**1. Introduction**

Silks are natural protein-like fibers produced by arthropods, such as spiders or silkworms. Domestic-species-producing silks have been used since antiquity. Certain species, such as domesticated silkworm *Bombyx mori*, have a central role within textile industry applications and more recently in biomedical applications [1–3]. *Bombyx mori* proteins have been intensively studied for their biocompatibility, great mechanical properties, tunable biodegradation process, easy processing, and favorable source supply. Silk is composed of two major proteins: silk fibroin (fibrous protein) and silk sericin (globular protein) [4–8]. Silk fibroin is the main protein with large usage in the biomedical field. Silk sericin was originally removed as it was associated with the general immune body response to silks [9–11]. Therefore, silk sericin was largely neglected as a biomaterial for medical applications. However, silk sericin has attracted the particular interest of researchers due to special properties such as antioxidant effect, UV protection, moisture adsorption, or antibacterial protection. Recent studies in the literature revealed that sericin is currently used in cosmetics, pharmaceuticals, wound dressing, drug delivery, or cell culture. The antioxidant properties allow sericin to stand against lipid peroxidation by scavenging reactive species or to suppress

**Citation:** Radu, I.-C.; Zaharia, C.; Hudit,a, A.; Tanas ˘ ˘ a, E.; Ginghin ˘a, O.; Marin, M.; G ˘al ˘at,eanu, B.; Costache, M. In Vitro Interaction of Doxorubicin-Loaded Silk Sericin Nanocarriers with MCF-7 Breast Cancer Cells Leads to DNA Damage. *Polymers* **2021**, *13*, 2047. https:// doi.org/10.3390/polym13132047

Academic Editor: Ki Hyun Bae

Received: 19 May 2021 Accepted: 19 June 2021 Published: 22 June 2021

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tumor genesis by reducing oxidative stress or inflammatory responses [12–14]. Furthermore, the antioxidant properties of silk sericin contributed to cancer applications due to the capability to reduce oxidative stress or suppressing cancer cytokines for skin and colon cancer [15,16]. In recent years, silk sericin was used in the development of scaffolds for regenerative medicine in wound healing or tissue engineering [6,17–19].

In this regard, silk sericin may favor and sustain migration, proliferation, or collagen type I production due to methionine amino acid [20–22]. Silk sericin promoted open wound healing and added to silver-zinc sulfadiazine cream prevented burn wound infection. It was also effective in healing second-degree burn wounds without serious inflammatory reactions [22]. Besides particular properties, which recommended silk sericin for various biomedical applications, the drug-delivery field represents the specific area in which different nanoparticle systems have been developed [23–30]. However, the future concept pathway is used to overcome the current limitations so that it sustains the continuous development of nanoparticle systems. Silk sericin proved the ability for self-assembling capacity by loading various active principles. The unique chemistry favored surface charge modification for DNA binding and active targeting by poly(ethylene glycol) (PEG) and folate for cancer management [24,26,31].

This unique ability offers the possibility to easily prepare nanoparticles for drug/ biomolecule delivery. These characteristics are tightly related to sericin chemistry and, in the last decades, studies showed new interesting insights on silk sericin structure. Therefore, silk sericin showed less amphiphilic character but higher hydrophilic character. This aspect was considered an impediment for self-assembling into nanoparticles, as compared to silk fibroin [32]. However, the synergistic effect of a proper preparation method, such as nanoprecipitation, and a specific precipitation agent may favor the preparation of silk sericin nanoparticles. Nanoparticles should display the desired characteristics such as size, morphology, and size distribution. Nanoprecipitation is a simple and fast method to produce nanoparticles from various types of polymers [33–38].

Doxorubicin is an anthracycline with therapeutic effects in a wide range of solid tumors, which still plays a major role in chemotherapy that induces apoptosis by causing DNA damage [39]. A great effort has been made to develop targeted nanodrug delivery systems due to their high therapeutic efficacy in cancer management. Polymeric nanoparticles are promising systems for drug delivery based on their nanometric size, high surface-areato-volume ratio, favorable drug release profiles, and targeting features that can promote their preferential accumulation in tumor tissue. Various preparation routes have been addressed in the literature in an attempt to show the interaction of doxorubicin with various polymeric systems [40,41]. In this regard, doxorubicin-loaded RGD-conjugated polypeptide nanoformulation was developed by an emulsion solvent evaporation method [42]. These zwitterionic biodegradable drug-loaded polypeptide vesicles showed great potential for cancer treatment having high drug loading content (45%) and loading efficiency (95%) [42]. An interesting approach was related to the encapsulation of doxorubicin by polymerizationinduced self-assembly methods [41]. Photopolymerization of various monomers in the presence of photocatalytic doxorubicin hydrochloride proved an interesting method to prepare drug-loaded polymeric nanoparticles with higher polymerization rates and good doxorubicin encapsulation efficiency [41]. Protein nanoparticles were also prepared by various routes to easily entrap and release various anticancer drugs [43]. They are prepared within biological systems, require fewer production steps, and show high biocompatibility and biodegradability, as compared to synthetic polymers. Protein nanoparticles have been prepared from various proteins including water-soluble proteins (bovine and human serum albumin, silk sericin) and partially soluble or insoluble proteins (silk fibroin, zein, and gliadin) [44]. TRAIL and Dox-loaded albumin nanoparticles showed great potential for synergistic apoptosis-based anticancer therapy [45].

This research study emphasizes the possibility to easily develop silk sericin nanoparticles by an optimized nanoprecipitation method. The novelty of the study arises from the development of sericin nanoparticles with a lower size distribution with respect to

the literature data. Furthermore, the nanoparticles' preparation is based on an optimized procedure that can be easily transferred being useful for other similar systems.

The present research study reports on the preparation and complex characterization of self-assembled silk sericin nanoparticles in the presence of acetone with secondary conformational changes. Doxorubicin loading in sericin nanoparticles and drug release behaviors were studied in neutral, acidic, and enzymatic media. DLS, SEM, and TEM were employed for morphological and structural characterization of the nanoparticles. The structural changes showed similar behavior to silk fibroin by revealing a special and stable physical crosslinking structure of nanoparticles. Moreover, we investigated the sericin particles' potential to reduce MCF-7 cells' viability and to induce DNA fragmentation when loaded with doxorubicin.
