Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

Figures & data

Table 1 Principal characteristics of used Ag NPs of different origins

Origin	aSize (nm)	bFunctional groups conjugated with the surface of Ag NPs
Chemical	30±10	None
Cryptococcus laurentii	35±10	–OH, –NH–, aliphatic CH, C=O, CN, CC, C-OH, C-O-C

Notes:

a Transmission electron microscopy

b Fourier transformed infrared characterizations.

Abbreviation: Ag NPs, silver nanoparticles.

Figure 1 Antiproliferative efficacies of biosynthesized Ag NPs produced by Cryptococcus laurentii at different concentrations.

Notes: MTT assay was used on MCF7, T47D, and MCF10-A. All values are expressed as the means of the difference between optical density at 480 and 690 nm ± standard deviation.

Abbreviations: Ag NPs, silver nanoparticles; OD, optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 2 Annexin V-FITC flow cytometry assay.

Notes: Negative annexin V populations are presented in P1 columns while positive populations are presented in P2 columns. Bar chart comparing effect of 0, 2.5, and 5 μg⋅mL⁻¹ Ag NPs produced by Cryptococcus laurentii after 12 hours’ incubation.

Abbreviations: FITC, fluorescein isothiocyanate; Ag NPs, silver nanoparticles.

Figure 3 Western-blot quantification of Bcl-2 and caspase-9 by ImageJ software.

Notes: To determine relative intensity values, the maximum value for each protein was assigned as 100%. Caspase-8 and caspase-3/7 activities were assigned by chemiluminescent assay following 6 hours’ incubation with 0, 1, and 2 mg⋅mL⁻¹ Ag NP concentrations.

Abbreviation: Ag NPs, silver nanoparticles.

Figure 4 Caspase-8 and caspase-3/7 activities were assigned by chemiluminescent assay following 12 hours’ incubation with 0, and 5 μg⋅mL⁻¹ Ag NP concentrations.

Abbreviation: Ag NPs, silver nanoparticles.

Figure 5 Fluorescent microscopy images of endocytosis assay using FITC-dextran.

Notes: MCF7, T47D, and MCF10-A incubated with FITC-dextran 10.000 kD (0.5 mg⋅mL⁻¹) at 37°C for 0, 4, and 8 hours. Nuclear fluorescence was obtained by DAPI.

Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Figure 6 Fluorescent microscopy images to identify Ag NPs and EEA1.

Notes: The colocalization between EEA1 and AgNPs was observed in the confocal analysis. The arrows show the points with colocalization. Nuclear fluorescence was obtained with DAPI. A and B are MCF7 cells; C are T47D cells.

Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; Ag NPs, silver nanoparticles.

Figure 7 Antiproliferative efficacy of biosynthesized Ag NPs produced by Cryptococcus laurentii at different concentrations after 12 hours.

Notes: MTT assay was used on dynasore treated and non-treated MCF7, T47D, and MCF10-A. All values are expressed as the means of the difference between optical density at 480 and 690 nm ± standard deviation.

Abbreviations: Ag NPs, silver nanoparticles; OD, optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure S1 Graphic representation of conjugation reaction between Ag NPs and FITC molecules.

Abbreviations: FITC, fluorescein isothiocyanate; Ag NPs, silver nanoparticles.

Figure S2 Absorption and emission spectrums of used fluorochromes.

Notes: DAPI: blue; FITC: green; and Alexa Fluor 568: red.

Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.

Figure S3 Antiproliferative efficacy of the Ag NPs produced by synthetic methods at different concentrations.

Notes: MTT assay was used on MCF7, T47D, and MCF10-A. All values are expressed as the means of the difference between optical density at 570 and 690 nm ± standard deviation.

Abbreviations: Ag NPs, silver nanoparticles; OD, optical density; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure S4 Typical figure of flow cytometry that shows annexin V expression between MCF7, T47D, and MCF10-A cell lines.

Notes: A, B, and C show the dot plot of cytometry analysis, the population 2 (P2) included the single epithelial cells. D, E, and F show the histogram plot of P2 that separates between P3 (positive annexin) and P4 (negative annexin). G, H, and I show the estatistic parameters of the three populations.

Abbreviations: FITC, fluorescein isothiocyanate; FSC-A, forward scatter area; SSC-A, side scatter area.

Figure S5 Endocytosis assay using FITC-dextran.

Notes: Fluorescent intensity of cells was measured in eight fields per sample and is presented as mean ± standard deviation.

Abbreviation: FITC, fluorescein isothiocyanate.

Table S1 Numeric table comparing cell viability of 0, 2.5, and 5 μg⋅mL⁻¹ Ag NPs produced by Cryptococcus laurentii and by synthetic methods

	0 μg/mL	SD	P-value	2.5 μg/mL	SD	P-value	5 μg/mL	SD	P-value
Ag NPs produced by Cryptococcus laurentii
MCF7	0.59	0.065	N/S	0.37	0.091	0.038	0.31	0.057	0.019
T47D	0.58	0.065	N/S	0.39	0.031	0.024	0.33	0.049	0.023
MCF10-A	0.55	0.048	N/S	0.54	0.081	N/S	0.51	0.094	N/S
Chemical Ag NPs
MCF7	0.56	0.071	N/S	0.31	0.091	N/S	0.20	0.074	N/S
T47D	0.57	0.063	N/S	0.37	0.068	N/S	0.22	0.056	N/S
MCF10-A	0.52	0.061	N/S	0.25	0.024	N/S	0.14	0.072	N/S

Notes: After 12 hours’ incubation. Values are expressed as the means of the difference between optical density at 570 and 690 nm; P-value is shown when the differences are statistically significant with respect to MCF10-A.

Abbreviations: Ag NPs, silver nanoparticles; N/S, nonsignificant; SD, standard deviation.

Table S2 Numeric table comparing effect of 0, 2.5, and 5 μg⋅mL⁻¹ of Ag NPs produced by Cryptococcus laurentii after 12 hours’ incubation

Negative and positive populations	0 μg⋅mL^−1			2.5 μg⋅mL^−1			5 μg⋅mL^−1
	MCF7	T47D	MCF10-A	MCF7	T47D	MCF10-A	MCF7	T47D	MCF10-A
P1	92.73	92.67	91.733	64	67.2	88.5	38.633	42.7	83.066
P2	7.27	7.33	8.2666	36	32.8	11.5	61.366	57.3	16.933

Note: Negative annexin V populations are presented in P1 row while positive populations are in presented P2 row.