Brain-Eating Amoebae: Silver Nanoparticle Conjugation Enhanced

Dec 5, 2017 - AFM analysis was carried out to determine size and morphology of synthesized drugs conjugated AgNPs. AFM results showed complete ...
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Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Brain-Eating Amoebae: Silver Nanoparticle Conjugation Enhanced Efficacy of Anti-Amoebic Drugs against Naegleria fowleri Kavitha Rajendran, Ayaz Anwar, Naveed Ahmed Khan,* and Ruqaiyyah Siddiqui Department of Biological Sciences, School of Science and Technology, Sunway University, Bandar Sunway 47500, Malaysia ABSTRACT: The overall aim of this study was to determine whether conjugation with silver nanoparticles enhances effects of available drugs against primary amoebic meningoencephalitis due to Naegleria fowleri. Amphotericin B, Nystatin, and Fluconazole were conjugated with silver nanoparticles, and synthesis was confirmed using UV−visible spectrophotometry. Atomic force microscopy determined their size in range of 20−100 nm. To determine amoebicidal effects, N. fowleri were incubated with drugs-conjugated silver nanoparticles, silver nanoparticles alone, and drugs alone. The findings revealed that silver nanoparticles conjugation significantly enhanced antiamoebic effects of Nystatin and Amphotericin B but not Fluconazole at micromolar concentrations, compared with the drugs alone. For the first time, our findings showed that silver nanoparticle conjugation enhances efficacy of antiamoebic drugs against N. fowleri. Given the rarity of the disease and challenges in developing new drugs, it is hoped that modifying existing drugs to enhance their antiamoebic effects is a useful avenue that holds promise in improving the treatment of brain-eating amoebae infection due to N. fowleri. KEYWORDS: Naegleria fowleri, silver nanoparticles, chemotherapy, primary amoebic meningoencephalitis, brain eating amoebae

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recent studies showed that nanoparticles enhance drug potency as they are effective drug carriers against Acanthamoeba castellanii.8 In this study, drugs conjugated with silver nanoparticles (AgNPs) were synthesized and characterized, and their anti-N. fowleri effects were determined.

rimary amoebic meningoencephalitis (PAM) is a severe, fulminating brain infection of the central nervous system instigated by the free living amoeba, Naegleria fowleri.1−4 Symptoms typically comprise stiff neck, severe headache, seizures, fever (38.5−41 °C), distorted mental status, and coma, leading virtually always to demise. Hundreds of cases of PAM have been reported worldwide, with fatality rates of more than 90%. PAM patients usually have a history of coming in contact with warm water via activities such as swimming, bathing, ritual ablution, and the use of neti pots.5 Most common therapeutic options available include intravenous Amphotericin B, combined with adjunctive regimens such as Rifampin and the azole antifungals. Nonetheless, these antimicrobials have severe systemic adverse effects such as nephrotoxicity, as they are administered intravenously. Moreover, intravenous administration leads to poor penetration of drugs into the central nervous system (CNS) due to high selectivity of the blood-brain barrier.6 Hence, high concentrations of drugs are needed to achieve minimum inhibitory concentration (MIC) at the target site for effective killing of parasites, but treatment is often halted due to adverse effects. Consequently, there is a crucial need to develop antimicrobials against this devastating infection. Given challenges in developing new drugs against largely ignored parasites, a feasible approach is to enhance efficacy of available antiamoebic compounds to allow their use at low concentrations. Nanoparticles have gained vast attention in recent years in the field of biotechnology, biomedical, textile, food, cosmetic and many other industries.7 The most common metal carriers for nanoparticles-based drug delivery systems include gold, silver and iron oxide due to their inertness and biocompatibility. Our © XXXX American Chemical Society

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RESULTS AND DISCUSSION Characterization of Drugs Conjugated with AgNPs Using UV−Vis and AFM. Nanomaterials-based drug delivery systems are generally associated with improvements in the pharmacokinetics and pharmacodynamics of cargo drugs.9 Various examples of nanomaterials such as liposomes and nanoparticles have been utilized in clinical applications and a lot more are under trials.9 Nanoparticles are sub-colloidal materials composed of metals, semiconductors, etc., which have been extensively used as drug delivery systems due to their high surface area for maximum drug loading. Drugs coated nanoparticles are recently shown to be more effective against various infectious diseases where the drugs had limitations due to resistance.10 Here, we determined the effects of AgNPs conjugation with Amphotericin B, Nystatin, and Fluconazole against N. fowleri. Drugs conjugated with AgNPs were synthesized successfully and characterized. All drugs coated AgNPs showed surface plasmon resonance (SPR) bands in the range of 400−450 nm, characteristic of medium sized AgNPs. Nystatin and Fluconazole Received: November 7, 2017 Accepted: December 1, 2017

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DOI: 10.1021/acschemneuro.7b00430 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 1. (A) Amoebicidal effects of silver nanoparticles (AgNPs) coated Amphotericin B (Amp B) were determined. Briefly, N. fowleri were incubated with different concentrations of Amp B for 24 h and viability determined by Trypan blue staining as described in Methods. The results revealed that as low as 1 μM Amp B-conjugated with AgNPs showed significant antiamoebic effects as compared with drug alone (*P < 0.05 using two sample t test; two tailed distribution). The results are representative of three independent experiments performed in duplicates. The data are presented as the mean ± standard error. Silver nanoparticle conjugation with Nystatin (B) and Fluconazole (C) enhanced amoebicidal properties at micromolar concentrations. The results are representative of several experiments performed in duplicates. The data are presented as the mean ± standard error. B

DOI: 10.1021/acschemneuro.7b00430 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 2. Representative effects of silver nanoparticles-conjugated Amphotericin B, Nystatin, and Fluconazole on parasite-mediated HeLa cell cytotoxicity. Briefly, amoebae (5 × 105 amoebae/0.5 mL/well) were pretreated with different concentrations of Ag-conjugated drugs and drugs alone for 20 h, and then added to HeLa cell monolayers for 24 h at 37 °C in a 5% CO2 incubator as described in Methods. The results showed that the drugsconjugated AgNPs inhibited parasite-mediated host cell damage. The results are representative of several experiments. (A) Host cells alone; (B) host cells + N. fowleri; (C) host cells + N. fowleri pretreated with Amphotericin B; (D) host cells + N. fowleri pretreated with AgNP-conjugated Amphotericin B; (E) host cells + N. fowleri pretreated with Nystatin; (F) host cells + N. fowleri pretreated with AgNP-conjugated Nystatin; (G) host cells + N. fowleri pretreated with Fluconazole; (H) host cells + N. fowleri pretreated with AgNP-conjugated Fluconazole.

coated AgNPs displayed sharper SPR bands as compared to Amp-AgNPs which suggests the difference in size distribution of nanoparticles, as rapid reduction methodologies (such as sodium borohydride used here) lack in size selectivity in synthesis. UV− vis spectra of Amp-AgNPs revealed maximum absorption at 424 nm. On the other hand, pure Amphotericin B revealed multiple absorption bands in the range of 280−400 nm11 implicit in its stabilizing interaction with AgNPs. Moreover, Nys-AgNPs and Flu-AgNPs displayed characteristic SPR band for AgNPs at 405 and 400 nm when compared to bands around 280−330 and 260 nm, respectively, for pure Nystatin and Fluconazole. AFM analysis was carried out to determine size and morphology of synthesized drugs conjugated AgNPs. AFM results showed complete agreement with UV−Vis spectrophotometric data.

Drugs coated AgNPs were found to be spherical and polydispersed in the wide size range of 10−90 nm. Drugs Conjugated with AgNPs Exhibited Increased Amoebicidal Effects against Naegleria fowleri Compared with Drugs Alone. Amoebicidal assays were performed to establish effects of drugs conjugated with AgNPs versus drugs alone on N. fowleri viability. The results revealed that drugs conjugated with AgNPs are more effective against N. fowleri as compared to drugs alone. Amphotericin B conjugated with AgNPs exhibited significant amoebicidal effects against N. fowleri at 2.5 μM, as the number N. fowleri was reduced from 9.2 × 105 to 2 × 105 (P < 0.05 using two sample t test and two-tailed distribution) (Figure 1). When amoebae were incubated with Amp B alone, the number of amoebae were reduced to 3.7 × 105 (P < 0.05 using two sample t test and two-tailed distribution) C

DOI: 10.1021/acschemneuro.7b00430 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 3. Pretreatment of N. fowleri with silver nanoparticles-conjugated Amphotericin B, Nystatin, and Fluconazole inhibited parasite-mediated HeLa cell cytotoxicity. Briefly, amoebae (5 × 105 amoebae/0.5 mL/well) were incubated with different concentrations of Ag-conjugated drugs and drugs alone and then added to HeLa cells monolayers for 24 h at 37 °C in a 5% CO2 incubator as described in Methods. The results showed that the drugsconjugated AgNPs significantly inhibited parasite-mediated host cell cytotoxicity compared with drugs alone (*P < 0.05 using two sample t test; two tailed distribution). The results are representative of several experiments performed in duplicates. The data are presented as the mean ± standard error.

limited treatment options available in the management and treatment of infections caused by N. fowleri, it is hoped that metal nanoparticles can be used as potential drug carriers for improved therapies in biological systems. In vivo studies and especially administration of these drugs coated AgNPs via different routes of administration such as intravenous, intranasal, and intrathecal for assessing their maximum biological potential are areas of interest for future studies.

(Figure 1). Moreover, Nys-AgNPs showed significant amoebicidal effects at micromolar concentrations compared with the drugs alone. The number of viable N. fowleri was reduced from 9.2 × 105 to 7.5 × 105 with Nys alone and 5.8 × 105 with AgNPsNys (P < 0.05 using two sample t test and two tailed distribution) (Figure 1). In contrast, Fluconazole conjugated AgNPs revealed limited amoebicidal effects (Figure 1). On the contrary, treatment with AgNPs alone had no effects on amoebae viability. Silver Nanoparticles-Conjugated Drugs Inhibited N. fowleri Mediated Host Cell Cytotoxicity. To determine whether AgNPs conjugated drugs inhibit amoebae-mediated host cell damage, assays were performed. When incubated with host cells, N. fowleri alone produced host cell damage (Figure 2). On the other hand, amoebae pretreated with antiamoebic drugs as well as drugs conjugated with AgNPs produced nominal host cell damage (Figure 2). We also used host cell cytotoxicity assays as a secondary screen to test their practicality as antiamoebic agents. N. fowleri produced 75% cytotoxicity to host cells, while pretreatment of amoebae with drugs-conjugated with AgNPs resulted in reduced host cells cytotoxicity (Figure 3). Since these drugs suffer from poor bioavailibility due to amphipathic nature and relatively large molecular size,10,11 the enhanced effects of their nanoconjugates are most likely to be due to increased bioavailibility and effective concentration at specific targets against N. fowleri. Size and surface properties play pivotal roles in the physicochemical effects of nanoparticles. The antimicrobial potential of AgNPs has been associated with multiple factors including generation of reactive oxygen species (ROS) and free radicals to alter DNA functions, and interaction with cell walls to transpose toxic ions.12 Future studies are needed to determine the translational value of these findings by testing drugs alone versus AgNPs-conjugated drugs in the treatment of PAM in vivo. In conclusion, these findings clearly showed that silver nanoparticles conjugation enhanced anti-amoebic effects of drugs likely due to their small size and high drug loading ability. Nys-AgNPs and Amp-AgNPs were found to be most effective against N. fowleri as compared with drug alone. As there are

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METHODS

Chemicals. Chemicals used in the synthesis of drugs coated nanoparticles include Silver nitrate, Sodium borohydride (Merck), Amphotericin B, Nystatin, and Fluconazole (Sigma-Aldrich). All chemicals used in the synthesis of drugs coated silver nanoparticles (AgNPs) were of analytical grade unless otherwise stated. Synthesis of Drugs Coated with AgNPs. AgNPs were conjugated with drugs; Amphotericin B (Amp), Nystatin (Nys), and Fluconazole (Flu) coated AgNPs were synthesized by procedure reported previously.7,8 Briefly, 5 mL (0.1 mM) Amphotericin B aqueous solution was stirred with 5 mL (0.1 mM) silver nitrate aqueous solution, followed by addition of freshly prepared 30 μL (4 mM) sodium borohydride aqueous solution as reducing agent. The color of solution turned yellowbrown instantly upon addition of sodium borohydride indicating the reduction of silver ions and formation of Amp-AgNPs. The reaction mixture was further stirred for 1 h to ensure complete utilization of starting materials and maximizing yield. Similar procedure was repeated for Nys-AgNPs and Flu-AgNPs by optimizing different volume/volume (v/v) ratios of silver solution and drugs. Stable Nys-AgNPs and FluAgNPs were obtained at respective volume ratio of silver to drug at 4:1 and 1:1, again the reducing agent was sodium borohydride. Unprotected bare AgNPs were also obtained by same procedure in the absence of drugs prior to use in biological assays. The synthesized nanoparticles were washed by deionized water after centrifuging at 10 000g for 20 min to remove excess unbound drug and side products from colloidal suspension. All synthesized nanoparticles were subjected to characterization using a UV−vis spectrophotometer (Evolution 300, Thermo Scientific), and atomic force microscopy (AFM) (Agilent 5500) as described previously.7,8 HeLa (Henrietta Lacks) Cell Lines and Culture Conditions. HeLa cervical cancer cells were obtained from ATCC (CCL-2) and D

DOI: 10.1021/acschemneuro.7b00430 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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cultured in RPMI-1640 supplemented with 10% fetal bovine serum 1% L-glutamine, 1% minimal essential media nonessential amino acid (MEM NEAA), and 1% penicillin−streptomycin. The cells were maintained in a 5% CO2 incubator at 37 °C. For cytotoxicity assays, HeLa cells were cultured in 24-well plates by inoculating 5 × 105 cells per well per mL and incubating at 37 °C in a 5% CO2 incubator, which resulted in the formation of complete monolayers within 48 h. Cultures of Naegleria fowleri. Naegleria fowleri isolated from the cerebrospinal fluid of a patient was obtained from American Type Culture Collection (ATCC 30174) and routinely cultured on HeLa cell monolayers as feeder layers at 37 °C containing 5% CO2. The amoeba consumed HeLa cells within 48 h and produced approximately 5 × 105 amoebae with 95% in the trophozoite form. Amoebicidal Assays. The amoebicidal potential of drugs alone, drugs conjugated with AgNPs, and AgNPs alone were determined as previously described.13 Briefly, 5 × 105 N. fowleri per well were incubated with various concentrations of drugs or relevant amounts of solvent and AgNPs alone in RPMI-1640 in 24-well plates. For controls, dH2O alone was used as a negative control while Amphotericin B was used as a positive control. Plates were incubated at 37 °C for 24 h. The viability of amoebae was determined by adding 0.1% Trypan blue to each well and counting live (nonstained) N. fowleri cells using a hemocytometer. The data are represented as the mean ± standard error of at least three independent experiments performed in duplicate. Host Cell Cytotoxicity Assays. Amoebae-mediated HeLa cells cytotoxicity was evaluated as described previously.14,15 Briefly, assays were performed in 96-well plates containing confluent HeLa monolayers. Amoebae (8 × 104) were treated with drugs conjugated with AgNPs, AgNPs alone, and drugs alone at different concentrations, followed by 20 h incubation at 37 °C. Samples were centrifuged at 5000g for 1 min to remove supernatant, and the pellet was resuspended in 200 μL of fresh RPMI before incubating with HeLa cell monolayers at 37 °C in a 5% CO2 incubator for 24 h and observed for cytotoxic effects. At the end of this incubation period, supernatants were collected and cytotoxicity was detected by measuring lactate dehydrogenase (LDH) release using Cytotoxicity Detection kit (Invitrogen) as follows: % cytotoxicity = (sample value − control value)/total LDH release − control value) × 100. Control values were obtained from host cells incubated in RPMI-1640 alone. Total LDH release was determined from HeLa cells treated with 1% Triton X-100 for 30 min at 37 °C. The basis of this assay is that cell supernatant containing LDH catalyzes the conversion of lactate to pyruvate, generating NADH and H+. In the second step, the catalyst (diaphorase, solution from kit) transfers H and H+ from NADH and H+ to the tetrazolium salt p-iodo-nitrotetrazolium violet (INT), which is reduced to formazon (dye), and absorbance is read at 492 nm.

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The authors declare no competing financial interest.

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REFERENCES

(1) Pugh, J. J., and Levy, R. A. (2016) Naegleria fowleri: Diagnosis, pathophysiology of brain inflammation, and antimicrobial treatments. ACS Chem. Neurosci. 7, 1178−1179. (2) Visvesvara, G. S. (2013) Infections with free-living amebae. Handb. Clin. Neurol. 114, 153−168. (3) Martinez, A. J., and Visvesvara, G. S. (1997) Free-living, amphizoic and opportunistic amebas. Brain Pathol. 7, 583−598. (4) Siddiqui, R., Ali, I. K., Cope, J. R., and Khan, N. A. (2016) Biology and pathogenesis of Naegleria fowleri. Acta Trop. 164, 375−394. (5) Siddiqui, R., and Khan, N. A. (2014) Primary amoebic meningoencephalitis caused by Naegleria fowleri: An old enemy presenting new challenges. PLoS Neglected Trop. Dis. 8, e3017. (6) Bhamra, R., Sa’ad, A., Bolcsak, L. E., Janoff, A. S., and Swenson, C. E. (1997) Behavior of amphotericin B lipid complex in plasma in vitro and in the circulation of rats. Antimicrob. Agents Chemother. 41, 886− 892. (7) Anwar, M. F., Yadav, D., Kapoor, S., Chander, J., and Samim, M. (2015) Comparison of antibacterial activity of Ag nanoparticles synthesized from leaf extract of Parthenium hystrophorus L in aqueous media and Gentamicin sulphate in vitro. Drug Dev. Ind. Pharm. 41, 43− 50. (8) Aqeel, Y., Siddiqui, R., Anwar, A., Shah, M. R., and Khan, N. A. (2016) Gold nanoparticle conjugation enhances the antiacanthamoebic effects of Chlorhexidine. Antimicrob. Agents Chemother. 60, 1283−1288. (9) Ho, B. N., Pfeffer, C. M., and Singh, A. T. K. (2017) Update on nanotechnology-based drug delivery systems in cancer treatment. Anticancer Res. 37, 5975−5981. (10) Xu, J., Zhang, S., Machado, A., Lecommandoux, S., Sandre, O., Gu, F., and Colin, A. (2017) Controllable microfluidic production of drugloaded PLGA nanoparticles using partially water-miscible mixed solvent microdroplets as a precursor. Sci. Rep. 7, 4794. (11) Radwan, M. A., AlQuadeib, B. T., Šiller, L., Wright, M. C., and Horrocks, B. (2017) Oral administration of amphotericin B nanoparticles: antifungal activity, bioavailability and toxicity in rats. Drug Delivery 24, 40−50. (12) Dakal, T. C., Kumar, A., Majumdar, R. S., and Yadav, V. (2016) Mechanistic basis of antimicrobial actions of silver nanoparticles. Front. Microbiol. 7, 1831. (13) Sissons, J., Alsam, S., Stins, M., Rivas, A. O., Morales, J. L., Faull, J., and Khan, N. A. (2006) Use of in vitro assays to determine effects of human serum on biological characteristics of Acanthamoeba castellanii. J. Clin Microbiol. 44, 2595−2600. (14) Khan, N. A., and Siddiqui, R. (2009) Acanthamoeba affects the integrity of human brain microvascular endothelial cells and degrades the tight junction proteins. Int. J. Parasitol. 39, 1611−1616. (15) Siddiqui, R., Abjani, F., Yeo, C. I., Tiekink, E. R., and Khan, N. A. (2017) The effects of phosphanegold(I) thiolates on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype. J. Negat. Results Biomed. 16, 6.

AUTHOR INFORMATION

Corresponding Author

*Mailing address: Department of Biological Sciences, School of Science and Technology, Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-8622. Ext: 7169. Fax: 60-(0)3-56358630. E-mail: [email protected]. ORCID

Naveed Ahmed Khan: 0000-0001-7667-8553 Author Contributions

R.S. and N.A.K. conceived the study. A.A. synthesized and characterized the materials under the supervision of N.A.K. K.R. planned and carried out experiments and analyzed the data under the supervision of R.S. K.R. prepared the first draft of the manuscript. R.S. corrected the original manuscript. All authors approved the final manuscript. Funding

This work was supported by the University Research Grant Scheme No. 2017-04, Sunway University, Malaysia. E

DOI: 10.1021/acschemneuro.7b00430 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX