Abstract
Purpose :
Acanthamoeba is a free-living amoeba known to cause rare but severe sight-threatening eye infection. The diagnosis of Acanthamoeba keratitis remains challenging, as early signs of this disease mimic other forms of keratitis. Our aim was to determine whether mannose-conjugated carbon quantum dots could be used to label Acanthamoeba trophozoites, and their safety on the L929 mouse fibroblasts.
Methods :
Mannose-conjugated carbon quantum dots (MCQDs) were synthesized using a reductive amination reaction. MCQDs were characterized using transmission electron microscopy, X-ray photo spectroscopy, and zeta sizer. MCQDs prepared at a weight ratio of 1 part of mannose to 10 parts of carbon quantum dots were incubated with five different stains of Acanthamoeba trophozoites at a concentration of 150, 200 and 300 µg/mL for 2 hours. Unbound MCQDs were washed by centrifugation and trophozoites resuspended in 1/4th Ringer’s solution. Trophozoites were examined for fluorescence under epifluorescence microscopy at an excitation wavelength of 460-490 nm and observed using an FITC filter with a 515-550 nm wavelength. Images were captured at 10X and 40X magnification. The fluorescence intensity was quantified using ImageJ. The cytotoxicity of the MCQDs was assessed using L929 mouse fibroblasts at concentrations ranging from 7.81 to 1000 µg/mL.
Results :
MCQDs were quasi spherical shape with size <10nm. All five strains of Acanthamoeba trophozoites exhibited bright fluorescence with MCQDs at 300 µg/mL. The fluorescence uptake percentage by no of trophozoites ranged between 56 to 70% with no statistical significance between the strains (p>0.05). There was a statistically significant difference noted in the overall fluorescence intensities among the Acanthamoeba strains tested (p<0.0001). Specifically, A. castellanii 112 showed a statistically significant difference in fluorescence intensities compared to all other strains except A. culberstoni (p<0.05). MCQDs exhibited low levels of cytotoxicity between 6-20% at concentrations ranging between 7.81 to 1000 µg/mL, which are below 30% cut off of ISO-10993-5:2009.
Conclusions :
MCQDs can be used to label five different Acanthamoeba strains and demonstrated minimal cytotoxicity on L929 mouse fibroblasts. These findings suggest that MCQDs have the potential to be further explored as a useful fluorophore for diagnosing Acanthamoeba keratitis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.