Formulation, characterization, and cellular toxicity assessment of tamoxifen-loaded silk fibroin nanoparticles in breast cancer

Figures & data

Figure 1. Chemical structure of (A) tamoxifen citrate and (B) silk fibroin.

Table 1. Physical characterization of blank SF-NPs, TC-SF-NPs, and lyophilized TC-SF-NPs.

Sample	Particle size (nm)	Polydispersity index	Zeta potential (mV)
Blank SF-NPs	172.2 ± 4.3	0.092 ± 0.02	−17.4 ± 1.1
TC-SF-NPs	186.1 ± 5.9	0.169 ± 0.01	−19.9 ± 1.4
Lyophilized TC-SF-NPs	195.1 ± 5.7	0.163 ± 0.01	−20.8 ± 1.9

Data represent mean ± SD of three independent experiments.

Figure 2. Surface morphology of TC-SF-NPs. (A) SEM image of TC-SF-NPs at X 100,000 (B) TEM of TC-SF-NPs. TC-SF-NPs: tamoxifen citrate silk fibroin nanoparticles; SEM: scanning electron microscopy; TEM: transmission electron microscopy.

Figure 3. Physicochemical characterization of TC-SF-NPs. (A) Fourier transform infrared spectra. (B) X-ray diffraction spectra. TC-SF-NPs: tamoxifen citrate silk fibroin nanoparticles.

Figure 4. In vitro release study of free TC and TC-loaded SF-NPs at different pH. TC: tamoxifen citrate; TC-SF-NPs: tamoxifen citrate silk fibroin nanoparticles.

Table 2. Stability study of optimized TC-SF-NPs at different temperatures and the humidity condition.

Stability condition	Time (month)	Particle size (nm)	Zeta potential (mV)	Initial amount of drug (mg)	Drug amount at the end of 3 months (mg)	% Degraded at the end of 3 months
25 ± 2 °C/60 ± 5% RH	0	186.12 ± 5.87	−19.21 ± 0.8	7.51 ± 0.12	7.42 ± 0.19	1.20
	1	187.31 ± 5.64	−19.42 ± 0.7
	3	189.24 ± 4.80	−20.02 ± 1.1
40 ± 2 °C/ 75 ± 5% RH	0	186.12 ± 5.87	−19.21 ± 0.6	7.51 ± 0.12	7.36 ± 0.13	1.99
	1	187.53 ± 6.25	−19.42 ± 0.9
	3	191.14 ± 4.86	−20.36 ± 1.3
5 ± 2 °C/ambient RH	0	186.12 ± 5.87	−19.21 ± 1.2	7.51 ± 0.12	7.46 ± 0.16	0.66
	1	187.63 ± 5.46	−19.42 ± 0.8
	3	187.54 ± 4.39	−20.16 ± 1.0

Data are given in mean ± SD (n = 3).

Figure 5. In vitro cytotoxicity of TC-SF-NPs against breast cancer cell lines. (A) MCF-7 and (B) MDAMB-231 cells were incubated with different concentrations (25–400 μg/ml) of free TC, SF-NPs or TC-loaded SF-NPs for 72 h. Untreated cells served as controls. The dose of TC in TC-SF-NPs group was equivalent to that of free TC group. Cell viability was assessed by MTT assay. Data represent mean ± SD. ^#p < .05 vs. TC-SF-NPs, ***p < .005 vs. SF-NPs. TC: tamoxifen citrate; SF-NPs: silk fibroin nanoparticles; TC-SF-NPs: tamoxifen citrate silk fibroin nanoparticles.

Figure 6. Cellular uptake of TC-SF-NPs by MCF-7 and MDAMB-231 cell lines. (A) Confocal microscopy images of MCF-7 and MDA-MB-231 cells treated with PI-labeled TC-SF-NPs for 24 and 48 h. (B) Flow cytometry quantitative analysis showing percentage of uptake of PI-labeled TC-SF-NPs by MCF-7 and MDA-MB-231 cells following 24 h incubation. Data are expressed in mean ± SD. **p < .01. TC: tamoxifen citrate; TC-SF-NPs: tamoxifen citrate silk fibroin nanoparticles.

Table 3. Effect of free TC and TC-loaded SF-NPs on the redistribution of growth-arrested MCF-7 and MDA-MB-231 cells in the different phases of the cell cycle.

Cell cycle phase	MCF-7 Cells			MDA-MB-231 Cells
	Control	Free TC	TC-SF-NPs	Control	Free TC	TC-SF-NPs
Sub G0/G1	11.48 ± 0.77	14.32 ± 0.81**	18.11 ± 0.94^**, #	10.72 ± 0.48	14.12 ± 1.08**	18.58 ± 1.03^**, #
G0/G1	36.10 ± 1.25	41.18 ± 1.71**	45.30 ± 2.07^**, #	34.88 ± 2.23	39.25 ± 1.98**	44.70 ± 2.95^**, #
S	25.31 ± 1.13	20.92 ± 1.23**	16.28 ± 0.65^**, #	24.45 ± 1.76	19.75 ± 0.98**	16.95 ± 1.13^**, #
G2/M	27.11 ± 1.45	23.58 ± 2.01**	20.31 ± 1.98^**, #	29.95 ± 2.56	26.88 ± 1.34**	19.77 ± 0.85^**, #

Data represent percentage of single-cell events (mean ± SD) in the four different phases. **p < .01 vs. control, ^#p < .05 vs. free TC.

Figure 7. Cellular apoptosis of breast cancer cells treated with TC-loaded SF-NPs. MCF-7 and MDA-MB-231 cells (3 × 10⁵ cells/well) were treated with either free TC or TC-SF-NPS for 24 h. Cellular apoptosis was evaluated by flow cytometry using Annexin V-FITC staining. TC: tamoxifen citrate; TC-SF-NPs: tamoxifen citrate silk fibroin nanoparticles.