*3.6. In Vitro Antitumor Activity Evaluation of DOX-Loaded Sericin Nanocarriers*

As described above, based on the nanoparticles' size and doxorubicin release profile, the 0.1% sericin formulation was employed in the in vitro biological investigations. To evaluate the viability of MCF–7 breast cancer cells after 6 h and 24 h of exposure to unloaded Ser NPs and DOX-loaded Ser NPs, the quantitative MTT assay was performed. Data were statistically analyzed and graphically represented in Figure 10 using GraphPad Prism 6 software.

**Figure 10.** Graphical representation of MCF-7 breast cancer cells viability after 6 h and 24 h of treatment with free and DOX-loaded Ser NPs (Dox Ser NPs vs. untreated control \*\*\*\* *p* ≤ 0.0001; Dox Ser NPs vs. Ser NPs ˆˆˆˆ *p* ≤ 0.0001).

Our data showed that after 6 h of treatment, none of the treatments induced cell viability alterations. Moreover, after 24 h of exposure to free Ser NPs, the viability of the MCF-7 cells remained similar to the control, demonstrating good biocompatibility of the pristine sericin nanocarriers. In contrast, after 24 h of treatment, the DOX-loaded Ser NPs significantly decreased the viability of the MCF-7 cells (\*\*\*\* *p* ≤ 0.0001). Moreover, MCF-7 breast cancer cell morphology was investigated by fluorescence microscopy after staining the cytoskeleton fibers with phalloidin–FITC and the cell nuclei with DAPI. The images captured are presented in Figure 11. No alterations were produced by the treatment with unloaded Ser NPs during 24 h, as compared with the untreated cells. In contrast, the treatment with DOX-loaded Ser NPs induced modifications in terms of actin filaments' organization and distribution in the cellular cytoplasm. Additionally, the fluorescence microscopy images captured in the samples treated with DOX-loaded Ser NPs revealed red fluorescence inside the MCF-7 cells. Considering that doxorubicin is well known as a red fluorescent chemical compound, this valuable observation indicates/proves that the DOX-loaded Ser NPs successfully enter the cells.

**Figure 11.** Fluorescence microscopy images of MCF-7 cells treated for 6 h and 24 h with free Ser NPs and DOX-loaded Ser NPs, as compared with an untreated MCF-7 monolayer: green fluorescence–phalloidin–FITC: actin filaments; blue fluorescence–DAPI: cell nuclei; red fluorescence–DOX). Scale bar: 50 μm.

Finally, to assess the genotoxic potential of the DOX-loaded Ser NPs treatment, the comet assay was performed. After fluorescence image processing and data analysis, the DNA damage profile in MCF-7 cancer cells after Ser NPs + DOX treatment was established based on the average length of the comet tails. As presented in Figure 12, the comet-like structures correlated with enhanced DNA migration were identified only in MCF-7 cells exposed for 24 h to the treatment with DOX-loaded Ser NPs. As DNA damage is a hallmark of apoptosis, our data suggest that DOX encapsulation in Ser NPs triggers apoptosis of MCF–7 breast cancer cells.

Sericin nanocarriers have previously been used for breast cancer management [28]. Mandal and Kundu showed that paclitaxel-loaded sericin nanocarriers induced apoptosis in MCF-7 breast cancer cells. Similarly, we demonstrated that DOX-loaded Ser NPs significantly decreased MCF-7 cells viability after 24 h of treatment and altered the morphology of the cells, as revealed by the fluorescent labeling of the cell's cytoskeleton.

Regarding DOX, the literature reports two potential mechanisms of action in the cancer cell: (i) the intercalation into DNA and disruption of topoisomerase-II-mediated DNA repair and (ii) the generation of free radicals producing damages to cellular membranes, DNA, and proteins [55]. Our data showed that the DOX-loaded Ser NPs induced DNA damage in MCF-7 cells, as compared with the pristine Ser NPs, probably due to the toxic effect of the delivered DOX.

**Figure 12.** (**a**) Graphical representation showing the comet tail length as an indicator of DNA damage in MCF–7 cell cultures treated for 6 h and 24 h with simple and DOX-loaded Ser NPs (Dox Ser NPs vs. untreated control \*\*\*\* *p* ≤ 0.0001; Dox Ser NPs vs. Ser NPs ˆˆˆˆ *p* ≤ 0.0001); (**b**) representative fluorescence micrographs of the comet-like structures in MCF–7 cell cultures treated for 6 h and 24 h with simple and DOX-loaded Ser NPs.

#### **4. Conclusions**

In conclusion, we obtained sericin nanoparticles with a size range between 15 and 40 nm. This dimensional range is below the usual range of the polymeric nanoparticles. The nanoprecipitation proved to be a suitable method for sericin nanoparticles' preparation, loading, and release. The advanced morphological investigation showed the size and size distribution of the nanocarriers with a direct positive influence on the biological investigation. Moreover, we also showed that the DOX-loaded Ser NPs significantly decreased MCF-7 cells viability, altered their morphology, and induced DNA damage, as compared with the unloaded Ser NPs.

**Author Contributions:** Conceptualization, I.-C.R., B.G., and C.Z.; methodology, I.-C.R., B.G., A.H., and C.Z.; validation, I.-C.R., B.G., A.H., and C.Z.; investigation, I.-C.R., E.T., B.G., A.H., O.G., M.M., and M.C.; writing—original draft preparation, I.-C.R., C.Z., A.H., and B.G.; writing—review and editing, B.G., O.G., and C.Z.; supervision, B.G. and C.Z.; project administration, C.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by a grant from the Ministry of Research, Innovation and Digitization, CNCS/CCCDI–UEFISCDI, Project Number PN-III-P4-ID-PCE-2020-1448, within PNCDI III.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** DLS and Circular dichroism were possible due to the European Regional Development Fund through Competitiveness Operational Program 2014–2020, Priority axis 1, Project No. P\_36\_611, MySMIS code 107066, Innovative Technologies for Materials Quality Assurance in Health, Energy and Environmental-Center for Innovative Manufacturing Solutions of Smart Biomaterials and Biomedical Surfaces–INOVABIOMED.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
