Zinc Oxide Nanoconjugates against Brain-Eating Amoebae

Abstract

Naegleria fowleri and Balamuthia mandrillaris are opportunistic protists, responsible for fatal central nervous system infections such as primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE) with mortality rates higher than 90%. Threatening a rise in cases is the increase in temperature due to global warming. No effective treatment is currently available. Herein, nanotechnology was used to conjugate Zinc oxide with Ampicillin, Ceftrixon, Naringin, Amphotericin B, and Quericitin, and the amoebicidal activity and host cell cytotoxicity of these resulting compounds were investigated. The compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT were found to reduce N. fowleri viability to 35.5%, 39.6%, 52.0%, 50.8%, 35.9%, and 69.9%, respectively, and B. mandrillaris viability to 40.9%, 48.2%, 51.6%, 43.8%, and 62.4%, respectively, when compared with their corresponding controls. Furthermore, the compounds reduced N. fowleri-mediated and B. mandrillaris-mediated host cell death significantly. Additionally, the compounds showed limited cytotoxicity against human cells; cell toxicity was 35.5%, 36.4%, 30.9%, 36.6%, and 35.6%, respectively, for the compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT. Furthermore, the minimum inhibitory concentrations to inhibit amoeba growth by 50% were determined for N. fowleri and B. mandrillaris. The MIC₅₀ for N. fowleri were determined to be 69.52 µg/mL, 82.05 µg/mL, 88.16 µg/mL, 95.61 µg/mL, and 85.69 µg/mL, respectively; the MIC₅₀ of the compounds for B. mandrillaris were determined to be 113.9 µg/mL, 102.3 µg/mL, 106.9 µg/mL, 146.4 µg/mL, and 129.6 µg/mL, respectively. Translational research to further develop therapies based on these compounds is urgently warranted, given the lack of effective therapies currently available against these devastating infections.

Keywords: Balamuthia mandrillaris; Naegleria fowleri; Zinc oxide; free-living amoebae; global warming; mortality; nanotechnology.

Figure 1

Conjugates of Zinc oxide showed significant amoebicidal properties against N. fowleri and B. mandrillaris. (a) At 100 µg/mL, significant amoebicidal effects of all compounds were recorded against N. fowleri after a 24 h incubation and (b) at 100 µg/mL, significant amoebicidal effects of all compounds were recorded against B. mandrillaris after a 24 h incubation. Data are illustrative of independent experiments and are presented as mean ± standard error. In addition, the p-values were determined through the conduction of a two-sample t-test, two tailed distribution; (*) is ≤ 0.05.

Figure 2

Limited cytotoxic activity was observed in human cells. Weak cytotoxic activity was determined upon the treatment of confluent monolayers of HeLa cells with the compounds at a concentration of 100 µg/mL.

Figure 3

All compounds led to a decrease in amoeba-mediated cytotoxicity in human cells. (a) All compounds reduced N. fowleri-mediated cytotoxic activity and (b) B. mandrillaris-mediated cytotoxic activity in human cells. After treatment with 100 µg/mL of the compounds for 2 h, 2 × 10⁵ amoebae were placed on HeLa cell monolayers overnight, and their cytotoxicity was measured the following day.