Examples of different phenotypes of zebrafish embryos at 24 h post fertilization (hpf) found in the bioassay described in the Section 2.6 ?Toxicity?. (a) Normally developed embryo; (b) embryo with underdeveloped eye, brain and impaired extensions of yolk sack and tail; and (c) dead embryo. Ear; eye; y: yolk; ye: yolk extension; V-s: v-shaped somite; U-s: abnormally-shaped somite; tb: tail bud; and de: decomposing embryo. Scale bar equals 1 mm.

PXRD patterns for Caf@CD obtained by three methods (1: cogrinding; 2: cogrinding with the addition of water drops; and 3: depositing from soln.). (a) ?-CD; (b) ?-CD; and (c) ?-CD series.

FT-IR spectra for Caf@CD obtained by three methods (1: cogrinding; 2: cogrinding with the addition of water drops; and 3: depositing from soln.). (a) ?-CD; (b) ?-CD; and (c) ?-CD series.

FT-IR spectra for Caf@CD obtained by three methods (1: cogrinding; 2: cogrinding with the addition of water drops; and 3: depositing from soln.). (a) ?-CD; (b) ?-CD; and (c) ?-CD series.

Raman spectra for Caf@CD obtained by three methods (1: cogrinding; 2: cogrinding with the addition of water drops; and 3: depositing from soln.). (a) ?-CD; (b) ?-CD; and (c) ?-CD series.

Raman spectra for Caf@CD obtained by three methods (1: cogrinding; 2: cogrinding with the addition of water drops; and 3: depositing from soln.). (a) ?-CD; (b) ?-CD; and (c) ?-CD series.

The most stable modeled structures: Caf@?-CD (upper); Caf@?-CD (middle); and Caf@?-CD (lower). Atoms: C (grey); H (white); O (red); and N (blue). The cyan and pink dashed lines represent possible hydrogen bonds and close contacts stabilizing the complexes, respectively.

The most stable modeled structures: Caf@?-CD (upper); Caf@?-CD (middle); and Caf@?-CD (lower). Atoms: C (grey); H (white); O (red); and N (blue). The cyan and pink dashed lines represent possible hydrogen bonds and close contacts stabilizing the complexes, respectively.

The effect of Caf@CDs on early development of the zebrafish embryo. (a) Images of zebrafish embryos after exposure to caffeine (Caf), CDs or Caf@CDs from 4 till 24 hpf. Embryos exposed to Caf@?-CD show underdeveloped eyes, ears and severely impaired convergent extension. Less severe morphological abnormalities were also present in embryos exposed to 5 mM Caf. Exposure to 5 mM ?-CD, 1.25 mM Caf@?-CD, or 5 mM Caf@?-CD was lethal to the embryos. Examples of live embryos at a most advanced developmental stage are shown. Scale bar, 1 mm. (b) Changes in the proportion between the dead, morphologically abnormal (affected), and unaffected (normal) embryos. The percentage of embryos which died upon treatment with Caf or Caf@CDs was significantly higher than that of untreated control. Also, pure ?-CD was more toxic than the other two CDs, whereas ?-CD and ?-CD had no effect on both the fish survival and their morphology. In order to reduce the number of animals, in the case of treatments which caused 100% rapid mortality, the experiments were repeated twice. Bars present mean values, n is the number of repetitions, each on 24 embryos. Mann-Whitney U test was performed on samples with ?3 biological replicas. Statistically significant differences (p < 0.01) were marked with different letters.