Importance of Theranostics in Rare Brain-Eating Amoebae Infections

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Importance of theranostics in rare brain-eating amoebae infections Ayaz Anwar, Ruqaiyyah Siddiqui, and Naveed Ahmed Khan ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00321 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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ACS Chemical Neuroscience

Importance of theranostics in rare brain-eating amoebae infections

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Ayaz Anwar, Ruqaiyyah Siddiqui, Naveed Ahmed Khan*

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

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Malaysia.

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Short title: Theranostics 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]

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Abstract Pathogenic free-living amoebae including Acanthamoeba spp., Balamuthia mandrillaris,

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and Naegleria fowleri cause infections to the central nervous system (CNS) which almost always

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prove fatal. The mortality rate is high with the CNS infections caused by these microbes despite

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modern developments in healthcare and antimicrobial chemotherapy. The low awareness,

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delayed diagnosis and lack of effective drugs are major hurdles to overcome these challenges.

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Nanomaterials have emerged as vital tools for concurrent diagnosis and therapy which are

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commonly referred to as theranostics. Nanomaterials offer highly sensitive diagnostic systems

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and viable therapeutic effects as a single modality. There has been good progress to develop

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nanomaterials based efficient theranostic systems against numerous kinds of tumors but this field

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is yet immature in the context of infectious diseases, particularly parasitic infections. Herein, we

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describe the potential value of theranostic applications of nanomaterials against brain infections

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due to pathogenic amoebae.

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Keywords: Theranostics; Nanomaterials; Infectious diseases; Parasites; Brain-eating amoeba.

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Theranostics’ are defined as a strategy consisting of therapeutic and diagnostic methods

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combined in one platform. Theranostic tools have acquired significant attention in the field of

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nanomedicine due to associated advantages such as the development of patient-specific

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personalized medicine producing improved outcomes at reduced costs, and fewer side effects.

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The basis of theranostic nanomedicine is nanotechnology that impacts medical research in both

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diagnosis and treatment. 1 It is anticipated that theranostic tools are on the verge of overcoming

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conventional diagnostic and therapeutic limitations. 2 There has been massive advancement in

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the progress of theranostic agents in oncology and neurodegenerative diseases, however there are

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limited reports against infectious diseases and even fewer in parasitology e.g., against

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leishmaniasis 3 and Malaria. 4, 5 Here, we propose that theranostic knowledge obtained from

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brain disorders can be extracted and applied against CNS infections due to pathogenic amoebae.

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The proposed theranostic strategies should be of potential value in reducing the disease burden

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associated with brain-eating amoebae by timely and sensitive diagnosis augmented with effective

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therapeutic capabilities (Fig. 1).

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Limitations in current clinical diagnostic tools

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Encephalitis due to pathogenic free-living amoebae either primary amoebic

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meningoencephalitis (PAM) or granulomatous amoebic encephalitis (GAE) are rare but deadly

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brain infections. 6, 7 These diseases are difficult to diagnose due to unawareness, limitations of

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diagnostic tools, and are challenging to treat due to the lack of effective drugs. Current diagnosis

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of brain-eating amoebae is based on the following: Real time PCR; Culture on monkey kidney;

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Agar plates culturing, however all of these techniques require a post-surgical sample from brain

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which is both painful and time taking. Notwithstanding the high fatality, pharmaceutical and

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healthcare industries have been failed to produce any drug against infections due to brain-eating 3 ACS Paragon Plus Environment


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amoebae. 8 One of the main challenges in the successful therapy of amoebal brain infection is the

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inefficacy of available antiamoebic drugs to cross the blood-brain barrier to reach the infection

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site and effectively target the residing parasite. The rationale behind nanotheranostics is to

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develop potential nanomaterials which can be used as biosensors for molecular image tracking in

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biological systems that can be further combined with controlled and site-specific drug release at

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the target tissues by locally inducing parameters such as pH, temperature, and enzymes to allow

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efficient killing of cells. There are various nanotheranostic agents based on their composition and

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mode of action. A few theranostic nanocarriers in the context of infectious and non-infectious

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diseases of brain are given below and summarized in Fig. 1.

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Metallic nanoparticles

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Nanoparticles lie in the range of 1-100 nm and consist of metals, semiconductors,

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polymeric, carbon-based, lipid-based. 9 Metallic nanoparticles are the most commonly used for

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widespread applications in medical and healthcare sciences. 10 The most common metallic

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nanoparticles are based on gold, silver and iron oxide. There are more than 35 FDA-approved

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nanoparticles currently used for imaging and therapy. 11-13 Most of these nanoparticles are used

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as drug delivery systems, while a few examples of their uses as MRI contrast agents are also

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reported.

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Gold nanoparticles (GNPs) are most studied nanomaterials in biomedicine due to their

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biocompatibility and tunable properties. GNPs are capable of conjugating and delivering

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biomolecules to specific targets whilst providing a theranostic approach simultaneously. 14 GNPs

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hybridized with other metals have also been utilized in theranostic-based nanomedicine. 15 The

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inert vehicles activated with surface functionalities provide them extensive applications in cancer

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and infectious disease. 16, 17 GNP-based molecular amplification and microassays have been 4 ACS Paragon Plus Environment

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developed to augment accurate and sensitive diagnosis of infectious diseases. 18 GNPs have high

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coefficient of X-ray absorption which is an essential requirement for a material to be used for

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computed tomography (CT) contrast agents, and radiotherapy sensitizers. 19 Various lateral flow

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assays (LFAs) have been developed based on the antibody-conjugated GNPs as detection probes.

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genome sequence, GNPs probes functionalized with oligonucleotide have been used with high

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efficiency and sensitivity. 21

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For the identification of pathogens containing unique nucleic acid markers as part of their

However, in the context of brain eating amoebae, not a single report can be found for

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theranostic applications of any of the aforementioned material. Promising theranostic application

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of multifunctional nanoparticles has been proposed for chronic neurodegenerative disorders such

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as Alzheimer’s, Parkinson’s disease and strokes. 22, 23 Liu et al. 24 reported nanoparticles-based

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iron chelation therapy against Alzheimer’s disease. The nanoparticles were found to be capable

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of crossing the blood-brain barrier. Cheng et al. 25 reported GNPs modified with transactivator of

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transcription (TAT) peptide, which are also demonstrated to be crossing the blood-brain barrier

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and efficiently delivering anticancer drug doxorubicin and contrast agent Gd3+ for enhanced

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malignant glioma. As these probes have been utilized in CNS disorders previously and have

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overcome the blood-brain barrier, there is a strong rationale to use these in the treatment of

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infections due to brain-eating amoebae. Research carried out by our group reports the potential

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of nanoparticles against infectious diseases against MDR bacteria and brain eating amoebae. For

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example, Chlorhexidine coated gold nanoparticles were shown to inhibit Acanthamoeba

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castellanii at much lower concentration as compared to the drug alone, moreover host cell

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cytotoxicity was significantly reduced. 26 These nanoparticles also inhibit Klebsiella pneumoniae

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biofilms as well as disrupted pre-formed biofilms. 27 More recently, the size of gold



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nanoparticles has shown antibiofilm potential due to Gram positive bacteria. 28 In another report,

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synthetic ligand dimethylamino pyridine (DMAP) propylthioacetate conjugated gold

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nanoparticles were shown to enhance the antibacterial activity of Pefloxacin against E. coli. 29

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Silver nanoparticles decorated with drugs such as Amphotericin B, Nystatin, and Fluconazole

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have shown to exhibit antiamoebic efficacy against brain-eating amoebae, A. castellanii and N.

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fowleri. 30, 31

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These reports show that metallic nanoparticles show potential as therapy for infections

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caused by brain-eating amoebae. We propose that due to the tunable physicochemical properties

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of metallic nanoparticles these should be conjugated with both targeting/imaging materials with

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effective drugs to produce theranostic effects. For example, anti-amoebal antibodies, florescence

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dyes such as Fluorescein isothiocyanate (FITC) or mannose to target mannose-binding protein

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adhesion of amoebae along with effective drugs such as Chlorhexidine, Pentamidine,

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Amphotericin B, azoles and other antiamoebic drugs.

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Magnetic nanoparticles

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Magnetic nanoparticles (MNPs) have a metallic core whose outer shell is generally

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conjugated with chemical functionalities. 32 MNPs are smaller than 100 nm, those can be

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composed of any material having some characteristics of magnetism, but mostly the metallic

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core is made up of iron oxide compounds. 33 There is a growing interest in the applications of

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MNPs for a wide variety of biomedical applications including the theranostics of cancer and

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infectious diseases. 34 Superparamagnetic iron oxide (SPIO) nanoparticles have been

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successfully utilized for staging and monitoring of treatment in a variety of cancers with MRI.

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MNPs have been widely studied against neurodegenerative diseases such as Alzheimer due to

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their tremendous potential to act as contrast agents in MRI. 35 6 ACS Paragon Plus Environment

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MNPs are known to produce magnetic hysteresis as a response to alternating current

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(AC) magnetic fields which results in hyperthermia (a localized thermal shock) ultimately

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leading to cell death. 36 Dai et al. 37 reported a multifunctional platform based on magnetic core

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plasmonic shell nanoparticles coated with methylene blue aptamer and a specific antibody for

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Salmonella DT104. This nanotheranostic system is shown to be useful for in vivo imaging of

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Salmonella DT104 followed by selective separation and photo-destruction of these MDR

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bacteria. 37 Park et al. 38 showed that MNPs could be used to eradicate Pseudomonas aeruginosa

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PA01 biofilms. More recently, Singh et al. 39 showed that up to 99 % reduction in the viability of

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Escherichia coli was observed as a result of magnetic hyperthermia using iron-based MNPs. In

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another report, Thomas et al. 40 showed iron-based MNPs stabilized by tiopronin, could diminish

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the 107 colonies of Staphylococcus aureus within 4 minutes of in vitro magnetic hyperthermia

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treatment. Magnetite-zinc oxide MNPs are also shown to exhibit an intrinsic bactericidal effect.

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pathogens via hyperthermia. However, the invasive nature of hyperthmal treatment is in some

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cases can also be non-selective to host cells therefore, careful design in development of these

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materials is of prime importance. Furthermore, proteins or nucleic acid conjugated MNPs can be

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used as specific probes for separation of pathogens to give theranostic benefits. Based on the

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above, it is evident that magnetic nanoparticles have been widely studied against CNS disorders

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and pathogenic bacteria and could be of value against brain-eating amoebae. We propose that

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combining the approaches of CNS targeting magnetic theranostics with effective antiparasitic

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agents would be an imperative step in the development of nanotheranostics against brain-eating

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amoebae infections.

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Quantum dots

Hence, it is anticipated that MNPs have potential in identifying, isolating and killing infectious



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Quantum dots (QDs) are semi-conducting materials with high luminescent properties of

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crystallinity. 42 They are made up from the II, III, IV, V, and VI group elements from the

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periodic table. Their quantum yield for emission depends on the electronic environment within

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the crystal, as well as on their size and shape. The semi-conducting inorganic core dictates the

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optical properties of the crystals, whereas the outer shell is commonly functionalized by a layer

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of organic molecules. 43

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Their high quantum yield and long luminescence lifetime are fundamental properties

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which have revolutionized bioanalytical measurements. QDs have been studied extensively for

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bio-imaging purposes since their discovery in the early 1990s. 44 QDs have also been utilized

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against CNS diseases due to their potential to cross the blood-brain barrier for example in

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encephalopathy associated with HIV. 45 Various QDs based bacterial detection systems,

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bioimaging platforms and cidal agents via photodynamic therapy have been reported. For

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example, Thakur et al. 46 developed ciprofloxacin conjugated carbon dots stabilized by gum

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arabica as a tool for the bioimaging of bacteria with high antimicrobial activity against a variety

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of Gram-positive and Gram-negative bacteria. Cheng et al. 47 reported antibodies conjugated

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QDs as fluorescence markers for simultaneous detection of three bacteria by using laboratory-

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based system. In another report, photoexcited QDs were used to kill a wide range of MDR

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bacteria. 48 The killing effect is shown to be controlled by the redox potential of photogenerated

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charge carriers that alters the redox states at cellular levels which ultimately leads to bacterial

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killing. 48

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Despite the toxic nature of metals generally used in the synthesis of QDs, the surface

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modifications and smart administration of QDs have paved their way into cancer theranostics. 49

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QD–aptamer–doxorubicin conjugate, have been used for simultaneous cancer imaging and 8 ACS Paragon Plus Environment

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traceable drug delivery. 50 In a recent report, QD-based micelles coated with anti-epidermal

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growth factor receptor (EGFR) nanobody, further loaded with aminoflavone for drug delivery,

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has been developed for triple negative breast cancer theranostics. 51 Ho et al. 52 highlighted

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aspects of imaging correlated with theranostics application of QDs. The major limitation in

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advancement of QDs to clinical applications is the use of toxic elements such as cadmium,

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arsenide or lead in their core. Therefore, not only the transmigration of therapeutic agents, but

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their safer administration strategies are required to develop effective theranostic tools. 53 The

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application of quantum dots in fluorescence mapping of sentinel lymph node could be promising

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due to less challenging local administration of QDs. 54 The ability of sensitive imaging of QDs

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due to high fluorescence is a tremendous opportunity to detect brain-eating amoebae at an early

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stage. Moreover, their applications against neurodegenerative diseases as mentioned above

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makes them ideal target for development of nanotheranostic tools against brain-eating amoebae

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and other infectious CNS pathogens. Hence, to make it possible QDs should be conjugated with

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specific antibodies against brain-eating amoebae and/or mannose due to its affinity towards

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mannose-binding proteins expressed on pathogenic Acanthamoeba, which further augmented

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with effective drugs for examples biguanides, amidines, and azoles and/or a mixture of these

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should produce excellent theranostic platforms against CNS infections caused by brain-eating

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amoebae. Furthermore, the luminescence ability of QDs may provide additional advantages for

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live imaging during diagnosis and therapy.

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Photo-triggered theranostic nanocarriers

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Theranostic probes that utilize photo-based techniques are of interest due to minimally

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invasive modalities. 55 A few examples of photo-triggered killing of MDR bacteria has been

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presented in previous sections. 48 Metal oxide nanoparticles such as TiO2, Fe2O3, ZnO etc. are



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frequently used against MDR microbes due to their photoactive nature and sensitivity to generate

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reactive oxygen species (ROS) while irradiated with UV light. Hence these nanoparticles can

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ideally be used as antimicrobial agents via photochemotherapy. Imran et al. 56 used Zinc doped

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TiO2 nanoparticles against Acanthamoeba via photodynamic therapy. Photo-triggered theranostic

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strategy include the advantage to customize the localized treatment. However, the limitations

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include high intensity of thermal dose in case of deeper tissues and prior information of the exact

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location of disease is needed to initiate treatment. 10

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Halas and West have pioneered photodynamic nanotheranostics against cancer. 57-60

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These systems work on the principle of photosensitization. For example, Khlebtsovet al. 61

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synthesized nanocage core of gold-silver alloy and shell made up of silica containing Yb–2,4-

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dimethoxyhematoporphyrin as near infrared (NIR) photosensitizer and used this multifunctional

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probe as tumor monitoring as well as simultaneous photodynamic and plasmonic heating

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therapy. In another report, NIR fluorescent heptamethine cyanine dye and Si-naphthalocyanine

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heating dyes containing non-plasmonic silica NPs have been shown for NIR fluorescence

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imaging and increased light absorption. 62 By direct tumor injection into mice, high fluorescence

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intensity and about 95% decrease in the viability of tumor was observed after irradiation with

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NIR laser. 62 Another novel nanoformulation for dual-imaging was prepared by Huang et al. they

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synthesized silica-modified gold nanorods conjugated with folic acid which can be

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simultaneously imaged in vivo using X ray or computed tomography (CT). 63 Photo-responsive

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materials such as Porphyrins have shown usefulness against infections caused by brain-eating

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amoebae. 64 Hence, Porphyrin and/or other photosensitizers based nanomaterials which can be

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further conjugated with specific antibodies, targeting agents and antiparasitic drugs can be of

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high value as theranostic tools against brain-eating amoebae. 10 ACS Paragon Plus Environment

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Liposomes Liposomes are clinically established nanomaterials that are used to deliver drugs, genes,

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vaccines, imaging agents, etc. 65 There are numerous examples of liposome-nanoparticles

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systems with varied characteristics that incorporate therapeutics and imaging capabilities. 66

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Similarly, liposome-QDs hybrid systems have also shown great potential in theranostics. 67

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Recently, Seleci et al. 68 reported liposome-QDs nanotheranostic agent to deliver topotecan.

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Some of parasitic diseases such as Malaria, Leishmania, Trypanosomiasis, Amoebiasis,

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Acanthamoeba keratitis etc. have also been targeted by liposomes. Serrano-Luna et al. 69 studied

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the virulence of Entamoeba histolytica by adding phosphatidylcholine-cholesterol liposomes in

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their culture medium and assessed various biochemical and biological functions of the parasite.

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The addition of liposomes in the culture medium is shown to produce no change in the virulence

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of these parasites against hamster liver as the original infectious E. histolytica strain, even

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without passage through hamster liver for more than one year. 69 Liposomal complexations of

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pentamidine with ergosterol produced enhancement in its antiamoebic potency against keratitis

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causing A. castellani. 70 More recently, a formulation of nanoemulsion from the plant extract of

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Pterocaulon balansae rich in coumarin was used for in vitro treatment of ocular Acanthamoeba

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keratitis. 71 In another report, therapeutic siRNA sequence loaded liposomes were shown to

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inhibit the encystment of Acanthamoeba by targeting glycogen phosphorylase. 72 Linolenic acid

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loaded liposomes with phosphatidylcholine and cholesterol have proved to be a potent

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antibacterial formulation against MDR Helicobacter pylori infection. 73 Muppidi et al. 74

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investigated the pharmacokinetics and biodistribution of vancomycin loaded liposomes within a

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mouse model of MRSA infection. Gao et al. 75 described the usefulness of nanoparticle-

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stabilized liposomes with hydrogel technology which was shown to be more effective and

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sustainable topical drug delivery system. It was demonstrated that this hydrogel formulation 11 ACS Paragon Plus Environment


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effectively releases nanoparticle-stabilized liposomes to S. aureus culture, those then pH

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dependently fuse with bacterial membrane. Interestingly, the topical administration of these

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hydrogel formulations did not cause skin toxicity even after prolonged application. 75

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Some of the conventional drugs loaded liposomes have also been approved and

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commercialized. These include Amphotec, and Abelcet containing liposomal Amphotericin B

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formulation for the treatment of fungal infections, 76 and Ambisome contain Amphotericin B

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against leishmaniasis. Another example of liposomal marketed therapy is Inflexal V which is

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used against influenza and consists of inactivated hemagglutinin of the influenza virus. 77 Some

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oncological examples of liposomal approved drugs are as follows; Doxil, a liposomal injection of

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doxorubicin is being used to treat multiple myeloma and ovarian cancer. 78 Onivyde, a liposome

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encapsulated irinotecan formulation being used to treat metastatic pancreatic cancer. 79

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Commercially available Amphotericin B liposomal formlations may be further conjugated with

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flouresent dyes and can potentially be used as theranostic platforms against brain infections

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caused by free-living amoebae for imaging cum therapeutic applications. Although infections due to pathogenic amoebae are rare, but due to the high abundance

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and prevalence of free-living amoebae are of global concern. 80 Their diagnosis and treatment

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currently suffers from lack of advancements as compared to other infectious and CNS diseases.

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infectious diseases caused by brain-eating amoebae to develop the missing link in timely and

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precise diagnosis merged with effective therapy for these devastating infections.

Here we highlighted some examples of nanotechnology driven theranostics and related them to

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Conclusion

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The theranostic approaches suggested here may play major role in reducing the

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devastating high mortality rate associated with rare infectious diseases caused by brain eating

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amoebae. There is an urgent need for development of smart materials based theranostics for

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improved laboratory and point-of-care testing. Nanomaterials have already shown promising

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theranostic applications in cancer, Alzheimers, Parkinson disease and bacteria imaging cum

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treatment. Moreover, they are anticipated to be a major breakthrough in parasitology especially

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against brain-eating amoebae. In our opinion, these suggested strategies have a strong rationale,

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and should yield a significant advancement in developing effective treatment of infections due to

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brain-eating amoebae as well as against other CNS pathogens.

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Acknowledgements: We are thankful to Sunway university for supporting this work.

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Funding: Not applicable.

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Authors’ contribution: NK proposed the concept. AA searched the literature and drafted the

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manuscript under the supervision of RS and NK. RS and NK corrected the manuscript. All

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authors read and approved the final version of the manuscript.

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Competing interests: None.

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505

506

Figure legends

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Fig. 1. Schematic depiction of proposed theranostic approaches against infections caused by

508

brain-eating amoebae. Some of the commonly utilized nanomaterials are highlighted which are

509

comprehensively discussed in the text. Nanomaterials due to their small size, unique

510

physicochemical properties, and readiness to drug delivery applications are suitable theranostic

511

platforms.


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Importance of Theranostics in Rare Brain-Eating Amoebae Infections

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