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

Abstract

The use of quantum dots (QDs) for nanomedicine is hampered by their potential toxicologic effects and difficulties with delivery into the cell interior. We accomplished an in vivo study exploiting Daphnia magna and Xenopus laevis to evaluate both toxicity and uptake of QDs coated with the membranotropic peptide gH625 derived from the glycoprotein H of herpes simplex virus and widely used for drug delivery studies. We evaluated and compared the effects of QDs and gH625-QDs on the survival, uptake, induction of several responsive pathways and genotoxicity in D. magna, and we found that QDs coating plays a key role. Moreover, studies on X. laevis embryos allowed to better understand their cell/tissue localization and delivery efficacy. X. laevis embryos raised in Frog Embryo Teratogenesis Assay-Xenopus containing QDs or gH625-QDs showed that both nanoparticles localized in the gills, lung and intestine, but they showed different distributions, indicating that the uptake of gH625-QDs was enhanced; the functionalized QDs had a significantly lower toxic effect on embryos' survival and phenotypes. We observed that D. magna and X. laevis are useful in vivo models for toxicity and drug delivery studies.

Keywords: blood–brain barrier; delivery; genotoxicity; membranotropic peptide; nanoparticles.

Figure 1

UV/Vis spectra of gH625-QDs and QDs alone. Abbreviations: QDs, quantum dots; UV/Vis, ultraviolet/visible.

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.

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.