eating amoeba Naegleria fowleri Kavitha

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Letter

trans-Cinnamic acid conjugated gold nanoparticles as potent therapeutics against brain-eating amoeba Naegleria fowleri Kavitha Rajendran, Ayaz Anwar, Naveed Ahmed Khan, Muhammad Raza Shah, and Ruqaiyyah Siddiqui ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00111 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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ACS Chemical Neuroscience

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trans-Cinnamic acid conjugated gold nanoparticles as potent therapeutics against brain-

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eating amoeba Naegleria fowleri

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Kavitha Rajendran1#, Ayaz Anwar1*#, Naveed Ahmed Khan1, Muhammad Raza Shah2,

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Ruqaiyyah Siddiqui1

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Subang Jaya 47500, Selangor, Malaysia.

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Sciences, University of Karachi, Karachi 75270, Pakistan.

Department of Biological Sciences, School of Science and Technology, Sunway University,

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological

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# Both authors contributed equally.

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*Address for correspondence:

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Dr. Ayaz Anwar, Department of Biological Sciences, School of Science and Technology,

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Sunway University, Selangor, Malaysia; E-mail: [email protected] Tel: +603-74918622

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Ext. 7119; Fax: +603-56358630.

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ABSTRACT

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Primary amebic meningoencephalitis (PAM), a deadly brain infection is caused by brain-

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eating amoeba Naegleria fowleri. The current first line of treatment against PAM is a mixture of

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amphotericin B, rifampin and miltefosine. Since, no single effective drug has been developed so

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far, the mortality rate is above 95 %. Moreover, severe adverse side effects are associated with

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these drugs. Nanotechnology has provided several advances in biomedical applications

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especially in drug delivery and diagnosis. Herein, for the first time we report antiamoebic

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properties of cinnamic acid (CA) and gold nanoparticles conjugated with CA (CA-AuNPs)

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against N. fowleri. CA-AuNPs were successfully synthesized by sodium borohydride reduction

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of tetrachloroauric acid. Size and morphology were determined by atomic force microscopy

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(AFM) while the surface plasmon resonance band was analyzed by ultraviolet-visible (UV-Vis)

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spectrophotometry for the characterization the nanoparticles. Amoebicidal and cytopathogenicity

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(host cell cytotoxicity) assays revealed that both CA and CA-AuNPs displayed significant anti-

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N. fowleri properties (P < 0.05), whereas nanoparticles conjugation further enhanced the anti-N.

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fowleri effects of CA. This study established a potential drug lead, while CA-AuNPs appear to

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be promising candidate for drug discovery against PAM.

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Key words: Brain eating amoeba; Naegleria fowleri; Natural compound; Cinnamic acid; Gold

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nanoparticles.

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Brain-eating amoeba N. fowleri causes lethal brain infection known as primary amoebic

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meningoencephalitis (PAM). PAM is a rare disease, but it has a 97% mortality rate. Brain-eating

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N. fowleri is a ubiquitous, free-living amoeba found worldwide predominately in water.1 The

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entry of N. fowleri to brain is noted to be intranasal upon inhalation of contaminated water. It

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attaches to the olfactory mucus influencing the olfactory bulb in the central nervous system

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(CNS).2,3 Upon infection, N. fowleri initiates a vigorous inflammation leading to hemorrhage and

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necrosis.4 N. fowleri has been known to be isolated from a variety of water sources including

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lakes, puddles, hot springs, swimming pools, sewage, but it has not been isolated from seawater

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due to the elevated levels of salinity.5 N. fowleri is cultivated on Henrietta Lacks (HeLa) cells,

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which destroys the confluent cell layer in 2-3 days,6 as compared to Acanthamoeba which can be

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grown in a variety of non-cellular media.5

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Current recommended drugs from the Centers for Disease Control and Prevention (CDC)

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include a combination of antibiotics azithromycin and rifampin, antifungal amphotericin B and

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fluconazole, and anti-cancer and anti-leishmanial drug miltefosine against PAM. However, the

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rate of success is very low, and specificity of these drugs remains a major issue.7,8. Recently,

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nanotechnology has offered innovative strategies to overcome a variety of biological limitations

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and is further projected to have a substantial impact on biomedical research.9 Nanoparticles are

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subcolloidal metallic particles within the size range of nanometer (nm) scale. For example, gold

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nanoparticles of size lower than 20 nm have been conjugated with diverse cellular functions

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targeting peptides which can infiltrate the cell membrane to reach in the nucleus.9,10 Gold

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nanoparticles hold significant value in the drug delivery and diagnostic applications due to their

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tunable physicochemical properties, strong optical absorption and high biocompatibility.11

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Nanoparticles are also known to enhance the bioactivity of drugs by high drug loading which 3 ACS Paragon Plus Environment


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eventually leads to increased concentration of drugs at target site. Furthermore, due to small size,

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nanoparticle can easily overcome the blood-brain barrier which further facilitate the efficacy of

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drugs used to combat brain-eating amoeba.12

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CA (trans-3-Phenylacrylic acid) is secondary metabolite obtained from a variety of

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plants. It is a biologically active organic acid with known properties against a broad spectrum of

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biological functions against humans as well as pathogens with low toxicity.13 CA and its related

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chemical derivatives have shown promising antimicrobial activities, hence they hold potential for

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drug development.14 In this contribution, we have studied the anti-N. fowleri activity of CA for

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the first time. Furthermore, CA-AuNPs were also synthesized, and their amoebicidal and host

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cells cytotoxicity was evaluated. The results of current study suggest that CA alone as well as

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CA-AuNPs exhibited antiamoebic effects, however AuNPs conjugation showed a significant

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increase in the antiamoebic effects of CA at higher concentration against N. fowleri.

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RESULTS AND DISCUSSION

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Surface plasmon resonance and morphological characteristics of CA-AuNPs

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The rapid reduction of gold salt in the presence of CA with sodium borohydride provide

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stable nanoparticles. The formation of CA-AuNPs was indicated by persistent color change from

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colorless to purplish-pink. UV-Visible analysis of CA-AuNPs showed the characteristic

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absorption band at 540 nm called surface plasmon resonance band as compared to CA alone

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which exhibits maximum absorption at 265 nm (Fig 1a). Bare AuNPs (unconjugated with any

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stabilizing agent) were also prepared by similar method but in the absence of CA, however, these

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were freshly prepared every time before usage to avoid their kinetic tendency to aggregates.

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These nanoparticles were centrifuged at 10000×g for 10 min to separate the larger aggregates,


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side products, and any excessive reagent. The pelleted nanoparticles were then re-dissolved in

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autoclaved deionized water. The size and morphological analysis of CA-AuNP was carried out

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by AFM. AFM analysis revealed that CA-AuNP were polydisperse in size and spherical in shape

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(Fig. 1b). Dynamic light scattering analysis confirmed the average diameter of CA-AuNPs to be

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89 nm.

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CA-AuNPs demonstrated enhanced anti-N. fowleri effects as compared to CA alone

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N. fowleri were treated with several concentrations of CA and CA-AuNPs ranging from

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2.5 to 50 µM for 24 h and the viability of the amoebae was confirmed by Trypan blue exclusion

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assay. CA significantly reduced the viability of N. fowleri as a function of increasing the

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concentration (*P˂0.05) (Fig 2). However, the findings proved that CA-AuNPs exhibited better

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amoebicidal effects against N. fowleri as compared to CA alone at 50µM only as CA-AuNPs

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showed significant difference in amoebicidal effects as the numbers of N. fowleri were limited to

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1.9 x 105 from 2.6 x 105 in comparison to CA alone (*P˂0.05) (Fig 2). On the other hand, AuNPs

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alone did not affect the viability of N. fowleri. Notably, CA-AuNPs also showed similar

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amoebicidal effects as standard drug amphotericin B, which also reduced the numbers of N.

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fowleri to 1.6 x 105 (*P˂0.05) (Fig 2). The results presented here are representatives of at least

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three experiments carried out in duplicates while the data is presented as the mean ± standard

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error. The antiamoebic effects of CA-AuNPs were found to be as potent as the positive control as

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highlighted above, which shows the translational value of these nanoparticles.

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CA-AuNPs suppressed the N. fowleri-mediated host cell cytotoxicity CA and CA-AuNPs inhibited the cytotoxicity of pre-treated N. fowleri against HeLa cell. 5 x 105 amoebae/well were treated with different concentrations of CA-AuNPs and CA for 2 h



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before incubation with HeLa cells as described in Methods. Next, the treated amoebae were

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placed on with HeLa monolayers and the cells were incubated for 24 h at 37°C in a 5% CO2

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incubator. Host cells cytotoxicity is a secondary screening to test whether CA-AuNPs are

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biologically capable to reduce the cell invasion of the parasite. Untreated N. fowleri showed 70%

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host cells toxicity, nevertheless, CA-AuNPs pretreated amoebae revealed significantly lower

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amoeba-mediated cytotoxicity of 25% (*P˂0.05) (Fig 3). A further confirmation was carried out

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by performing revival assay whereby the treated amoebae were further incubated for 24 h on the

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host cells to test their survival rate by staining with 0.4 % aqueous solution of Trypan blue. The

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results showed that CA as well as CA-AuNPs prevented parasite-based host cell damage, and the

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monolayer was undamaged as compared to N. fowleri or AuNPs alone (Fig 4). The results

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illustrate data from several experiments performed in duplicate.

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CA-AuNPs caused no cell cytotoxicity

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The biological tolerance of CA and CA-AuNPs was also determined by measuring

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cytotoxicity using Lactate dehydrogenase (LDH) assay. Briefly, HeLa cells were challenged with

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several concentrations of test samples at 37ᵒC for 24 hours. Following this incubation, the cell

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cytotoxicity was determined by estimation of LDH release measured by absorption values

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obtained at 492 nm on plate reader. The results demonstrated that CA and CA-AuNPs did not

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exhibit cytotoxic effects against human cells.

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Previous studies have shown that delivery with AuNPs enhanced the efficacy of drugs at

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the desired cellular target by preventing drug loss.11 Research on other protists have shown that

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the CA derivatives showed promising in vitro activity against Leishmania by affecting the

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mitochondria, hence, making it an efficient choice for the development of chemotherapy.15 CA is

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also known to downregulate oncogenes by altering the protein synthesis which leads to growth 6 ACS Paragon Plus Environment

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inhibition.16 In our previous study, CA-AuNPs showed amoebicidal and antibacterial effects

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against Acanthamoeba castellanii, and both Gram-negative (E. coli K1) and positive bacteria

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(MRSA) while protecting the functioning and growth of host cells.17 However, the exact mode of

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action of antiamoebic activity of CA-AuNPs is yet unknown and needing experimental

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explanations along with the in vivo evaluation before they can be recommended clinically.

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In conclusion, because of their small size and ability to enhance the antiamoebic effects

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by high drug loading, CA-AuNPs are useful alternatives for drug discovery against N. fowleri.

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Since, the current approaches in drug discovery against rare but deadly infection caused by

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brain-eating amoebae are limited, naturally occurring CA and its gold nanoconjugates hold

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tremendous potential for in vivo studies which will be examined in future studies.

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METHODS

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Chemicals

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Reagents that were used in the synthesis of CA-AuNPs were of scientific grade.

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Tetrachloroauric acid and sodium borohydride (NaBH4) were purchased from Merck chemicals,

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while CA and any other chemicals were obtained from Sigma-Aldrich unless stated otherwise.

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Synthesis of CA-AuNPs

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Gold nanoparticles were stabilized by CA after reducing gold solution with NaBH4 used

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as rapid reducing agent as described previously.17 Briefly, 1:1 (v/v) mixture of equimolar

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aqueous solution of CA and tetrachloroauric acid (0.1 mM) was magnetically stirred for 15 min.

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Following that, 20 µL of stoichiometrically excessive NaBH4 aqueous solution (5 mM) was

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added into above reaction mixture. The color of reaction mixture instantly changed from pale

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yellow to purplish-pink indicating the formation of CA-AuNPs. The colloids were further stirred 7 ACS Paragon Plus Environment


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for 2 h and were kept at 4 oC for 1 day to ensure the stability. These nanoparticles were then

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subjected to instrumental analysis for characterization by UV-Vis spectrophotometry (Evolution

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201, Thermo Scientific), and AFM (Agilent 5500) as reported previously.18

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HeLa cell cultures

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HeLa cells were procured from ATCC (CCL-2) and were routinely cultured in Roswell

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Park Memorial Institute (RPMI)-1640. The medium was further supplemented with growth

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nutrients and antibiotics as; 1% Penicillin-Streptomycin, 1% L-glutamine, 1% non-essential

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amino acid, and 10 % fetal bovine serum. The cells were grown in T75 tissue culture flasks at

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37°C in 5% CO2 incubator. For cytotoxicity analysis, HeLa cells were cultivated on 24-well

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plates by seeding half million cells/well, and the plates were incubated to obtain the monolayer

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of cells. Confluent HeLa cells monolayers was obtained between 24 to 48 h incubation which

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was routinely observed under light microscope.

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N. fowleri cultures

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A clinical strain of N. fowleri (ATCC 30174) isolated from of a PAM infected patient

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was routinely cultured in T75 culture flasks at 37°C with 5% CO2 on HeLa cells as nutrition as

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described previously.19 The amoebae were grown on HeLa cells to confluency within 48 h and a

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populace of approximately 5 x 105 trophozoites of amoebae was achieved.

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Amoebicidal activity

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Amoebicidal properties of CA and CA-AuNPs were evaluated as previously described.20

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5×105 N. fowleri trophozoites were treated with various concentrations of CA-AuNPs, CA and

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bare AuNPs and appropriate controls in RPMI-1640 in 24-well plates. Negative controls were set

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up using dH2O without adding any other drug compound whereas standard drug, amphotericin B


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treated N. fowleri were taken the positive control. The amoebae were incubated for 24 h at 37°C.

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Next, Trypan blue (0.1%) was used to differentiate the viabile and dead N. fowleri, and alive

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(non-stained) numbers of amoebae were tallied using a hemocytometer. The data is represented

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as the mean ± standard error of various experiments carried out in duplicate.

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N. fowleri-mediated host cell cytotopathogenicity

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Amoeba-based host cell cytotoxicity was measured against HeLa cells as described

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previously.21 Briefly, 8 x 104 N. fowleri trophozoites were incubated with different

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concentrations of CA-AuNPs, CA and bare AuNPs alone for 2 h as pre-treatment. Next, the

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samples were centrifuged at 5000 x g for 5 min, supernatant was aspirated, and the pellet was re-

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dissolved in 200 μL fresh media to remove the excessive drug, and unwanted cellular materials.

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These samples were then incubated with HeLa monolayers for 24 h at 37°C in a 5% CO2

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incubator. After the incubation time, supernatant was withdrawn from each well and mixed with

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equal volume of LDH detection kit (Invitrogen). The LDH release was measured by determining

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absorbance at 492 nm on plate reader. The percent cytotoxicity was computed by using

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following equation: % cytotoxicity = (test sample value – negative control value) / positive

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control value – negative control value) × 100. The negative control values were acquired from

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untreated cells incubated in only RPMI-1640, while cells totally damaged by Triton X-100 for

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maximum LDH release were considered as positive control.

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FUNDING

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This work is supported by Sunway University, Malaysia. AUTHOR CONTRIBUTIONS



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KR and AA synthesized the nanoparticles. MRS analysed the characterization by UV-vis

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and AFM. NK and RS established the idea and supervised the study. KR and AA conducted the

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bioassays and prepared the first draft of manuscript. NK and RS corrected and finalized the

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manuscript for submission. The manuscript was submitted with the endorsement of all authors.

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NOTES

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Authors declare no competing interests. REFERENCES 1. Capewell, L.G., Harris, A.M., Yoder, J.S., Cope, J.R., Eddy, B.A., Roy, S.L., Visvesvara,

199

G.S., Fox, L.A.M., Beach, M.J. (2015) Diagnosis, clinical course, and treatment of

200

primary amoebic meningoencephalitis in the United States, 1937-2013. J. Pediatric

201

Infect. Dis. Soc. 4, e68–e75.

202 203 204

2. Heggie, T.W. (2010) Swimming with death : Naegleria fowleri infections in recreational waters. Travel Med. Infect. Dis. 8, 201–206. 3. Herman, E.K., Greninger, A.L., Visvesvara, G.S., Marciano-Cabral, F., Dacks, J.B.,

205

Chiu, C.Y. (2013) The mitochondrial genome and a 60-kb nuclear DNA segment from

206

Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis. J.

207

Eukaryot. Microbiol. 60, 179–191.

208

4. Jarolim, K.L., McCosh, J.K., Howard, M.J., (2002) The role of blood vessels and lungs in

209

the dissemination of Naegleria fowleri following intranasal inoculation in mice. Folia

210

Parasitol. (Praha). 49, 183–188.

211 212

5. Schuster, F.L. (2002) Cultivation of pathogenic and opportunistic free-living amebas. Clin. Microbiol. Rev. 15, 342–354.


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213 214 215


6. Siddiqui, R., Ali, I.K.M., Cope, J.R., Khan, N.A. (2016) Biology and pathogenesis of Naegleria fowleri. Acta Trop. 164, 375–394. 7. Debnath, A., Nelson, A.T., Silva-Olivares, A., Shibayama, M., Siegel, D., McKerrow,

216

J.H. (2018) In Vitro efficacy of Ebselen and BAY 11-7082 against Naegleria fowleri.

217

Front. Microbiol. 9, 414.

218 219 220

8. Siddiqui, R., Khan, N.A. (2012) Biology and pathogenesis of Acanthamoeba. Parasit. Vectors 5, 6. 9. Daraee, H., Eatemadi, A., Abbasi, E., Aval, S.F., Kouhi, M., Akbarzadeh, A. (2016)

221

Application of gold nanoparticles in biomedical and drug delivery. Artif. Cells, Nanomed.

222

Biotechnol. 44, 410–422.

223

10. Doubrovsky, V.A., Yanina, I.Y., Tuchin, V. V. (2010) Inhomogeneity of photo-induced

224

fat cell lipolysis. Saratov Fall Meet. 2010 Opt. Technol. Biophys. Med. XII. Ed. by

225

Tuchin, Val. V.; Genina, Elina A. Proc. SPIE, Vol. 7999, Artic. id. 79990M, 9 pp.

226

(2010). 7999, 79990M–79990M.

227

11. Pissuwan, D., Niidome, T., Cortie, M.B. (2011) The forthcoming applications of gold

228

nanoparticles in drug and gene delivery systems. J. Control. Release 149, 65–71.

229 230 231

12. Oyelere, A. (2008) Gold nanoparticles: From nanomedicine to nanosensing. Nanotechnol. Sci. Appl. 1, 45–66. 13. Kanaani, J., Ginsburg, H. (1992) Effects of CA derivatives on in vitro growth of

232

Plasmodium falciparum and on the permeability of the membrane of malaria-infected

233

erythrocytes. Antimicrob. Agents Chemother. 36, 1102–1108.

234 235

14. Sova, M. (2012) Antioxidant and antimicrobial activities of CA derivatives. Mini Rev. Med. Chem. 12, 749–67.



Page 12 of 20

236

15. Masic, A., Hernandez, A.M.V., Hazra, S., Glaser, J., Holzgrabe, U., Hazra, B., Schurigt,

237

U. (2015) CA bornyl ester derivatives from valeriana wallichii exhibit antileishmanial in

238

vivo activity in leishmania major-infected BALB/c Mice. PLoS One 10, 1–20.

239

16. Zhang, J., Xiao, A., Wang, T., Liang, X., Gao, J., Li, P., Shi, T. (2014) Effect and

240

mechanism of action of CA on the proliferation and apoptosis of Leukaemia cells.

241

Biomed. Res. 25, 405–408.

242

17. Anwar, A., Siddiqui, R., Shah, M.R., Khan, N.A. (2018) Gold nanoparticle-conjugated

243

CA exhibits antiacanthamoebic and antibacterial properties. Antimicrob. Agents

244

Chemother. 62, e00630-18.

245

18. Anwar, A., Shah, M.R., Muhammad, S.P., Afridi, S., Ali, K. (2016) Thio-pyridinium

246

capped silver nanoparticle based supramolecular recognition of Cu(i) in real samples and

247

T-lymphocytes. New J. Chem. 40, 6480–6486.

248

19. Rajendran, K., Anwar, A., Khan, N. A., Siddiqui, R. (2017) Brain-eating amoebae: Silver

249

nanoparticle conjugation enhanced efficacy of anti-amoebic drugs against Naegleria

250

fowleri. ACS Chem. Neurosci. 8, 2626-2630.

251

20. Sissons, J., Kim, K.S., Stins, M., Alsam, S., Khan, N.A., Jayasekera, S. (2005)

252

Acanthamoeba castellanii induces host cell death via a Phosphatidylinositol 3-Kinase-

253

dependent mechanism. Infect. Immun. 73, 1–6.

254

21. Siddiqui, R., Jarroll, E.L., Khan, N.A. (2009) Balamuthia mandrillaris: Staining

255

properties of cysts and trophozoites and the effect of 2,6-dichlorobenzonitrile and

256

calcofluor white on encystment. J. Eukaryot. Microbiol. 56, 136–141.

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FIGURE LEGENDS 12 ACS Paragon Plus Environment

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Figure 1. (a) shows the UV-visible spectrum of CA-AuNPs, a surface plasmon resonance band

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at 540 nm was observed, which indicates the successful formation of CA-conjugated AuNPs

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while (b) shows the AFM image of CA-AuNPs. CA-AuNPs were found to be spherical in shape

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and polydispersed in size.

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Figure 2. The amoebicidal effects of CA and gold nanoparticles (AuNPs) coated CA were

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determined. Briefly, N. fowleri were incubated with different concentrations of CA for 24 h and

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viability determined by Trypan blue staining as described in Methods. The results revealed that

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as low as 2.5µM CA and CA-conjugated with AuNPs showed significant antiamoebic effects as

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compared with untreated amoeba (*P˂0.05 using 2 sample t test; two tailed distribution). The

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results are representative of three independent experiments performed in duplicate. The data are

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presented as the mean ± standard error.

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Figure 3. Pre-treatment of N. fowleri with CA and nanoparticles-conjugated CA inhibited

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parasite-mediated HeLa cell cytotoxicity. Briefly, amoebae (5 x 105 amoebae/0.5 ml/well) were

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incubated with different concentrations of Au-conjugated CA and CA alone and then added to

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HeLa cells monolayers for 24 h at 37°C in a 5% CO2 incubator as described in Methods. The

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LDH determination results showed that the drugs-conjugated AuNPs significantly inhibited

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parasite-mediated host cell cytotoxicity compared with drugs alone (*P˂0.05 using 2 sample t

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test; two tailed distribution). The results are representative of three independent experiments

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performed in duplicate. The data are presented as the mean ± standard error.

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Figure 4. The qualitative results also showed that CA and CA-conjugated AuNPs inhibited

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parasite-mediated host cell damage. The results are representative of several experiments, and

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the images are recorded after staining with 0.4 % aqueous solution of Trypan blue at 250 X

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magnification. The results revealed the staining of the parasite as well as the host cells. (a) Host 13 ACS Paragon Plus Environment


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cells alone; (b) Host cells + N. fowleri; (c) Host cells + N. fowleri + 2.5µM CA; (d) Host cells +

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N. fowleri + 5µM CA; (e) Host cells + N. fowleri + 10µM CA; (f) Host cells + N. fowleri +

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25µM CA; (g) Host cells + N. fowleri + 50µM CA; (h) Host cells + N. fowleri + 2.5µM CA-

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AuNPs; (i) Host cells + N. fowleri + 5µM CA-AuNPs; (j) Host cells + N. fowleri + 10µM CA-

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AuNPs; (k) Host cells + N. fowleri + 25µM CA-AuNPs; (l) Host cells + N. fowleri + 50µM CA-

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AuNPs; (m) Host cells + N. fowleri + 25µM Au alone; (n) Host cells + N. fowleri + 50µM Au

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alone; (o) Host cells + N. fowleri + solvent alone. Panels i-l show the staining of parasites but not

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host cells as in those treatments no significant damage to host cells was observed, however the

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impact on the parasites is clear.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41


A

0.15

Absorbance

Page 15 of 20

0.1

B

0.05

0 400

450

500

550

600

650

Wavelength (nm)

ACS Paragon Plus Environment


* 1.20E+06

Number of viable Amoeba

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 16 of 20

1.00E+06 8.00E+05 6.00E+05

*

4.00E+05 2.00E+05 0.00E+00

+ N. fowleri ACS Paragon Plus Environment

Page 17 of 20

100.00

% Host cell Cytotoxicity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41


80.00

60.00

40.00

*

*

*

*

20.00

0.00

+ Environment N. fowleri ACS Paragon Plus

A

1 2 3 4 5 6 7 8 9 10 11D 12 13 14 15 16 17 18 19 20 21 22 23 24 25 G 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

B


C

Host cells

E

F

H

I

N. fowleri

Host cells


Page 18 of 20

Page 19 of 20 J 1 2 3 4 5 6 7 8 9 10 11 M 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

K


N

L

O


ACS Chemical Neuroscience CA-AuNPs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Host cells + N. fowleri

Page 20 of 20

Host cells + N. fowleri + CA-AuNPs ACS Paragon Plus Environment

eating amoeba Naegleria fowleri Kavitha

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