figure 1. Steps involved in the synthesis of mannose porphyrin, as described in “Materials and
Methods”. Briefly, benzaldehyde and pyrrole was mixed in propionic acid to yield tetra-phenyl
porphyrin. It was then nitrated in the presence of sodium nitrite and mono-nitrated phenyl
porphyrin was formed. Mono-nitrated phenyl porphyrin when neutralized in the presence of
ammonium hydroxide yielded mono-amino phenyl porphyrin. Finally the product was mixed
with mannose and yielded mannose-conjugated porphyrin. The purity of intermediate
compounds synthesized at each step was checked using NMR and Mass spectrometry. For
mannose-conjugated porphyrin, mass spectrometry was not performed due to its high polarity.
Figure 2. (A) NMR spectroscopy performed after the synthesis of mono-nitrated phenyl
porphyrin as described in figure 1, and (B) mass spectrometry performed after the synthesis of
mono-nitrated phenyl porphyrin.
Fiogure 3. (A) NMR spectroscopy performed after the synthesis of mono-amino phenyl
porphyrin as described in figure 1, and (B) mass spectrometry performed after the synthesis of
mono-amino phenyl porphyrin.
Figure 4. (A) NMR spectroscopy performed after the synthesis of mannose-conjugated
porphyrin as described in figure 1. Mass spectrometry of mannose-conjugated porphyrin was not
performed due to its high polarity. (B) UV-visible absorption spectrum of mannose-conjugated
porphyrin showed that it absorbed light of wave length ranging from 425-450 nm.
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Figure 5. Mannose-conjugated porphyrin exhibited amoebicidal effects against A. castellanii.
Briefly, amoebae (106) were incubated with and without 10 µM and 50 µM of mannose-
conjugated porphyrin and exposed to blue light as described in “Materials and Methods”.
Following this, plates were incubated for 24 h and amoebae were enumerated using a
hemocytometer. Treatment of A. castellanii with mannose-conjugated porphyrin exhibited
significant amoebicidal effects (P<0.01, using 2 sample T-test; two-tailed distribution) compared
with amoebae incubated alone. In contrast, controls did not show any effect. The data are
presented as the mean ± standard error of at least three independent experiments performed in
duplicate.
Figure 6. A. castellanii was incubated with and without 50 µM of mannose-conjugated
porphyrin and representative images were obtained at 100X (A) and at 400X (B) magnification
using a phase-contrast inverted microscope.
Figure 7. (A) A. castellanii (5 x 105) pre-incubated with and without 50 µM of mannose-
conjugated porphyrin, exposed to light, and their cytotoxic potential was determined using
human brain microvascular endothelial cells (BMEC) as described in “Materials and Methods”.
When pre-treated with mannose-conjugated porphyrin, A. castellanii showed limited host cell
cytotoxicity (P<0.01, using 2 sample T-test; two-tailed distribution) compared with non-treated
amoebae. A. castellanii incubated with porphyrin, mannose, DMSO and chloroform: methanol
also induced host cell cytotoxicity. However, compounds and corresponding solvents alone had
no effect on human BMEC cytotoxicity. The data are presented as the mean ± standard error of
at least three independent experiments performed in duplicate. (B) A. castellanii (5 x 105) were
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co-incubated with human BMEC and 50 µM of mannose-conjugated porphyrin and exposed to
light as described in “Materials and Methods”. Co-incubation of 50 µM mannose-conjugated
porphyrin with amoeba + human BMEC reduced cell cytotoxicity, albeit at lower levels
compared with pre-treatment assays. The results represent the mean ± standard error of three
independent experiments performed in duplicate.
Figure 8. Mannose-conjugated porphyrin inhibited excystation of A. castellanii cysts. A.
castellanii cysts were scraped from non-nutrient agar plates and incubated with 50µM mannose
porphyrin and exposed to light as described in “Materials and Methods”. Following this
incubation, cysts were incubated in fresh growth medium, PYG for up to 72 h. After this
incubation A. castellanii trophozoites were enumerated using a hemocytometer. The dotted line
indicates the original inoculum. When treated with 50 µM mannose-conjugated porphyrin, A.
castellanii cysts did not re-emerge as actively growing viable trophozoites. Viable A. castellanii
trophozoites emerged in control wells, as well as with all other compound and solvents tested.
The results are presented as the mean ± standard error of at least three independent experiments
performed in duplicate.
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