RETRACTED ARTICLE: Antiamoebic activity of synthetic tetrazoles against Acanthamoeba castellanii belonging to T4 genotype and effects of conjugation with silver nanoparticles

Article abstract of DOI:10.1007/s00436-020-06694-4

Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines

Full text of DOI:10.1007/s00436-020-06694-4

Parasitology Research
https://doi.org/10.1007/s00436-020-06694-4
TREATMENT AND PROPHYLAXIS - ORIGINAL PAPER
Antiamoebic activity of synthetic tetrazoles
against Acanthamoeba castellanii belonging to T4 genotype
and effects of conjugation with silver nanoparticles
Areeba Anwar1 & Yim Pei Yi1 & Itrat Fatima2 & Khalid Mohammed Khan2,3 & Ruqaiyyah Siddiqui4
Naveed Ahmed Khan4 & Ayaz Anwar1
&
Received: 28 January 2020 /Accepted: 15 April 2020
#
Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous
Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexi-
dine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to
be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with
nanoparticles have shown significantly increased activity against Acanthamoeba castellanii. In this study, two functionally
diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1H-tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1H-
tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti-Acanthamoeba effects at different
concentrations ranging from 5 to 50 μM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs)
to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70 nm
respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were sub-
jected to anti-Acanthamoeba studies. Amoebicidal assay showed that at concentration 10 μM and above, T2 showed promising
antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have
inhibited differentiation activity against Acanthamoeba castellanii. Conjugation of T1 and T2 to AgNP also increased efficacy of
tetrazoles as anti-Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of
treatment compounds to A. castellanii. Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less
toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity
assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2
alone, suggesting that T2-conjugated AgNP is an effective and safe anti-Acanthamoeba agent. The use of a synthetic azole
compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii. However, mech-
anistic and in vivo studies are needed to explore further translational values.
.
.
.
Keywords Acanthamoeba Antiamoebic Tetrazole Nanoparticles
Section Editor: Julia Walochnik
3
* Ayaz Anwar
Department of Clinical Pharmacy, Institute for Research and Medical
Consultations (IRMC), Imam Abdulrahman Bin Faisal University,
Dammam 31441, Saudi Arabia
ayazanwarkk@yahoo.com
1
4
Department of Biological Sciences, School of Science and
Department of Biology, Chemistry and Environmental Sciences,
College of Arts and Sciences, American University of Sharjah,
Sharjah 26666, United Arab Emirates
Technology, Sunway University, 47500 Subang Jaya, Selangor,
Malaysia
2
H. E. J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan

Parasitol Res
Introduction
Acanthamoeba cysts (Garajová et al. 2019; Anwar et al.
2019).
Acanthamoeba, an opportunistic pathogen, is commonly
found in soil and water (Köhsler et al. 2016). A blinding
infection of the eye known as Acanthamoeba keratitis (AK)
is caused by the invasion of Acanthamoeba in the eye where it
causes chronic ulceration of the cornea which often leads to
permanent sight damage. AK usually occurs in contact lens
wearing individuals where the malpractice of contact lenses is
the main cause, such as wearing contact lenses while swim-
ming activities (Siddiqui and Khan 2012). Another human
disease caused by Acanthamoeba is granulomatous amoebic
encephalitis (GAE), where the pathogen invades the central
nervous system (CNS) and causes the formation of granulo-
mas (Siddiqui and Khan 2012). GAE is known to be an op-
portunistic infection as it occurs mainly in immunocompro-
mised individuals (Slater et al. 1994). This disease is rare but
highly fatal as statistics show 95% of recorded patients die
even when they are given different antimicrobial treatments
(Kulsoom et al. 2014). Besides these two infections,
Acanthamoeba also causes the lung, kidney, and skin to be
compromised by the pathogen, where trophozoites and cysts
usually surround the walls of blood vessels. Acanthamoeba
may feed on skin tissue and cause severe skin lesions in im-
munocompromised patients as well (Schuster and Visvesvara
2004).
Acanthamoeba can exist as a free-living unicellular micro-
organism in nature or as a parasite inside a host.
Acanthamoeba is known to feed on bacteria in their environ-
ments and support the growth of several other pathogens, such
as Mycobacterium, Chlamydia, and certain viruses (Khan
2006). The pathogen undergoes asexual reproduction by di-
viding through binary fission (Band and Mohrlok 1973).
There are two phases in the life cycle of Acanthamoeba:
growth phase trophozoites and cellular differentiation to form
cysts. The two types of cellular differentiation are known as
encystment and excystment (Weisman 1976). Excystment
leads to Acanthamoeba trophozoites from cysts which is the
active form and is described as having protruding spine-like
structure known as acanthopodia which are important for
movement, engulfing bacteria, and surface adhesion.
Encystment, on the other hand, leads to Acanthamoeba cysts
from trophozoites, which are the resistant form of the patho-
gen, when it is subjected to a harsh environment such as star-
vation, dryness, and extreme temperatures (Khunkitti et al.
1998). Cysts are described as double walled with a wrinkled
ectocyst (outer layer) and an endocyst (inner layer) which
varies in shape. At the site of contact between the ectocyst
and endocyst, pores covered by small lids (operculum) are
present (Siddiqui and Khan 2012). These cyst walls are made
up of polysaccharides of glucose and galactose; hence, poly-
saccharides biosynthesis pathway is considered as an impor-
tant target for the development of treatments against
The current recommended treatment for AK is biguanide
such as chlorhexidine together with a diamidine and added
antibiotics if the bacterial infection was involved (Lorenzo-
Morales et al. 2015). Chlorhexidine releases a cation that in-
teracts with the cell membrane, hence increasing cell perme-
ability and ultimately leading to cell death through leakage of
ions and cytoplasmic disruption (Sharma et al. 2013).
Although chlorhexidine is effective against A. castellanii, im-
mediate and aggressive treatment with the biguanide is needed
to make the treatment effective (Lamb et al. 2015).
Chlorhexidine also exhibits undesirable effects when treated
against AK such as mature cataracts and iris atrophy (Aqeel
et al. 2016). Other anti-protozoa drug currently used are azoles
such as metronidazole, which is notably effective against
amoebic infections in the human gastrointestinal tracts caused
by Entamoeba histolyca (Cano et al. 2014). However, the
issue of metronidazole-resistant protozoa has also been
brought into light and the drug is known to cause undesirable
side effects such as headaches and dry mouth and is potential-
ly toxic (Bendensky et al. 2002). There are reports of using a
mixture of metronidazole, chlorhexidine, and neomycin sul-
fate that led to rapid resolution of inflammation in AK
(Xuguang et al. 2003). Other than metronidazole,
voriconazole is also found to be effective in treatment of
AK; however, prolonged treatment with the drug was required
to prevent relapse (Tu et al. 2010). Next, ketoconazole was
found to be effective against a brain abscess caused by
Acanthamoeba when treated together with a mixture of anti-
biotics (Nampoothiri et al. 2018). However, ketoconazole has
previously shown high hepatotoxicity and endocrine dysreg-
ulation, which lead to the ban of the azole in the Australian
and European markets as well as strict regulations in America
(Gupta and Lyons 2015). Previously, several triazole antifun-
gal agents were tested against Acanthamoeba castellanii and
Acanthamoeba polyphaga by inhibiting the sterol 14-
demethylase (CYP51) (Lamb et al. 2015). It was found that
Acanthamoeba is more related to algae and plants than mam-
malian ancestries; therefore, existing antifungal azoles were
not very effective anti-Acanthamoeba agents due to the low
sequence identity of Acanthamoeba and fungal CYP51 as
shown by NCBI-BLASTP (Lamb et al. 2015).
Besides known azole drugs, synthetic azoles are also of
widespread interest for drug development against numerous
microbes. Tetrazoles are five-membered aromatic ring com-
pounds with four nitrogen and one carbon atom. This planar
structure makes the compound electron rich and allows the
compound to form weak bonds with enzymes or biological
receptors (Lingling et al. 2013). This characteristic also allows
tetrazoles to interact and stabilize functional molecules that
were formed through combination of multiple small mole-
cules, showing many potentials in the pharmaceutical industry

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(Dai et al. 2015). Besides, tetrazoyl fragments have been de-
scribed as the non-toxic and non-metabolized analogues of
carboxylic and cis-amide groups (Dai et al. 2015). Certain
derivatives of tetrazoles have also shown strong antifungal
activities through inhibition of ergosterol synthesis such as
tetrazole derivatives which contain hydrazone and thiazoline
portion shows high efficacy in inhibiting certain Candida,
Aspergillus, and Fusarium species (Łukowska-Chojnacka
et al. 2016). Several types of synthetic chromone-tetrazoles
have also shown to be effective against Entamoeba
histolytica, the causative agent of amoebic diarrhea and dys-
entery (Cano et al. 2014). Among membrane-acting agents,
alkylphosphocholines such as miltefosine have been found to
be effective against AK and GAE (Tavassoli et al. 2018;
Barisani-Asenbauer et al. 2012; Webster et al. 2012).
Furthermore, synthetic analogues of alkylphosphocholines
have also been reported to exhibit potent antiamoebic effects
against Acanthamoeba spp. (Timko et al. 2015; Garajová et al.
2014).
Nanoparticles may be the solution to multidrug-resistant
pathogens as they have shown to increase the efficacy of com-
pounds against various microbes (Egger et al. 2009).
Researchers are interested in silver nanoparticles because sil-
ver was used as an antimicrobial agent against bacteria (Bello-
Vieda et al. 2018). Besides that, conjugation of natural prod-
ucts, plant extracts, and contact lens solutions to silver nano-
particles have been shown to increase the in vitro effectivity of
their payloads against Acanthamoeba spp. (Anwar et al. 2020;
Padzik et al. 2018; Padzik et al. 2019). The mechanism of
silver nanoparticles against microbes is still not fully grasped,
but several mechanisms have been proposed. It is said that
silver nanoparticles alter cell wall and cell membrane which
in turn increases cell permeability (Bello-Vieda et al. 2018).
Previously, antifungal drugs amphotericin B, nystatin, and
fluconazole conjugated silver nanoparticles showed signifi-
cantly increased activity against Acanthamoeba castellanii
(Anwar et al. 2018). Developing a novel tetrazole silver nano-
particle with increased efficacy compared with the respective
tetrazole alone against the human pathogen Acanthamoeba is
the overall aim of this current study. In this study, we tested the
antiamoebic properties of two novel synthetic tetrazoles
against A. castellanii belonging to the T4 genotype and the
effects of silver nanoparticles conjugation on the efficacy of
these compounds.
borohydride (NaBH4) was obtained from Merck, RPMI-
1640 was obtained from Gibco, and absolute DMSO was
obtained from Thermo Scientific.
Acanthamoeba castellanii cultures
A clinical isolate obtained in 1984 from a keratitis patient from
India, A. castellanii of T4 genotype (ATCC 50492) was pur-
chased from American Tissue Culture Collection (ATCC).
The Amoeba cells were cultured in 10 ml of PYG medium
consisting of 0.75% yeast extract, 0.75% protease peptone,
and 1.5% glucose in T-75 cm2 culture flasks (Aqeel et al.
2016).
Henrietta Lacks cell cultures
HeLa cells (CCL-2) were obtained from American Tissue
Culture Collection (ATCC). The cells were cultured in
RPMI-1640 supplemented with 1% (2 mM) L-glutamine,
1% penicillin-streptomycin, 10% fetal bovine serum, and
1% minimal essential media nonessential amino acid (MEM
NEAA). The cells were incubated in a CO2 incubator at 37 °C
and 95% humidity. Two milliliters (0.25%) of trypsin was
used to detach the cells which enabled the inoculation of cells
into 96-well plates.
Synthesis and characterization of T1 and T2
The tetrazoles tested in this study are 5-(3-4-
dimethoxyphenyl)-1H-tetrazole and 1-(3-methoxyphenyl)-5-
phenoxy-1H-tetrazole, represented by T1 and T2 respectively
(Fig. 1).
T1 and T2 were synthesized by click reaction of aromatic
nitriles with sodium azide in the presence of ammonium chlo-
ride to afford T1 and T2 as given in Scheme 1 (Fatima et al.
2018).
Materials and methods
Chemicals
Different aromatic nitriles were purchased from different
chemical suppliers including Alfa-Aesar, Merck, Sigma-
Aldrich, and TCI. Silver nitrate (AgNO3) and sodium
Fig. 1 The chemical structures of 5-(3-4-dimethoxyphenyl)-1H-tetrazole
(T1) and 1-(3-methoxyphenyl)-5-phenoxy-1H-tetrazole (T2)

Parasitol Res
described by Sissons et al. (2006). In 24-well plates, 5 × 105
A. castellanii trophozoites were treated with 5 μM, 10 μM,
25 μM, and 50 μM of T1 and T2 respectively as well as 5 μM
and 10 μM of tetrazole-conjugated AgNPs and AgNPs alone
in RPMI-1640. Chlorhexidine served as the positive controls
and RPMI-1640 alone as the negative control. Appropriate
amounts of DMSO and water were also plated as solvent
controls. After a 24-h incubation period at 30 °C, a trypan blue
exclusion assay was carried out to calculate the estimated
average well count for each duplicate.
Scheme 1 Synthesis of Tetrazoles T1 and T2 (Fatima et al. 2018)
Characterization of T1 and T2
5-(3′,4′-Dimethoxyphenyl)-1H-tetrazole (T1)
Yield: 88%; m.p. 207–208 °C; 1H-NMR: (300 MHz, DMSO-
d6): δH 16.62 (s, NH), 7.63 (s, 1H, H-2′), 7.62 (d, 1H, J6′,5′
=
Encystation assays
8.4 Hz, H-6′), 7.18 (d, 1H, J5′,6′ = 8.4 Hz, H-5′), 3.84 (s, 3H,
3′-OCH3), 3.83 (s, 3H, 4′-OCH3); 13C-NMR (100 MHz,
DMSO-d6): δc 151.1 (C-3′), 149.1 (C-4′), 120.1 (C-6′),
112.1 (C-2′), 110.0 (C-5′), 55.7 (4′-OCH3), 55.6 (3′-OCH3);
IR (KBr, cm−1): 3477 (N–H), 1609 (C=N), 1144 (C–O); EI-
MS: m/z (rel. abund. %), 206 [M]+ (1), 191 (4), 182 (100), 152
(50), 136 (90); HREI-MS: m/z calcd for C9H10N4O2 [M]+
206.0804; found 206.0791.
In 24-well plates, 5 × 105 A. castellanii trophozoites were
treated with 5 μM, 10 μM, 25 μM, and 50 μM of T1 and
T2 respectively as well as 5 μM and 10 μM of tetrazole-
conjugated AgNPs and AgNPs alone. All treatment was done
in encystation medium comprised of 5 mM MgCl2 and 10%
glucose. Chlorhexidine served as the positive controls and
PBS containing an encystation medium served as the negative
control. Appropriate amounts of DMSO and water were also
plated as solvent controls. After a 96-h incubation period at
30 °C, 0.1% SDS was added to dissolve trophozoites to enable
the counting of cysts using a hemocytometer.
Methyl2-[(1-phenyl-1H-1,2,3,4-tetraazol-5-yl)oxy]phenyl
ether (T2)
Yield: 84%; m.p. 249–251 °C; 1H-NMR: (300 MHz, DMSO-
d6): δH 7.84 (d, 2H, J2′,3′ = J6′,5′ 7.5 Hz, H-2′,6′), 7.68 (m, 3H,
H-3′,4′,5′), 7.42 (dd, 1H, J4″,5″ = 9, Hz J4″,3″ = 8.4 Hz, H-4″),
7.13 (m, 1H, H-5″), 7.07 (d, 1H, J6″,5″ = 7.8 Hz, H-6″), 6.93
(d, 1H, J3″,4″ = 8.1 Hz, H-3″), EI-MS: m/z (rel. abund. %), 268
(M+, 4), 238 (36), 225 (4), 117 (100); HREI-MS: m/z calcd for
C14H12O2N4 [M]+ 268.0960; found 268.0941.
Excystation assay
Preparation of cysts was done few weeks prior to this exper-
iment by plating healthy A. castellanii trophozoites on non-
nutrient agar plates for at least 14 days before it was scraped
and collected. 1 × 105 cysts were treated with 5 μM, 10 μM,
25 μM, and 50 μM of T1 and T2 respectively as well as 5 μM
and 10 μM of tetrazole-conjugated AgNPs and AgNPs alone
in PYG. Chlorhexidine served as the positive controls and
PYG alone as the negative control. Appropriate amounts of
DMSO and water were also plated as solvent controls. After a
96-h incubation period at 30 °C, a trypan blue exclusion assay
was carried out to calculate the estimated average well count
(trophozoites only) for each duplicate.
Synthesis of tetrazole-conjugated AgNPs
Tetrazoles were weighed and dissolved in absolute DMSO to
prepare 10-mM stocks. The 10-mM stocks of the tetrazoles
were diluted with sterile water to prepare 0.5-mM and 0.1-mM
stocks which was used to plate concentrations 5 μM, 10 μM,
25 μM, and 50 μM in each assay. Synthesis of tetrazole-
conjugated AgNPs was done by adding 2 ml of 0.1 mM tet-
razole to 2 ml of 0.1 mM silver nitrate(aq) solution and mixed
on a magnetic stirrer. Sodium borohydride (4 mM, 20 μl) was
added to reduce the silver ions to enable conjugation of tetra-
zole to AgNP (Anwar et al. 2018). The successful formation
was indicated by the color change of the mixture from color-
less to yellow. Further analysis such as UV-Vis spectropho-
tometry and FT-IR spectroscopy was done to confirm the for-
mation and stabilization of tetrazole-conjugated AgNPs.
Host cell cytotoxicity assays
In 96-well plates, HeLa cells were treated with 5 μM, 10 μM,
25 μM, and 50 μM of T1 and T2 respectively as well as 5 μM
and 10 μM of tetrazole-conjugated AgNPs and AgNPs alone
in RPMI-1640. Chlorhexidine served as the positive treatment
and RPMI-1640 alone as the negative treatment. The positive
control and negative control were achieved in quadruplicates
by treating HeLa cells with 1% Triton X-100 (100% cell
death) and incubating in RPMI-1640 alone respectively.
After the 24-h incubation period, the supernatant of each well
was transferred to microcentrifuge tubes. Lactate dehydroge-
nase enzyme (LDH) assay was carried out using a cytotoxicity
Amoebicidal assays
The antiamoebic properties of tetrazoles alone and tetrazole-
conjugated AgNPs were tested in duplicates for this assay, as

Parasitol Res
detection kit to measure absorbance reading at 490 nm. %
cytotoxicity was calculated as (sample absorbance-negative
control absorbance)/(positive control absorbance-negative
control absorbance) × 100 (Aqeel et al. 2015).
DLS confirmed that the average size of nanoconjugates to
be 52 and 70 nm. Figure 2b shows the T1-conjugated nano-
particles while Fig. 2c shows T2-conjugated nanoparticles as
compared with compounds alone. Both FT-IR spectra show
that the stabilization of nanoparticles was achieved by nitro-
gen of the tetrazole rings.
Host cell cytopathogenicity assays
In 24-well plates, 5 × 105 A. castellanii trophozoites were
treated with 5 μM, 10 μM, 25 μM, and 50 μM of T1 and
T2 respectively as well as 5 μM and 10 μM of tetrazole-
conjugated AgNPs and AgNPs alone in RPMI-1640.
Chlorhexidine served as the positive treatment and RPMI-
1640 alone as the negative treatment. After a 2-h incubation
period, the treated trophozoites were centrifuged at 3000 rpm
for 10 min and resuspended in 200 μl RPMI-1640 to retrieve
viable trophozoites. The treated trophozoites were used to
treat HeLa cells in 96-well plates at 37 °C for 24 h. The
positive control and negative control were achieved in qua-
druplicates by treating HeLa cells with 1% Triton X-100
(100% cell death) and incubating in RPMI-1640 alone respec-
tively. After the 24-h incubation period, the supernatant of each
well was transferred to microcentrifuge tubes. Lactate dehydro-
genase enzyme (LDH) assay was carried out using a cytotox-
icity detection kit to measure absorbance reading at 490 nm. %
cytopathogenicity was calculated as (sample absorbance-
negative control absorbance)/(positive control absorbance-
negative control absorbance) × 100 (Aqeel et al. 2015).
Increased antiamoebic activity was observed
in tetrazole-conjugated AgNPs compared
with tetrazoles alone
To determine the antiamoebic activity of tetrazoles alone and
conjugated with AgNPs against A. castellanii, amoebicidal
assays were carried out for each drug in duplicates for three
times. Figure 3a shows the antiamoebic effect of T1 and T2
for all concentrations as compared with the negative control
(5 × 105 trophozoites in RPMI-1640 alone). T1 did not exhibit
significant amoebicidal activity while T2 showed significant
and dose-dependent antiamoebic activity from 10 to 50 μM
(*P < 0.05). The antiamoebic effect increased significantly
when concentration was increased from 10 to 25 μM. IC50
of T2 was estimated to be within 10 μM to 25 μM. Figure 3b
shows that both tetrazole-conjugated AgNP had higher
antiamoebic activity compared with respective tetrazoles
alone. However, only 10 μM T2-AgNP showed statistical
significance when compared with negative control (5 × 105
trophozoites in RPMI-1640 alone), solvent control (AgNPs
alone), and T2 alone (**P < 0.01). Figure 3b shows that the
T2-AgNPs showed a 50% reduction in the viability of
A. castellanii at 10 μM which corresponds to the IC50. This
indicates that the conjugation of T2 to AgNP significantly
increased the antiamoebic properties of T2 at 10 μM.
Statistical analysis
The results were shown as mean standard error of the three
experiments performed in duplicates for each compound. The
significance of the differences between the averages of negative
and sample well counts were determined by using a two-sample
t test with a two-tailed distribution, done on Microsoft Excel.
Encystation of A. castellanii was inhibited
with tetrazoles and nanoparticles
To determine the ability of the tetrazoles and AgNP-conjugated
tetrazoles to prevent A. castellanii from converting from tropho-
zoites to its dormant and more resistant form, cysts, encystation
assay was carried out in duplicates for three times. Figure 4a
shows that overall inhibition of encystation was higher for T2
compared with T1. Inhibition of encystation in T1 showed sta-
tistical difference for 10 μM (**P < 0.01), 25 μM (*P < 0.05),
and 50 μM (***P < 0.001). Inhibition of encystation of T2
showed higher significance for all concentrations
(***P < 0.001). From Fig. 4b, it can be observed that both
T1-AgNP and T2-AgNP exhibited higher inhibition activity
compared with respective tetrazoles. However, only T1-AgNP
showed statistical significance as compared with the negative
control (5 × 105 trophozoites in encystation medium), solvent
control (AgNPs alone), and T1 alone (**P < 0.01). This indi-
cates that the conjugation of T1 to AgNP significantly increased
the inhibition of encystation at 5 μM.
Results
Analyzing the conjugation of tetrazoles with silver
nanoparticles with UV-Vis spectrophotometry,
dynamic light-scattering, and Fourier-transform
infrared spectroscopy
To ensure the conjugation of T1 and T2 to silver nanoparticles,
the synthesized compounds were analyzed using UV-Vis
spectrophotometry. From Fig. 2, the maximum absorption
for T1-AgNP and T2-AgNP was observed at 417 nm and
398 nm respectively. Both maximum absorptions were within
the range of the peak shown by AgNP which was expected to
have a surface plasmon resonance (SPR) band at the range of
350 nm to 450 nm (Anwar et al. 2018). This shows that both
tetrazoles have been successfully conjugated to the AgNP.

Parasitol Res
Fig. 2 a The UV-Vis spectra for
AgNP, T1-AgNP, and T2-AgNP.
Maximum absorptions observed
for AgNP, T1-AgNP, and T2-
AgNP were at 393 nm, 417 nm,
and 398 nm respectively. b and c
Comparative FT-IR spectra for
T1-AgNP and T2-AgNP with T1
and T2 respectively
1
0.8
0.6
0.4
0.2
0
300
400
500
600
700
800
-0.2
Wavelength (nm)
T1-AgNP
AgNP
T2-AgNP
a
FT-IR (AgNP-T1 & T1)
120
100
80
60
40
20
0
3900
3400
2900
2400
1900
1400
T1
900
400
Wavenumbers
AgNP-T1
b
FT-IR (AgNP-T2 & T2)
120
100
80
60
40
20
0
3900
3400
2900
2400
1900
1400
T2
900
400
Wavenumbers
AgNP-T2
c
Excystation of A. castellanii was inhibited
with tetrazoles and nanoparticles
trophozoites, excystation assay was carried out in duplicates
for three times. Figure 5a shows that both T1 and T2 have
shown significant inhibition activities compared with the neg-
ative control (cyst alone) (**P < 0.01). However, activities of
T1 at 25 μM and 50 μM, as well as T2 at 50 μM, showed
To determine the ability of tetrazoles alone and conjugated to
AgNP to prevent A. castellanii cyst from reverting back to

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Fig. 3 a Antiamoebic activity of
T1 and T2 alone. b Antiamoebic
activity of T1 and T2 conjugated
with a silver nanoparticle. The
assay was carried out by
incubating each compound at
concentrations 5 μM, 10 μM,
25 μM, and 50 μM with 5 × 105
trophozoites in RPMI-1640 at
30 °C for 24 h. Trypan blue ex-
clusion assay was used to estimate
the number of viable cells in each
well and the average well count
was calculated for each duplicate
6
5
4
3
2
1
0
*
*
*
μM
a
6
5
4
3
2
1
0
*
**
μM
b
statistical significance when compared with solvent control
(0.025% DMSO (*P < 0.05)). At 25 μM, T1 shows higher
effectiveness compared with T2. However, the effectiveness
of T1 plateaued when concentration was increased to 50 μM.
IC50 of T1 is estimated to be between 5 μM and 10 μM. From
Fig. 5b, both tetrazole-conjugated AgNPs are shown to be
slightly more effective compared with respective tetrazoles
alone at concentration 5 μM. However, only 5 μM T2-
AgNP showed statistical significance when compared with
the negative control (1 × 105 cysts), solvent control (AgNPs
alone), and T2 alone (*P < 0.05). This indicates that the con-
jugation of T2 to AgNPs significantly increases the inhibition
of excystation at 5 μM.
AgNPs exhibited the highest % cytotoxicity (18.6% and 14.8%)
among all samples. However, all samples, including T1-
conjugated AgNPs, showed lower % cytotoxicity compared with
chlorhexidine (39% cytotoxicity) at 10 μM. Solvents used to
prepare tetrazoles and NPs showed lower % cytotoxicity com-
pared with Triton X and chlorhexidine as well.
Tetrazole-conjugated AgNPs significantly decreased
cytopathogenicity of A. castellanii
To evaluate the A. castellanii-mediated cytotoxicity against
HeLa cells after being treated with tetrazole-conjugated
AgNPs and tetrazoles alone, cytopathogenicity assay was car-
ried out in duplicates. Figure 7 shows that both T1 and T2
alone significantly decreased % cytopathogenicity of
A. castellanii (*P < 0.05) compared with untreated
A. castellanii (89%) only. T2-conjugated AgNPs exhibited
significantly lower % cytopathogenicity compared with T2
alone (*P < 0.05).
Tetrazole-conjugated AgNPs and tetrazoles alone
exhibited low human cell cytotoxicity
To evaluate the cell-damaging effects of tetrazole-conjugated
AgNPs and tetrazoles alone on human HeLa cells, cytotoxicity
assay was carried out in duplicates. From Fig. 6, T1-conjugated

Parasitol Res
Fig. 4 a The effects of T1 and T2
on the encystation of
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
A. castellanii. b The effects of T1-
AgNP and T2-AgNP on the
encystation of A. castellanii com-
pared with T1 and T2 alone as
well as AgNPs alone. The assay
was carried out by incubating
each compound with 5 × 105 tro-
phozoites in PBS at 30 °C for
96 h. A total of 0.1% SDS was
used to dissolve remaining tro-
phozoites and the cysts were
counted to calculate average well
count for each duplicate.
*
**
***
***
***
*** ***
Statistical significance of the in-
hibition effects was determined
using two-sample t test with two-
tailed distribution, done on
μM
a
Microsoft Excel
1.8
1.6
1.4
1.2
1
*
0.8
0.6
0.4
0.2
0
*
*
**
*
μM
b
Discussion
conjugated compounds and functioning as a drug carrier
(Zazo et al. 2016).
The major problems in GAE treatment are the formation of
cysts and difficulty of drug delivery through the blood-brain
barrier (BBB) due to the tight junctions; therefore, it is impor-
tant to research for a formulation that can better penetrate the
BBB (Anwar et al. 2018). Currently, one of the biggest chal-
lenges in research is delivering a drug to the site of treatment
and release the drug at a specific rate to ensure maximum
potency; thus, researchers are interested in developing effi-
cient drug delivery systems (Kim et al. 2009). Drugs are usu-
ally low in potency due to low solubility which leads to ag-
gregation, as well as short half-lives which causes the drug to
degrade before reaching the target site (Parveen et al. 2012).
Nanoparticles have much to offer in drug delivery as they
allow the transport of drugs to specific target sites, sustained
release of drugs, and are highly stable (Parveen et al. 2012).
Metal nanoparticles such as AgNP were described to increase
bioavailability and efficacy by reducing the size and shape of
Azoles are usually used as antifungal agents with a
mode of actions such as ergosterol and CYP51 inhibition
(Chopra and Khuller 1983; Hitchcock et al. 1990). Azoles
were used in treating Acanthamoeba as ergosterol was
found to be present on the membrane of the pathogen
(Cabello-Vílchez et al. 2014). A study was also done by
Lamb et al. (2015), where several antifungal agents such
as fluconazole, itraconazole, and voriconazole were used
to inhibit CYP51 of Acanthamoeba. However, treatment
was not effective as Acanthamoeba and fungal CYP51
have low sequence identity as shown by NCBI-BLAST
(Lamb et al. 2015). Existing treatments are also not effec-
tive against all strains of Acanthamoeba; therefore, it is
crucial to perform mechanistically and in vivo studies to
better understand the receptors and molecules that play a
role in the pathogenesis of Acanthamoeba (Lorenzo-
Morales et al. 2015).

Parasitol Res
Fig. 5 a The effectiveness of T1
and T2 on inhibiting the
9
8
7
6
5
4
3
2
1
0
excystation of A. castellanii. b
The effectiveness of T1-AgNP
and T2-AgNP on inhibiting the
excystation of A. castellanii com-
pared with T1 and T2 alone. The
assay was carried out by incubat-
ing each compound with 5 × 105
cysts in PYG at 30 °C for 96 h.
The viable trophozoites were
counted with trypan blue exclu-
sion assay and the average well
count was calculated for each
duplicate
**
**
**
μM
a
9
8
7
6
5
4
3
2
1
0
*
μ M
b
As UV-Vis results (Fig. 2) show, T1 and T2 were success-
fully conjugated to AgNPs as both T1-AgNP and T2-AgNP
showed SPR band at 417 nm and 398 nm which is within the
expected SPR band range of 350 nm to 450 nm, indicating the
formation of bonds between ligand (tetrazole) and AgNPs
(Anwar et al. 2018). This study is focused on the development
Fig. 6 Tetrazole-conjugated
AgNPs and tetrazoles alone
100
exhibited low host cell
cytotoxicity. The assay was
80
carried out by treating HeLa cells
with 5 μM, 10 μM, 25 μM, and
60
50 μM of T1 and T2 respectively
as well as 5 μM and 10 μM of
40
tetrazole-conjugated AgNPs and
AgNPs alone in RPMI-1640
20
0
followed by incubation for 24 h at
37 °C in a 5% CO2 incubator.
After the 24-h incubation period,
the supernatant of each well was
transferred to microcentrifuge
tubes and lactate dehydrogenase
enzyme (LDH) assay was carried
out to determine % cytotoxicity
μM

Parasitol Res
100
80
60
40
20
0
*
*
μM
Fig. 7 T2-conjugated AgNPs significantly decreased %
cytopathogenicity of A. castellanii. This assay was carried out by
treating 5 × 105 A. castellanii trophozoites with 5 μM, 10 μM, 25 μM,
and 50 μM of T1 and T2 respectively as well as 5 μM and 10 μM of
tetrazole-conjugated AgNPs and AgNPs alone in RPMI-1640. After a 2-h
incubation period, the treated trophozoites were used to treat HeLa cells in
96-well plates at 37 °C for 24 h. After the 24-h incubation period, the
supernatant of each well was transferred to microcentrifuge tubes and
lactate dehydrogenase enzyme (LDH) assay was carried out to determine
% cytopathogenicity
of anti-A. castellanii agent using two new synthetic tetrazoles
and tetrazole-conjugated AgNPs. Antiamoebic assays showed
(Figs. 3, 4, and 5) that at concentration 10 μM and above, T2
showed promising antiamoebic activities compared with T1
while encystation and excystation assays reveal that both T1
and T2 inhibited differentiation activity of Acanthamoeba
castellanii. The use of T1 and T2 can allow the prevention
of encystation of trophozoites, which makes the pathogen
more susceptible to the antiamoebic effects of the tetrazoles
or other drugs (Dart et al. 2009). Conjugation of T1 and T2 to
AgNP also successfully increased the antiamoebic activities.
This may be due to the increased bioavailability as AgNP
allows better delivery of treatment compounds to
A. castellanii by altering the shape, size, and surface properties
of nanoparticles (Anwar et al. 2018). Host cell cytotoxicity
and cytopathogenicity assays were also carried out to evaluate
the host cell cytotoxicity as there is a lack of high-efficacy
treatment with little adverse effects in treating AK and GAE.
Cell cytotoxicity assay (Fig. 6) revealed that tetrazoles and
AgNPs are significantly less toxic towards host cells com-
pared with chlorhexidine which is known to cause undesirable
side effects. Cytopathogenicity assay also revealed that T2-
conjugated AgNPs reduced cytopathogenicity of
A. castellanii compared with T2 alone (Fig. 7), suggesting that
T2 is an effective anti-Acanthamoeba agent.
to the increased bioavailability as AgNP allows better delivery of
treatment compounds to A. castellanii. Metal nanoparticles can
potentially accumulate in the liver; therefore, the degradation
process of nanoparticles should be addressed. It is crucial to
further study the mechanism of pathogenesis of Acanthamoeba
to better understand the crucial receptors that play a role in the
process. This would ultimately lead to a more targeted and effec-
tive treatment for Acanthamoeba infections. However, animal
studies should be performed to validate these results.
Author’s contributions The study was conducted by team effort of all
authors. The manuscript was submitted with the consent of all authors.
Funding information This work is supported by the Sunway University,
Malaysia (GRTIN-RRO-98-2020), and the Pakistan Academy of
Sciences for providing financial support Project No. (5-9/PAS/440).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval Not required.
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