Zinc Oxide Nanoconjugates against Brain-Eating Amoebae

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.

As stated earlier, amoebae are located everywhere in the environment; hence, a concept referred to as "One Health" comes into play. One health considers the interconnection between the environment, humans, and animals indicating that the health each of these elements influences that of the others [27,28].
Recently, nanotechnology has evolved and attracted the interest of the scientific community due to possibility to manipulate various physio-chemical properties of different compounds [29,30]. Specifically, nanoparticles are gaining interest due to their small size which allows their use for targeted delivery, which in turns can enhance the potency of drugs and reduce their toxicity [31]. Zinc is an essential metal element, believed to have various biochemical and physiological functions. In fact, Zinc ions are capable of causing oxidative stress and damage proteins, DNA, and lipids, inhibiting the function of respiratory enzymes and promoting free radical formation [32]. Interestingly, the use of Zinc oxide can be traced back to 2000 BC when it was used as a form of ointment to treat boils and injuries [33,34]. Moreover, Zinc oxide is greatly selective and is a semi-conductor. Researchers have tested Zinc oxide for its antibacterial properties. According to the results obtained, nano-Zinc oxide exhibited potent antibacterial effects [32,35]. In fact, a group of researchers have tested Polypyrrole-Zinc oxide against the free-living amoeba A. castellanii. This nanoconjugate exhibited potent amoebicidal effects against A. castellanii [31]. In this study, Zinc oxide nanoparticles were conjugated with ampicillin, Amphotericin B, Naringin, Quercetin, and Ceftrixon and tested against N. fowleri and B. mandrillaris. The aim of the study was to evaluate the activity of the compounds via various assays including amoebicidal, cytotoxicity and cytopathogenicity tests to determine their anti-amoebic properties.

Materials and Methods
The preparation of ZnO nanoparticles and beta-cyclodextrin-capped ZnO NPs was performed as previously described [30]. Briefly, the compounds Quercetin (QT), Naringin (NAR), and beta-Cyclodextrin (BCD) were obtained from Sigma-Aldrich, whereas the compounds Amphotericin (AMB), Ampicillin (AMPi) and Ceftriaxone (CFT) were obtained from Merck; solutions were prepared using deionized water [30]. The Zinc oxide nanoparticles were produced using the direct precipitation method, utilizing Zinc acetate dihydrate and NaOH as precursors [30].

Henrietta Lacks (HeLa) Cervical Cancer Cells
Henrietta Lacks (HeLa) Cervical Cancer Cells (HeLa) cells (ATCC, CCL-2, Singapore) were acquired from the American Type Culture Collection (ATCC) to allow the conduction of various assays such as cytotoxicity and cytopathogenicity assays, as well as a feeder layer to maintain N. fowleri and B. mandrillaris cultures on [4]. These cells were grown and preserved through their cultivation in complete medium. The complete medium was the Roswell Park Memorial Medium (RPMI) containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% minimum-essential-medium amino acids, and 1% L-glutamine [4]. Furthermore, flasks containing the cells were kept at 37 • C in an incubator with 5% CO 2 and 95% humidity [4].

Naegleria fowleri Culture
The HB1 ATCC 30174 strains of N. fowleri were obtained from the ATCC [4]. The amoebae were placed on HeLa cell monolayers which acted as a food source for the amoebae [4]. Additionally, the amoebae were maintained in a humidified incubator with 5% CO 2 and 95% humidity at a temperature of 37 • C [7]. After 48 h on the feeder layer, the amoebae had consumed the HeLa cell monolayer; hence, they increased in number up to approximately 5 × 10 5 , of which 95% were in trophozoite form [7].

Balamuthia mandrillaris Culture
The ATCC 30174 strains of B. mandrillaris were obtained from the ATCC [4]. The amoebae were placed on HeLa cell monolayers which acted as a food source [4]. Additionally, the amoebae were maintained in a humidified incubator with 5% CO 2 and 95% humidity at a temperature of 37 • C [7]. After 48 h on HeLa cells, the amoebae had consumed the HeLa cell monolayer; hence, they increased in number up to approximately 5 × 10 5 , of which 95% were in trophozoite form [7].

Amoebicidal Assay
We determined the antiamoebic effects of the examined compounds using amoebicidal assays [4]. In total, 2 × 10 5 amoeba were placed in a 96-well plate, obtaining a final volume of 200 µL. Next, the amoebae were treated with the drug combinations ZnO-CD, ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-NAR, ZnO-CD-AMB, and ZnO-CD-QT at 100 µg/mL, after which each 96-well plate was placed in a humidified incubator with 95% humidity and 5% CO 2 at 37 • C for 24 h.
Accurate results were ensured through the addition of positive and negative controls. Amoebae in RPMI alone served as the negative control, whereas amoebae treated with 0.25% SDS served as the positive control. Live and dead amoeba were identified through the addition of 0.1% methylene blue; the number of viable amoebae was determined by counting the living cells using a hemocytometer [4]. Additionally, compounds with significant activity were identified by conducting a Student's t-test with a two-tailed distribution [4]. Moreover, the MIC 50 values of the compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-NAR, ZnO-CD-AMB, and ZnO-CD-QT were determined using concentrations of 50 µg/mL, 100 µg/mL, and 150 µg/mL [36].

Cytotoxicity Assay
HeLa cells were cultured in 96-well plates and treated with the compounds ZnO-CD, ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-NAR, ZnO-CD-AMB, and ZnO-CD-QT at 100 µg/mL. Each 96-well plate was then placed in a humidified incubator with 95% humidity and 5% CO 2 at 37 • C for 24 h [4]. The following day, the supernatant was collected, and the toxicity of the compounds in the cells was determined through the use of a cytotoxicity detection kit. The kit measured the quantity of lactate dehydrogenase (LDH) release [37]. Accurate results were ensured by comparison to the positive and negative controls. Untreated HeLa cells represented the negative control, whereas HeLa cells treated with 1% Triton X-100 represented the positive control.
Finally, the cytotoxic properties were quantitatively determined with the formula: (absorbance of the medium from cells treated with the drugs − absorbance of the medium from negative control cells)/(absorbance of the medium from positive control cells − absorbance of the medium from negative control cells) × 100 [4].

Cytopathogenicity Assay
The amoebae-mediated host cell death was determined by treating 2 × 10 5 amoebae with the compounds ZnO-CD, ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-NAR, ZnO-CD-AMB, and ZnO-CD-QT at 100 µg/mL. Each plate was then placed in the incubator for 2 h at 37 • C with 5% CO 2 and 95% humidity. After 2 h, the treated amoebae were placed on confluent HeLa cell monolayers cultured in 96-well plates [4]. The plates containing the treated amoebae and HeLa cells were incubated for 24 h [4]. After the incubation, the supernatant was collected, and the compounds' cytotoxicity was determined [4].
Accurate results were ensured by the comparison with the negative and positive controls. The amoebae on the cells served as the negative control, while the cells treated with Triton X-100 served as the positive control [4].

Statistical Analysis
The data presented show the mean ± standard error of two different independent experiments carried out in duplicates for all assays. A two-tailed distribution t-test was conducted to determine the statistical significance of the results. Additionally, the p values were determined to further examine and elaborate the results [4].

Limited Cytotoxic Activity Was Observed against Human Cells
To determine the toxicity of the compounds, the lactate dehydrogenase (LDH) assay was conducted using human cells. The toxicity of the compounds against human cells was determined through the LDH assay. At a concentration of 100 µg/mL the compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT showed toxicity levels of 35.5%, 36.4%, 30.9%, 36.6%, and 35.6%, respectively, compared to the positive control (Triton X-100-treated cells, which showed 100% toxicity), as reported in Figure 2. Furthermore, based on the data obtained from the LDH assay and in accordance with the ISO 10993-5, the compounds appeared to possess weak cytotoxic activity, since it was  Table 2). According to the International Organization for Standardization (ISO) 10993-5, if cell viability is between 60% and 80%, then a compound has limited cytotoxic activity [38,39].
was conducted using human cells. The toxicity of the compounds against human cells was determined through the LDH assay. At a concentration of 100 µg/mL the compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT showed toxicity levels of 35.5%, 36.4%, 30.9%, 36.6%, and 35.6%, respectively, compared to the positive control (Triton X-100-treated cells, which showed 100% toxicity), as reported in Figure 2. Furthermore, based on the data obtained from the LDH assay and in accordance with the ISO 10993-5, the compounds appeared to possess weak cytotoxic activity, since it was between 30.9% and 36.6% (Figure 2, Table 2). According to the International Organization for Standardization (ISO) 10993-5, if cell viability is between 60% and 80%, then a compound has limited cytotoxic activity [38,39].

All Compounds Promoted a Decrease in Amoeba-Mediated Cytotoxicity in Human Cells
The impact of the compounds on amoeba-mediated host cell death was determined through cytopathogenicity assays. As described earlier, the amoebae were pre-treated with the compounds, following which they were introduced to the human cells. After 24 h, the amoeba-mediated host cell death was determined, and the compounds were found to reduce amoeba-mediated cytotoxicity in human cells.

Discussion
Since nanoparticles are believed to enhance drug efficiency, in previous work, various drugs were conjugated with different nanoparticles and evaluated against free-living amoebae. This method appeared efficient. In a previous study, Guanabenz was conjugated with silver and gold nanoparticles. The conjugated drug was found to possess significant amoebicidal activity against N. fowleri and A. castellanii [40]. Additionally, it was found that when Diazepam, Phenobarbitone, and Phenytoin were conjugated with silver nanoparticles, they exhibited greater effects against A. castellanii and N. fowleri compared to the drugs alone [41]. Although nanotechnology has only recently gained popularity, Zinc oxide was used as a form of treatment against boils and injuries thousands of years ago. In fact, the use of Zinc oxide dates back to 2000 BC [33]. Hence, Zinc-oxide has been evaluated against various microorganisms to determine its antimicrobial activity. For example, previous works tested drugs conjugated with Zinc oxide nanoparticles against a range of multidrugresistant bacteria and were found to possess anti-bacterial activity [30]. Furthermore, due to the antibacterial properties Zinc-oxide nanoparticles possess, they have been considered for use in food packaging to fight foodborne diseases [42]. Additionally, Zinc oxide nanoparticles have been found to possess antifungal activity against Botrytis cinerea and Penicillium expansum [43]. Moreover, Zinc oxide nanoparticles are also believed to be able to preserve food and nutritional supplements by acting as antimicrobials [44].
In this study, Zinc oxide-conjugated compounds were tested for their amoebicidal properties. Through amoebicidal assays, the antiamoebic properties of the tested compounds were determined. The compounds ZnO-CD-AMPi, ZnO-CD-CFT, ZnO-CD-Nar, ZnO-CD-AMB, and ZnO-CD-QT were found to exhibit significant amoebicidal effects against both N. fowleri and B. mandrillaris; the greatest reduction in amoeba viability was achieved with the compound ZnO-CD-AMPi, which reduced B. mandrillaris viability to 40.9% and N. fowleri viability to 35.5% with respect to a negative control. The smallest antiamoebic effects were exhibited by the conjugated compounds ZnO-CD-Nar against N. fowleri, with a reduction of amoeba viability to 52% with respect to the control (100% viability), and ZnO-CD-QT, which showed the smallest anti-amoebic effects against B. mandrillaris, reducing amoeba viability to 62.4% with respect to the control; nonetheless, the effects were significant according to the statistical analysis we conducted. Moreover, the compounds were found to reduce amoeba-mediated host cell death. This was determined through cytopathogenicity assays. It was found that ZnO-CD-AMB promoted the greatest reduction in N. fowleri-mediated host cell pathogenicity, whereas ZnO-CD-NAR induced the greatest reduction in B. mandrillaris-mediated host cell pathogenicity. Additionally, all compounds were shown to possess limited cytotoxicity when tested against human cells, as human cell viability in the presence of these compounds was greater than 60%. Moreover, the MIC 50 of the compounds were determined. The lowest concentration necessary to inhibit N. fowleri growth by 50% was determined for ZnO-CD-AMPi, whereas, for B. mandrillaris, the lowest concentration necessary to inhibit amoeba growth by 50% was found for ZnO-CD-CFT.
The possible mechanism of action of these compounds in B. mandrillaris and N. fowleri are not known. However, Zinc oxide nanoparticles are believed to exhibit antimicrobial activity through the alteration of the cell wall and membrane permeability by the production of reactive oxygen species in bacteria [42]. Additionally, in a study conducted on the bacterium Campylobacter jejuni, it was reported that a possible mechanism of action of ZnO against the bacterium is based on interactions between the compound and cell surfaces, which may be direct or electrostatic in nature. Additionally, ZnO nanoparticles may undergo cellular internalization resulting in the production of active oxygen species in the cells [45]. The compounds may also be inducing apoptosis within the amoebae, as they were previously found to induce apoptosis in human cancer cells [46]. However, this is the subject of future studies.
The examined compounds should be tested against the cyst form of the amoebae, to determine their effects against B. mandrillaris and N. fowleri cysts. In vitro studies are necessary to determine the mechanism of action of these drugs. Next, in vivo studies need to be accomplished to understand the effects of these compounds in animal models, and translational research is urgently warranted in order to develop the much needed effective anti-amoebic drugs. These nanoconjugates could also be used as disinfectants against amoebae in water storage tanks, especially in developing countries where there is often scarcity of water; future, work is needed in this respect.

Conclusions
To conclude, Zinc oxide was conjugated with Ampicillin, Ceftrixon, Naringin, Amphotericin B, and Quericitin and was tested against amoebae to evaluate the antiamoebic properties of these conjugates. The compounds were found to exhibit significant amoebicidal properties against both Naegleria fowleri and Balamuthia mandrillaris, while exhibiting limited cytotoxic activity in human cells. Future studies will include the testing of the compounds against amoebae cysts, as well as the determination of their mechanisms of action; in vivo studies should be carried out in parallel. Moreover, the addition of these nanoconjugates to water storage tanks should be investigated.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.