trans-Cinnamic Acid Conjugated Gold Nanoparticles as Potent

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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,

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

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

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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.,

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

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

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Eukaryot. Microbiol. 60, 179–191.

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

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

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Parasitol. (Praha). 49, 183–188.

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


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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,

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

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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

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



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

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

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

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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

trans-Cinnamic Acid Conjugated Gold Nanoparticles as Potent

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