The use of nanoparticles both commercially and pharmaceutically has increased over the past decade, including fluorescent quantum dot nanoparticles (QD) in biochemical research for in vivo imaging. Previous studies have reported the toxic effects of nanoparticles, but their effects on larval metamorphosis and animal development and growth have not been thoroughly examined. Additionally, the method of uptake of nanoparticles by larval systems is unknown. Amphibian larvae are an ideal model for assessing toxicity because of their sensitivity to environmental contaminants and rapid and easily observable developmental stages. We used Anaxyrus fowleri tadpoles to investigate QD (≤ 25 nm diameter) integration into larvae and possible deleterious effects on their growth and development. Tadpoles (A. fowleri; n = 5/group) were placed in 24-well plates containing 1 mL of distilled water and increasing concentrations of QD (0, 1, and 2 nM) 72 h post-hatch. The fluorescence emission of QD in wells was detected at various time points (1, 2, 24, 48, and 72 h) using the in vivo imaging system (IVIS). A subset of tadpoles was killed (MS-222) and sectioned for histopathology. Remaining tadpoles were monitored throughout development. Fluorescence emission of QD in sectioned tadpoles was visualised using an EVOS Cell Imaging System. Developmental metrics of living tadpoles were recorded until metamorphosis. Fluorescence intensity between controls and dosage groups were analysed by ANOVA-1, followed by Student’s l.s.d. test to evaluate the effects of QD concentration and exposure time. The threshold of significance was P < 0.05. The rate of incorporation of QD into tadpoles was determined using the equation y = C + Ao*2(–x/t1/2), where t1/2 is the half-life of QD remaining in solution. The IVIS imaging revealed a rapid decrease of QD fluorescence (total flux) signals from the aqueous tadpole environment. Decreases in fluorescence occurred within 1 h post-exposure and appeared dose and time dependent, with signal nearly gone within 48 h. Half-life of total flux (time necessary for tadpoles to absorb half of the QD in solution) is 20.75 h (R2 = 0.92) and 2.54 h (R2 = 0.96) for 1 nm and 2 nm QD in solution, respectively. The EVOS imaging revealed integration of QD and localization into tadpole tissues. Fluorescence was exclusively found within the mouth, gills, and sections of the intestinal lumen of exposed tadpoles within the first hour. Dose-dependent increases in fluorescence within tissue were observed at each time-point. No signal was observed in controls. In remaining live tadpoles, QD treated tadpoles were smaller in size [t(34) = 2.35, P = 0.024] than controls. Findings reveal that (1) A. fowleri tadpoles integrate and accumulate nanoparticles, without detectable excretion within 72 h post-exposure, and (2) nanoparticles impede normal tadpole development. Ongoing studies are determining the effects of QD exposure on complete tadpole metamorphosis.
The work was supported by USDA-ARS Biophotonics Initiative grant #58–6402–3-018.
Nanotechnology: from In Vivo Imaging System to Controlled Drug Delivery
