HomeScienceAmoebicidal activity of cationic carbosilane dendrons derived with 4-phenylbutyric acid against Acanthamoeba...

# Amoebicidal activity of cationic carbosilane dendrons derived with 4-phenylbutyric acid against Acanthamoeba griffini and Acanthamoeba polyphaga trophozoites and cysts

### Acanthamoeba spp. clinical strains

A. polyphaga 2961 (kindly supplied by Dr. E. Hadas, Poznan University of Medical Sciences, Poland) and A. griffini MYP2004 (isolated by our research group, University of Alcalá, Spain) were used16. A. polyphaga 2961 was grown at 32 °C in Peptone Yeast Glucose broth supplemented with Bacto Casitone (PYG + B) and A. griffini MYP2004 was grown at 37 °C in CERVA16,17. The strains were maintained by weekly media changes.

### Dendritic compounds and standard treatment drugs used

Different generations of cationic carbosilane dendrons derived using 4-phenylbutyric acid (PBA) at the ArCO2Gn(SNMe3I)m focal point (n = 1, m = 2; n = 2, m = 4 and n = 3, m = 8) were studied and are referred to as ArCO2G1(SNMe3I)2 (1), ArCO2G2(SNMe3I)4 (2) and ArCO2G3(SNMe3I)8 (3), respectively (Fig. 1). The focal point is the location from which the reactive peripheral groups emerge to form the hyperbranched wedge. These systems were prepared as described by Lozano-Cruz et al., 2020. To perform combination assays, chlorhexidine digluconate (CLX) (Sigma-Aldrich Ltd., St. Louis, MO, USA) was considered the reference drug3.

### Amoebicidal activity assays against trophozoites

The three dendrons, ArCO2G1(SNMe3I)2 (1), ArCO2G2(SNMe3I)4 (2) and ArCO2G3(SNMe3I)8 (3) were evaluated against A. polyphaga 2961 and A. griffini MYP2004 trophozoites. Assays were prepared in 96-well microtiter plates treated with Poly-L-Lysine (CellStar, Greiner Bio-one).

Firstly, the inoculum was adjusted to 10,000 trophozoites (A. polyphaga 2961) and 15,000 trophozoites (A. griffini MYP2004) per well, based on a previous strain growth analysis performed in our laboratory. For this purpose, a Fuchs-Rosenthal© counting chamber (Optic Labor) and 0.2% Congo Red in distilled water (Congo Red, Sigma Aldrich) were used under an optic microscope (Carl Zeiss)17.

Secondly, the dendrons were twofold serially diluted from stock solutions to reach final concentrations ranging from 2 to 512 mg/L and 100 μL of each concentration were added to each well. Then, 100 μL of the adjusted trophozoite solution (2 × PYG + B and 2 × CERVA for A. polyphaga 2961 and A. griffini MYP2004, respectively) were also added. Plates were incubated at 32 °C for A. polyphaga 2961 and 37 °C for A. griffini MYP2004. Manual counting using the 0.2% Congo Red exclusion assay was performed at 24 and 48 h of treatment and percent viability was defined as: % viability = (mean treated/mean control) × 100. The minimum trophozoite amoebicidal concentration (MTAC) was defined as the lowest concentration of test solution that produced a complete reduction in trophozoite viability18.

Each drug concentration was tested in triplicate and in at least two independent experiments. A control well containing the amebae with no treatment and control wells of medium were included. All microtiter plates were sealed with Parafilm®. These procedures apply to all tests carried out in this study.

### Cysticidal activity assays

Cysts were obtained from ameba cultures in logarithmic phase under optimal growing conditions. The medium was replaced with Neff’s encystment medium and flasks were agitated for 9 days at room temperature17. A. polyphaga 2961 and A. griffini MYP2004 cyst assays were run in 96-well microtiter plates (Deltalab).

The inoculum was adjusted in Neff’s encystment medium, and 10,000 cysts were inoculated per well for both strains. The dendrons were twofold serially diluted to final concentrations ranging from 2 to 512 mg/L; 100 μL of each concentration were mixed with 100 μL of adjusted cyst solution. The dendron solutions were removed after 24 and 48 h of treatment, then each well was washed with 1 × PBS (10x, Sigma Aldrich, St. Louis, MO, USA) and culture medium was added (1 × PYG + B for A. polyphaga 2961 and 1 × CERVA A. griffini MYP2004). Plates were incubated at optimal growing conditions (32 °C for A. polyphaga 2961 and 37 °C for A. griffini MYP2004). Wells were observed three times per week with an inverted microscope (Motic AE21) for 21 days to visualize excystment and determine the minimum cysticidal concentration (MCC), defined as the lowest concentration that completely inhibits excystment and trophozoite growth18.

### Combined treatment against trophozoites

To perform these assays, the procedure was the same as for the amoebicidal activity assays described above. For this purpose, the checkerboard method was used19. The most effective dendron was tested in combination with CLX in a final volume of 200 μL (1:1 ratio). Required concentrations were achieved by serial dilution. Dendron concentrations ranged from 0.5 to 16 mg/L (< MTAC), while CLX concentrations ranged from 0.5 to 4 mg/L (< MTAC). Manual counting was performed after 24 and 48 h of incubation under optimal growing conditions to determine trophozoite viability compared to the untreated control.

To determine synergy, the fractional inhibitory concentration index (FICI) was calculated:

$$\textFICI \left( \textcombination \right) \, = \, [\textMTAC_(\textDendron + \text CLX) /\text MTAC_{\left( \textDendron \right)} \left] \, + \, \right[\textMTAC_{\left( \textCLX + \text Dendron \right)} /\text MTAC_(\textCLX) ]$$

The result was interpreted as synergistic when FICI was $$\le$$ 0.5, additive when 0.5 $$<$$ FICI $$\le$$ 1, indifferent when 1 $$<$$ FICI $$<$$ 4 and antagonistic when FICI $$\ge$$ 415.

### Combined treatment against cysts

Experiments were prepared as explained above for the cysticidal activity assays. Briefly, 100 μL of adjusted cyst inoculum was added to each microtiter plate well. The combination of the most effective dendron in concentrations ranging from 2 to 64 mg/L (MCC) and CLX, in concentrations from 1 to 8 mg/L, were added to each well (50 μL of each compound). After 24 and 48 h of treatment, the compounds were discarded. Wells were then washed twice with 1 × PBS (10x, Sigma Aldrich, St. Louis, MO, USA) and fresh medium was added. Finally, plates were incubated under optimal growing conditions. They were observed three times per week using an inverted microscope (Motic AE21) for 21 days to determine excystment. To determine synergy, the FICI was calculated as previously described.

### Cytotoxicity assay in HeLa cells

HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum and 1% of an antibiotic mixture. The inoculum was adjusted to 1 × 104 cells per well in a 24-well microtiter plate. Cells were grown for 3–4 days at 37 °C with 5% CO2 to reach confluence17. Then, the medium was discarded and 400 μL of the most effective dendron and CLX concentrations diluted in DMEM medium were added.

After 24 and 48 h of treatment, the medium was discarded once again, and the wells were washed twice with 1 × PBS (10x, Sigma Aldrich, St. Louis, MO, USA). After that, 500 μL of fresh medium and 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added (Sigma-Aldrich Ldt). The plates were incubated at 37 °C for 4 h. Then, the medium was discarded and 500 μL of DMSO were added20. Absorbance values were determined at the 570 nm wavelength using a spectrophotometer (BioTek Instruments Inc. Model: ELX 800).

Viability was calculated as (absorbance mean of treated/absorbance mean of non-treated) × 100. Values higher than 90% viability were considered non-cytotoxic, while values between 75 and 90% were considered low cytotoxicity. If viability descended to the range of 60–75%, it was deemed a moderate cytotoxic level. High cytotoxicity was established when viability values were lower than 60%21.

### Scanning electron microscopy (SEM)

SEM study was performed to evaluate the impact of the most effective dendron, CLX and combinations of these as previously described by our group16. To perform these studies, 200 μL of adjusted trophozoite suspension were placed on a glass coverslip. After 1 h of incubation under optimal growing conditions (32 °C for A. polyphaga 2961 and 37 °C for A. griffini MYP2004), the medium was discarded and treated for 24 or 48 h. Then, the medium was discarded, the wells were washed twice with PBS, and the fixative solution (2% glutaraldehyde and 1% CaCl2 in Milloning’s solution (NaH2PO4·H2O and NaOH)) was added for 1 h. Afterwards, the coverslips were washed with washing solution. The samples were dehydrated in increasing concentrations of ethanol (30, 50, 70, 95 and 100%) and finally in anhydrous acetone. Desiccation was performed in a critical point drying apparatus (Polaron E-3000), and the coverslips were mounted on aluminium stubs and coated with gold (Polaron E-5000/5100). The samples were examined using a Scanning Electron Microscope (JSM-IT500, JEOL) at the Medicine and Biology Research Support Center (University of Alcalá).

### Statistical analysis

For statistical analysis and graph generation, Microsoft Excel (Microsoft Office 365, Microsoft, Redmond, Washington, USA) and GraphPad Prism 8® (GraphPad Software, San Diego, California, USA) were used. Two-way ANOVA and One-way ANOVA (followed by Dunnett’s multiple comparisons test) were performed, and significance was established at p < 0.05. IC50 was obtained on GraphPad Prism 8® a using non-linear regression analysis22.

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