Daphnia magna and Xenopus laevis as in vivo models to probe toxicity and uptake of quantum dots functionalized with gH625

Figures & data

Table 1 Size measured by DLS, expressed as z-average and PDI

NPs	Average size (nm)	PDI
QDs	120.90±2.37	0.35±0.11
gH625-QDs	171.40±1.27	0.35±0.05

Note: Data are expressed as mean ± SD of three separate experiments for each of the two batch formulations, with at least 13 measurements for each.

Abbreviations: NPs, nanoparticles; PDI, polydispersity index; QDs, quantum dots; SD, standard deviation; DLS, dynamic light scattering.

Figure 1 UV/Vis spectra of gH625-QDs and QDs alone.

Abbreviations: QDs, quantum dots; UV/Vis, ultraviolet/visible.

Table 2 Mean effective concentration from toxicity tests (95% CI shown in brackets) against exposure to QDs and gH625-QDs for 48 h

Sample	EC50 (95% CI) nM	EC20 (95% CI) nM	EC10 (95% CI) nM
QDs	1.5 (0.01–0.16)	ND	ND
gH625-QDs	ND	4.4 (2.5–4.5)	1.0 (0.6–1.01)

Abbreviations: CI, confidence interval; EC₅₀, concentration incurring 50% effect of test species; QDs, quantum dots; ND, not determined.

Figure 2 Immobility of Daphnia magna exposed to QDs and gH625-QDs for 48 h; dashed line represents confidence intervals.

Notes: The concentrations reported are relative to QDs (the concentration of 10 nM QDs corresponds to a peptide concentration of 43 µM). Data are reported as mean ± SEM (n=9). Bonferroni post hoc test following two-way ANOVA versus the QDs group. All the data present significant differences (P<0.001).

Abbreviations: ANOVA, analysis of variance; QDs, quantum dots; SEM, standard error of the mean.

Figure 3 Comet assay. Histogram of DNA damage of Daphnia magna added to QD, with or without gH625. Results are expressed as mean ± SD (P<0.05).

Abbreviations: QD, quantum dot; SD, standard deviation; Ctl, control.

Figure 4 Observed heart rate of Daphnia magna after exposure to QDs and QDs-gH625. The results are relative to average of beats ± SD.

Abbreviations: bpm, beats per min; QD, quantum dot; SD, standard deviation; Ctl, control.

Figure 5 Fluorescence image of Daphnia magna after exposure (30′, 60′, 90′, 6 h and 24 h) to QDs and QDs-gH625 (5 nM).

Abbreviations: QD, quantum dot; Ctl, control.

Figure 6 Response of Daphnia magna to oxidative stress on exposure to QDs and gH625-QDs for 24 h.

Notes: (A) ROS production by dichlorofluorescein fluorescence. Data are given as fluorescence values (mean ± SEM [n=9]). *P<0.05, **P<0.01 and ***P<0.001, Tukey post hoc test following one-way ANOVA versus the control group (Ctl). (B) CAT activity is expressed as units* mg⁻¹ of protein mean ± SEMs (n=9). *P<0.05, **P<0.01 and ***P<0.001, Bonferroni post hoc test following two-way ANOVA versus the QDs group. (C) SOD activity is given as percentage of SOD inhibition compared to the control mean ± SEMs (n=9). *P<0.05, **P<0.01 and ***P<0.001, Bonferroni post hoc test following two-way ANOVA versus the QDs group. All the data show significant differences (P<0.05) compared to the control group (Ctl).

Abbreviations: ANOVA, analysis of variance; CAT, catalase; QD, quantum dot; ROS, reactive oxygen species; SD, standard deviation; SEM, standard error of the mean; SOD, superoxide dismutase.

Table 3 Embryotoxic effects in Xenopus laevis

	Ctl	QDs	gH625-QDs
Utilized embryos (n)	63	63	63
Dead embryos (n)	13	10	13
Living larvae (n)	50	53	52
Mortality (%)	20	16	18

Note: The concentration used was 117 nM.

Abbreviations: Ctl, control; QDs, quantum dots.

Figure 7 Embryotoxic parameters in Xenopus laevis.

Notes: (A) Mortality evaluation. The mortality distributions were evaluated by nonparametric Mantel–Cox test, which gave P>0.05. (B) Growth retardation analysis. The growth retardation was evaluated with ANOVA statistical test, which gave P>0.05. (C) Observed heart rate of Xenopus laevis after exposure to QDs and QDs-gH625. The heartbeat was evaluated with ANOVA statistical test, which gave P>0.05. (D) Analysis of dorsal pigmentation. The dorsal pigmentation was evaluated with ANOVA statistical test, which gave P>0.05. Data are reported as mean ± SD.

Abbreviations: ANOVA, analysis of variance; QDs, quantum dots; SD, standard deviation; Ctl, control.

Figure 8 gH625-QDs confocal localization in stage 46 Xenopus laevis embryos.

Notes: (A) Control section. (B, C) Optical sections of primordium of lung treated with (B) naked QDs and (C) gH625-QDs. QDs localize in the form of widespread dots (B, asterisks) in contrast gH625-QDs, which are disposed in small fluorescent areas (C, arrows). In the intestine (D–F), gH625-QDs are visible only in some stretches: st, du and il. Hindbrain shows a slight localization of gH625-QDs (G, asterisks). (H) QDs are barely visible in the gills (I) gH625-QDs have a distribution similar to the gills (arrows). (J–L) QDs and gH626-QDs are not visible in the heart. Bars: (A–C, G, I) =20 µm; (E, F, K, J, L) =50 µm; (H) =100 µm; (D) =200 µm.

Abbreviations: du, duodenum; il, ileum; QDs, quantum dots; st, stomach.