Fig 1.
TEM characterization of iron oxide nanoparticles.
A) TEM image by low magnification with inserted size distribution of IONPs, B) HRTEM image that shows the ultrastructure and morphology of Fe3O4 nanoparticles.
Table 1.
The properties of the bioengineered spider silk proteins MS1, MS2 and EMS2 predicted on the basis of their amino acid sequences.
Fig 2.
SEM images and EDXS quantitative results of selected elements of MS1, MS1/IONP spheres, MS2, MS2/IONP spheres, EMS2, and EMS2/IONP spheres.
A) Spheres were prepared by mixing 2 M potassium phosphate at pH 8 with the indicated version of silk (2.5 mg/mL) in the presence or absence of iron oxide nanoparticles (5 mg/mL). B) EMS2 and EMS2/IONP spheres were formed by mixing 2 M potassium phosphate at pH 8 with a silk solution (1 mg/mL) in the presence or absence of the iron oxide nanoparticles (5 mg/mL); scale bar– 4 μm.
Fig 3.
Size analysis of the EMS2 and EMS2/IONP spheres.
For the A) size and B) size distribution analyses, the spheres were prepared by mixing a silk solution (1 mg/mL) and iron oxide nanoparticles (5 mg/mL) with 2 M potassium phosphate at pH 8. B) Whiskers show the minimal and maximal sizes of the spheres, and the line inside the box shows the median size. ***indicates statistical significance with p < 0.0001.
Fig 4.
Secondary structure analysis of the EMS2 and EMS2/IONP spheres.
A) The secondary structure composition of the EMS2 and EMS2/IONP spheres investigated on the 1st day of storage. B, C) Analysis of the secondary structure composition of the (B) EMS2 and (C) EMS2/IONP spheres during storage on the 1st, 7th and 14th day. The secondary structure content of both types of particles was calculated after Fourier self-deconvolution of the amide I region of the FTIR spectra. The mean and error bars indicating the standard deviations are shown. The experiment was repeated three times. * indicates statistical significance with: * p < 0.05; ** p < 0.01; *** p < 0.001.
Table 2.
The iron content and zeta potential of the spheres.
Fig 5.
Magnetic measurements of the IONPs and silk/ iron oxide spheres (EMS2/IONP).
A) Zero-field-cooled (ZFC) and field-cooled (FC) magnetization curves for IONPs (upper) and EMS2/IONP spheres (bottom); B) Hysteresis loops at T = 5 and 300 K for IONPs (upper) and EMS2/IONP (bottom) spheres.
Fig 6.
The loading efficiency and release kinetics of doxorubicin (Dox).
A) Spheres were loaded with Dox using a post-loading method. B, C) From the B) EMS2 and C) EMS2/IONP spheres, doxorubicin was released at 37°C in PBS buffers at pH values of 7.4, 6 and 4.5 over 7 days. The time points for the drug release measurements were after 1 and 3 h of incubation on day 1 and then every 24 h. The means and standard deviations of three independent experiments are shown. * indicates statistical significance with p < 0.0001; *** indicates statistical significance with p < 0.001.
Fig 7.
The SEM images of A) EMS2/IONP spheres B) EMS2/IONP spheres loaded with doxorubicin and C) EMS2/IONP spheres after the release of doxorubicin.
EMS2/IONP spheres were prepared by mixing 2 M potassium phosphate at pH 8 with silk (1 mg/mL) and iron oxide nanoparticles (5 mg/mL) and then were loaded with Dox by the post-loading method. Next, Dox was released from EMS2/IONP spheres for 7 days at pH of 4.5; scale bar– 1 μm.
Fig 8.
Cytotoxicity study by MTT assay.
The NIH3T3 fibroblasts were cultured in the presence of EMS2 and EMS2/IONP spheres for 72 h. Spheres were produced using an initial silk concentration of 1 mg/mL and 2 M potassium phosphate (pH 8) in the presence or absence of IONPs. The MTT reduction was calculated with reference to the non-treated cells. Error bars show the standard deviations of the mean of three independent experiments.