Toxicity in vitro and in Zebrafish Embryonic Development of Gold Nanoparticles Biosynthesized Using Cystoseira Macroalgae Extracts

Figures & data

Table 1 Several Reaction Conditions Tested for the Biosynthesis of Au@CT

g Extract/mL H2O	[Au] (mM)
0.2	0.1
	0.2
	0.3
	0.4
	0.5
0.1	0.1
	0.2
	0.3
	0.4
	0.5

Abbreviation: [Au], gold concentration.

Figure 1 UV-Vis spectra of gold nanoparticles synthesized from C. tamariscifolia (Au@CT) under different reaction conditions (A and B). Time course absorbance measurements of Au@CT synthesized with CT extract at 0.1 g/mL and with 0.4 mM of gold (C).

Abbreviation: AU, arbitrary units.

Figure 2 Low magnification TEM images of gold nanoparticles synthesized with (A) 0.2 g/mL and (B) 0.1 g/mL of C. tamariscifolia extract and 0.4 mM of gold, and their corresponding size distribution histogram.

Figure 3 (A) HRTEM image of Au@CT; (B) Fourier capture showing the polycrystalline nature of Au@CT; (C) amplification of the selected area showing interplanar distance of the Au@CT crystalline structure, with the calculated d-spacing and corresponding Miller index.

Figure 4 (A) DF-STEM images showing Z-contrast, (B) elemental mapping image, (C) EDX and (D) EELS spectra of Au@CT.

Abbreviation: AU, arbitrary units.

Figure 5 FTIR spectra of CT extract and Au@CT, compared to fucoidan.

Abbreviation: AU, arbitrary units.

Table 2 In vitro Antioxidant Activity of Algae Extracts and Their AuNPs

	CT Extract	Au@CT	CB Extract	Au@CB
Reducing activity (mg ascorbic acid/g algae)	1783.6±60.2^a	1344.2±60.7^b	583.1±17.1^c	352.3±15.2^d
Total phenolic content (mg gallic acid/g algae)	9.26±0.34^a	6.15±0.21^b	2.41±0.09^c	2.47±0.21^c
DPPH scavenging activity (EC50 mg/mL)	3.66±0.02^a	3.13±0.11^a	12.65±0.18^b	30.20±0.80^c

Notes: Values represent average±standard error (n=3); For the same row, different superscript letters are significantly different (One-way ANOVA and Tukey’s multiple comparisons test, P<0.05).

Figure 6 MTT data on exposure of CB and CT extracts (A and B, respectively), and the correspondent Au@CB and Au@CT (C and D, respectively) to mammalian (L929, mouse) fibroblast cells, for 24 and 48 h.

Notes: Error bars represent mean ± SEM of three independent experiments. Different letters indicate significant differences among (nominal) test concentrations of the algae extracts and their AuNPs, independently.

Figure 7 Cumulative survival of zebrafish embryos exposed to CB and CT extracts (A and B, respectively), and the corresponding Au@CB and Au@CT (C and D, respectively), for 80 h_pf.

Table 3 Statistical Analysis of Lethal and Sub-Lethal Effects Detected on Zebrafish Embryonic Development Upon Exposure of CB or CT Extracts and Their AuNPs

Embryonic Development Parameters	Exposed to
	hpf	CB Extract	CT Extract	Au@CB	Au@CT
Epiboly	8	F(3,73)=2.923;P<0.05	F(3,76)=5.413;P<0.05	F(3,74)=2.291;P=0.085	F(3,74)=3.467;P<0.05
Spontaneous movements	32	χ^2=11.962;DF=3;P<0.05	χ^2=67.948;DF=3;P<0.05	χ^2=18.724;DF=3; P<0.05	χ^2=4.919;DF=3;P=0.178
Head-trunk angle	32	F(3,69)=1.203;P=0.315	F(2,53)=1.741;P=0.185	F(3,68)=3.110;P<0.05	F(3,71)=4.693;P<0.05
Pupil surface (co-variance: eye surface	32	F(3,69)=5.768;P<0.05	F(2,53)=3.890;P<0.05	F(3,67)=1.010;P=0.394	F(3,71)=5.038;P<0.05
Yolk volume (co-variance: egg volume)	32	F(3,69)=9.525;P<0.05	F(3,62)=99.641;P<0.05	F(3,70)=23.020;P<0.05	F(3,71)=1.638;P=0.188
Heart rate	32	F(3,53)=28.919;P<0.05	F(2,27)=13.867;P<0.05	F(3,55)=20.984;P<0.05	F(3,36)=6.041;P<0.05
Heart rate	56	F(3,46)=6.512;P<0.05	F(2,27)=1.570;P=0.226	F(3,49)=83.760;P<0.05	F(3,36)=20.95;P<0.05
Hatching rate	56	F(3,8)=2.505;P=0.133	F(2,9)=6.387;P<0.05	F(3,8)=8.497;P<0.05	F(3,12)=9.229;P<0.05
Yolk extension (co-variance: LT)	56	F(3,54)=0.392;P=0.759	F(2,21)=0.214;P=0.809	F(3,43)=1.257;P=301	F(2,25)=2.203;P=0.131
LT	56	F(3,55)=14.283;P<0.05	F(2,22)=0.641;P=0.536	F(3,44)=80.863;P<0.05	F(2,26)=2.292;P=0.121
Free swimming	80	χ^2=26.329;DF=3;P<0.05	F(2,9)=0.285;P=0.758	χ^2=75.243;DF=3; P<0.05	F(3,12)=0.475;P=0.705
Cumulative survival rate	80	χ^2=3.996;DF=3;P=0.262	χ^2=62.762;DF=3;P<0.05	χ^2=5.735;DF=3;P=0.125	χ^2=0.317;DF=3;P=0.957

Figure 8 Yolk sac and pericardial edemas (A and B, respectively) detected on zebrafish embryos exposed to CB and CT extracts, and the corresponding Au@CB and Au@CT, at 32 and 56 h_pf, respectively.

Notes: Error bars represent mean ± standard deviation for two replicates, of two independent experiments. Different letters indicate significant differences among algae extracts and their AuNPs, as compared to control (one-factor ANOVA, P<0.05). It should be noted that the highest (nominal) test concentration is not represented due to the significant embryonic developmental delay recorded.

Figure 9 Wound-healing assay data for pre-incubation of CB and CT extracts (A and B, respectively), and the corresponding Au@CB and Au@CT (C and D, respectively), in mammalian (L929, mouse) fibroblast cells.

Notes: Error bars represent mean ± SEM of three independent experiments. Different letters indicate significant differences among (nominal) test concentrations of the algae extracts and their AuNPs, independently.

Figure 10 Wound-healing assay data for post-incubation CB and CT extracts (A and B, respectively), and the correspondent Au@CB and Au@CT (C and D, respectively), in mammalian (L929, mouse) fibroblast cells.

Notes: Error bars represent mean ± SEM of three independent experiments. Different letters indicate significant differences among (nominal) test concentrations of the algae extracts and their AuNPs, independently.