Clinically-approved drugs against CNS diseases as potential

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Clinically-approved drugs against CNS diseases as potential therapeutic agents to target brain-eating amoebae Ayaz Anwar, Kavitha Rajendran, Ruqaiyyah Siddiqui, Muhammad Raza Shah, and Naveed Ahmed Khan ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00484 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

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ACS Chemical Neuroscience

Clinically-approved drugs against CNS diseases as potential therapeutic agents to target brain-eating amoebae

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Ayaz Anwar1*, Kavitha Rajendran1, Ruqaiyyah Siddiqui1, Muhammad Raza Shah2, Naveed

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Ahmed Khan1

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

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Malaysia. 2: HEJ Research Institute of Chemistry, International Center for Chemical and

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Biological Sciences, University of Karachi, Karachi, Pakistan.

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[email protected]; [email protected]; [email protected];

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[email protected]; [email protected]

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Short title: CNS drugs against brain-eating amoebae

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*Corresponding address: Department of Biological Sciences, School of Science and Technology,

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Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-8622. Ext: 7169. Fax: 60-

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(0)3-5635-8630. E-mail: [email protected]; [email protected]

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Abstract Central nervous system (CNS) infections caused by free-living amoebae such as

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Acanthamoeba species, Naegleria fowleri etc. are rare but fatal. A major challenge in the

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treatment against the infections caused by these amoebae is the discovery of novel compounds

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that can effectively cross the blood-brain barrier to penetrate CNS. It is logical to test clinically-

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approved drugs against CNS diseases for their potential antiamoebic effects since they are

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known for effective blood-brain barrier penetration and effect eukaryotic cell targets. The

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antiamoebic effects of clinically available drugs for seizures targeting gamma-amino butyric acid

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(GABA) receptor and ion channels were tested against Acanthamoeba castellanii (A. castellanii)

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belonging to the T4 genotype and Naegleria fowleri (N. fowleri). Three such drugs namely;

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Diazepam (Valium), Phenobarbitone (Luminal), Phenytoin (Dilantin) and their silver

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nanoparticles (AgNPs) were evaluated against both trophozoites and cysts stage. Drugs alone

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and drugs conjugated silver nanoparticles were tested for amoebicidal, cysticidal and host-cells

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cytotoxicity assays. In vitro amoebicidal assay showed potent amoebicidal effects for Diazepam,

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Phenobarbitone, and Phenytoin-conjugated AgNPs as compared to drugs alone against A.

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castellanii and N. fowleri. Nanoparticles were synthesized by sodium borohydride reduction of

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silver nitrate with drugs as capping agents. Drugs conjugated nanoconjugates were characterized

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by ultraviolet-visible (UV-vis), and Fourier transform infrared (FT-IR) spectroscopies, and

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atomic force microscopy (AFM). Furthermore, both drugs and drugs conjugated AgNPs showed

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compelling cysticidal effects. Drugs conjugations with silver nanoparticles enhanced their

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antiacanthamoebic activity. Interestingly, amoeba-mediated host cells cytotoxicity was also

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significantly reduced by drugs alone as well as their nanoconjugates. Since, these drugs are being

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used to target CNS diseases, their evaluation against brain-eating amoebae seems feasible due to

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advantages such as; permeability of the blood-brain barrier; established pharmacokinetics and 2 ACS Paragon Plus Environment

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dynamics; FDA approval etc. Given the limited availability of effective drugs against A.

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castellanii, the clinically available drugs tested here present potential for further in vivo studies.

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Keywords: Acanthamoeba; Naegleria; Diazepam (Valium); Phenobarbitone (Luminal);

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Phenytoin (Dilantin); Nanoparticles.

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Introduction

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Infectious diseases involving CNS due to brain-eating amoebae; Acanthamoeba species,

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Balamuthia mandrillaris, and Naegleria fowleri are rare but almost always result is mortality.1

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Acanthamoeba castellanii (A. castellanii) is a facultative parasite and most frequently causes a

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painful, sight intimidating infection, Acanthamoeba keratitis, and devastating granulomatous

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amoebic encephalitis.2 Acanthamoeba keratitis most commonly affects contact lens wearers.3 A.

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castellanii life cycle consists of two forms; reproductive trophozoite, and sturdy cysts.2 Whereas,

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Naegleria fowleri causes deadly primary amoebic encephalitis (PAM).4,5 The current

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management and treatment of the brain-eating amoebae infections include combination of

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multiple drugs e.g., chlorhexidine, polyhexamethylene biguanides and propamidine with

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ketoconazole, neomycin, amphotericin B, milfetosine, sulfadiazine etc. There are some

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limitations to the use of these drugs including; resistance of cysts to uptake the drugs hence risk

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of recurrence, host-cells cytotoxicity due to prolonged treatment, etc.6 Albeit challenges in the

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discovery of novel drugs and approvals for clinical use, re-purposing of clinically-approved

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drugs against new diseases targets is a useful strategy. The discovery of novel compounds which

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can penetrate blood-brain barrier and target amoebae residing in CNS is indispensable, hence, to

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test the clinically-approved drugs for CNS diseases against brain-eating amoebae has a practical

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rationale.



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Nanoparticles are effective drug delivery vehicles associated with increasing the

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bioavailability of their cargoes.7 Their small size ensures specific target transportation and

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minimal side effects at lower concentration.7 Silver nanoparticles (AgNPs) are anticipated as

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next generation class of antimicrobials due to their known affinity towards DNA of pathogens

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and production of reactive oxygen species.8 There are several reports on antimicrobial activities

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of AgNPs in particular against bacteria.9 Currently, AgNPs uses in the topical burn ointments,

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wound healing materials for dressings in injuries, and their impregnation in medical devices such

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as catheters, grafts etc., are subject of vast interest.10-12 However, their utility against protists

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infectious diseases is limited. AgNPs are recently developed as viable alternatives in treatment

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against brain-eating amoebae, A. castellanii and N. fowleri.13-15

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Herein we demonstrated the effects of clinically available drugs; Diazepam,

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Phenobarbitone, and Phenytoin on A. castellanii and N. fowleri. Diazepam is clinically used to

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treat anxiety disorder, and sometimes used with other drugs to control seizures.16 The oral dosage

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of Diazepam ranges from 2-10 mg 2-3 times a day depending on severity of disease.17

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Phenobarbitone is an anticonvulsant drug which is mainly used to control seizures in epileptic

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and bipolar patients.18 The recommended dosage of Phenobarbitone is 15-18 mg/kg via

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intravenous administration for epileptic patients.19 Phenytoin is another

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anticonvulsant/antiepileptic drug taken orally in the form of capsule with the dosage of 30 mg

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once daily.20 All selected drugs are FDA approved, and are in the WHO list of essential

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medicines needed in health system. Besides using these drugs alone, we also tested whether the

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conjugation of silver nanoparticles with these drugs enhanced their bioactivities against brain-

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eating amoebae. Testing these CNS targeting drugs and their silver nanoconjugates against A.

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castellanii and N. fowleri resulted in antiamoebic therapy. All of drugs and nanoparticles showed 4 ACS Paragon Plus Environment

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remarkable amoebicidal and cysticidal properties and reduced amoeba-mediated host cells

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cytotoxicity.

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Results and discussion

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CNS infectious diseases caused by brain-eating amoebae such as Granulomatous amoebic

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encephalitis (GAE), Primary amoebic encephalitis (PAM) etc. are rare, and due to lack of

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knowledge difficult to diagnose as well as no single available effective therapeutic option make

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them lethal in almost all cases.21,22 GAE due to A. castellanii is a fatal infection of brain and

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spinal cord generally affecting the immunocompromised patients.23 GAE symptoms typically

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progress over several weeks to months. Once the CNS is affected, the symptoms may include;

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headaches and fever, stiff neck, nausea, tiredness, lack of focus and body coordination, seizures,

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etc.1 PAM due to N. fowleri is a severe, fulminating infection of the CNS.5 The therapeutic

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agents used to treat and manage infections caused by brain-eating amoebae include

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chlorhexidine, amphotericin B, voriconazole, rifampicin, pentamidine, polyhexamethylene

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biguanide, azithromycin, miltefosine and/or a mixture of these drugs, but they have limitations

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due to their reduced bioavailability and minimum inhibitory concentration (MIC) at the site of

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infection, off-site cytotoxicity and limited potency against cysts.24 Therefore, there is an urgent

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need to find novel approaches for development of new and effective therapeutic alternatives, as

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well as administration strategies.

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Herein, we selected three drugs clinically used against CNS diseases including

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Diazepam, Phenobarbitone, and Phenytoin (benzodiazepine, barbiturate, and hydantoin

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respectively) to evaluate their activity against brain-eating amoebae. Their brief mode of action

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in humans is summarized in Table 1. Benzodiazepines bind to benzodiazepine receptors resulting

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in relaxation of muscles, and cause calming effects which effects motor coordination and 5 ACS Paragon Plus Environment


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memory, and show anticonvulsant activity.25 Benzodiazepine receptors are known to be coupled

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to gamma-aminobutyric acid-A (GABAA) receptors, which effects GABA by increasing its

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affinity for the receptor. 25 This binding causes opening of the chloride ions channel, resulting in

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a hyperpolarization on the cell membrane which control seizures and leads to stabilization.25

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Phenytoin acts on sodium ion channels at the neuronal cell membrane, and limits spreading of

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seizure activity. As a result of promoting sodium efflux from neurons, it controls the threshold

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against excitability caused by excessive stimulation capable of reducing gradient of sodium on

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the membrane.26 Phenobarbitone is another promoter of GABAA receptors, which has the

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potential of increasingseizure threshold and reducing the spread of seizure. It is also known to

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inhibit calcium channels, which aids in a decrease in excitatory transmitter release.27 Diazepam

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is known to penetrate blood-brain barrier much faster than Phenobarbitone and Dialantin.28 A.

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castellanii is known to have specific binding sites with benzodiazepines in mitochondrial

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membrane with proteins similar as mammalian cells called mitochondria benzodiazepine

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receptors (MBR).29 Thus, the alteration in mitochondrial activity by proteins-Diazepam

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interactions with amoebae may be the reason for such a huge reduction in its viability. Moreover,

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MBR are also associated with several other functions such as; importance in steroids

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biosynthesis, control of mitochondrial respiration, cells proliferation, transfer of calcium ions

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etc.29 Hence the mode of action of effects of Diazepam against brain-eating amoebae is proposed

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to be a multifactor action of above features. Phenobarbitone and Phenytoin are effective

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sodium/calcium channels inhibitors which play pivotal role in the metabolism functioning of

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brain-eating amoebae and their genotype modification30,31 which may be responsible for

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inhibition of these processes. Since the usage of anticonvulsant drugs such as those used in this

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study is common in amoebic encephalitis management, we anticipate that these findings provide


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a novel strategy to develop the potent amoebicidal agents combined with their known antiseizure

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effects which can be used to minimize the suffering of patients. Based on above discussion, it

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was logical to test the antiamoebic activity of these drugs and their nanoconjugates.

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Characterization of Diazepam, Phenobarbitone, and Phenytoin conjugated Silver

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nanoparticles via UV-visible spectrophotometry, AFM, and FT-IR spectroscopy.

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Drugs conjugated AgNPs were analyzed by UV-vis, FT-IR spectroscopy, and AFM for

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characterization. Diazepam conjugated silver nanoparticles (DZP-AgNPs), Phenobarbitone

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conjugated silver nanoparticles (PBT-AgNPs), and Phenytoin conjugated silver nanoparticles

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(PTN-AgNPs) were successfully stabilized by these drugs. UV-vis spectra of DZP-AgNPs, PBT-

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AgNPs, and PTN-AgNPs showed a representative surface plasmon resonance (SPR) band for

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AgNPs at 415, 420 and 428 nm respectively (Fig. 1a), which suggest the formation of

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nanoconjugates. AFM analysis was performed for determination of size and morphology of

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AgNPs conjugated with drugs. All nanconjugates of drugs were found to be spherical with a

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wide size distribution (10-200 nm) due to rapid reduction by using sodium borohydride (Fig. 1b-

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d). The comparative FT-IR spectral analysis of Phenytoin sodium with the PTN-AgNPs was

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carried out to identify the stabilizing functional groups in drug as a representative example. The

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FTIR spectrum of phenytoin sodium showed the characteristic signals of functional groups

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present in PTN. The peak at 3318 cm-1 was due to N-H stretch while peak at 1397 cm-1 was due

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to N-H bending. The peaks at 3066, 1592 and 1492 cm-1 were due to aromatic ring stretching

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vibration. The C=O of amide group appeared as a split peaks at 1674 and 1687 cm-1.32 After the

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formation of PTN-AgNPs, the N-H stretching shifted from 3318 to 3449, whereas the N-H

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bending signal was observed with slight red shift at 1383 cm-1. These results clearly indicate the

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involvement of N-H functionality present in the hydantoin ring in the stabilization of AgNPs. 7 ACS Paragon Plus Environment


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Silver nanoparticles conjugation with Diazepam, Phenobarbitone, and Phenytoin significantly

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enhanced their amoebicidal effects against A. castellanii

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Amoebicidal assays were carried out at various concentrations, the result (Fig. 2)

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revealed that CNS drugs conjugated with AgNPs exhibited significant amoebicidal potency

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against A. castellanii as compared to CNS drugs when used alone (P < 0.05, two-sample T test

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and two-tailed distribution). On the other hand, neither AgNPs alone nor DMSO used as solvent

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control showed any effects. All drugs nanoconjugates inhibited the viability of A. castellanii to

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104 as compared to controls and initial inoculum i.e., 105.

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Diazepam, Phenobarbitone, and Phenytoin alone and their nanoconjugates both inhibited

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encystation of A. castellanii

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The potential of conjugation of AgNPs with Diazepam, Phenobarbitone, and Phenytoin

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on encystment of A. castellanii was evaluated to establish the contrasting encystation

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differentiation as compared to drugs alone. The encystation media induced formation of cysts

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and 4.13x104 number of cysts were enumerated without any drugs (Fig. 3). However, the

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addition of drugs and their nanoconjugates, resulted in significant reduction in encystation of

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trophozoites (P < 0.05, two-sample T test and two-tailed distribution). The difference between

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encystation inhibition capabilities can be observed only in the case of Diazepam and

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Phenobarbitone with respect to their nanoconjugates with latter being more effective, whereas

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Phenytoin and PTN-AgNPs both produced same effects.

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Diazepam, Phenobarbitone, Phenytoin, and their AgNPs conjugates inhibited excystation

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To test the cysticidal effects of drugs nanoconjugates, cysts were prepared as described in materials and methods section and were incubated with various concentrations of drugs and 8 ACS Paragon Plus Environment

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drugs coated nanoconjugates in growth medium PYG. In the absence of CNS drugs, A.

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castellanii cysts emerged as viable trophozoites (Fig. 4). Contrary, both drugs and their AgNPs

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showed significant inhibition in excystation of A. castellanii cysts (P < 0.05, two-sample T test

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and two-tailed distribution). However, the enhanced effects of nanoparticles conjugation on

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drugs were clearly evident at 10 µM concentration. On the other hand, AgNPs alone had limited

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effects on excystation.

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CNS drugs and their nanoconjugates reduced host cells cytopathogenicity of A. castellanii

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A. castellanii mediated host cells cytotoxicity was also evaluated by cytopathogenicity

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assays using LDH determination as a measure of cells damage. Triton X-100 treated cell

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cytotoxicity was considered as 100% while all other cytotoxicity measured by LDH assay were

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plotted comparatively to this positive control (Fig. 5). A. castellanii alone produced more than

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80% host cell cytotoxicity, whereas Chlorhexidine pretreated amoeba showed no damage to

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cells. Instead, A. castellanii pretreated with drugs alone and drugs conjugated AgNPs caused

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significant reduction in host cell cytotoxicity at 10 µM and showed minimal cell damage (P

Clinically-approved drugs against CNS diseases as potential

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