*2.4. Drug Release Behavior*

In vitro drug release behaviors of doxorubicin-loaded sericin nanoparticles were investigated in different pH conditions and enzymatic medium at 37 ◦C. Briefly, 5 mL of drug-loaded nanoparticles (4 wt.%) in phosphate buffer saline solution (PBS, pH 7.45) was placed in a tubular cellulose membrane, followed by immersion in flasks with a fixed volume (40 mL). The flasks were further incubated in an orbital mixer (Benchmark Scientific) at 300 rpm, and 37.0 ± 0.5 ◦C. 5 mL of PBS dialysate was collected at predetermined time intervals and then investigated by UV–VIS spectroscopy (SHIMADZU UV-3600 instrument). To maintain a constant volume, after each collection, 5 mL of fresh PBS were added to every flask. A similar procedure was performed for an acidic medium (pH = 3). Protease type XIV from *Streptomyces griseus* and protease α-chymotrypsin from bovine

pancreas were used for enzymatic drug release test. Enzymes were added in a suspension of drug-loaded NPs for 1 h prior to release test, and the drug release behavior followed the above-mentioned protocol. The enzyme activity was 8 U/mL. The release efficiency was calculated as follows:

$$RE\ \left(\%\right) = \frac{amount\ of\ released\ DOX}{amount\ of\ the\ loaded\ DOX} \times 100\tag{1}$$

The encapsulation efficiency was calculated with the following equation:

$$EE\left(\%\right) = \frac{amount\ of\ the\ loaded\ DOX - amount\ of\ unloaded\ DOX}{amount\ of\ the\ loaded\ DOX} \times 100\tag{2}$$

**Scheme 1.** Workflow of sericin nanoparticle preparation.

#### *2.5. Characterization Methods*

#### 2.5.1. FTIR–ATR Analysis

FTIR–ATR investigation was performed using a Bruker Vertex 70 FTIR spectrophotometer with an attenuated total reflectance (ATR) accessory. FTIR spectrophotometer used 32 scans and a resolution of 4 cm−<sup>1</sup> in mid-IR region 4000–600 cm<sup>−</sup>1. Sericin nanoparticles SER 1% (*w*/*v*), loaded with doxorubicin and aqueous mixtures of enzyme–doxorubicin, were analyzed.

#### 2.5.2. Morphological Characterization

Scanning electron microscopy (SEM) analysis was performed to reveal the main features of sericin nanoparticles, including aggregates' size, shape, or morphology. Silk sericin nanoparticles were investigated by a Quanta Inspect F scanning electron microscopy device equipped with a field emission gun (FEG) with 1.2 nm resolution and with an X-ray energy-dispersive spectrometer (EDS).

Transmission electron microscopy (TEM) analysis was performed using a TECNAI F30 G2 S-TWIN microscope operated at 300 kV in an energy-dispersive X-ray analysis (EDAX) facility. Formulations of sericin nanoparticles with 1% (*w*/*v*) and 0.1% (*w*/*v*) were evaluated by TEM.

### 2.5.3. Dynamic Light Scattering (DLS)

The size distribution of the nanoparticles was evaluated by dynamic light scattering in a static domain using a Malvern Zetasizer Nano instrument. Nanoparticles prepared from all sericin concentrations were subjected to DLS investigation (0.1, 0.25, 0.5, and 1% (*w*/*v*)).

The average molecular weight of silk sericin was determined by DLS in a static domain using a Malvern Zetasizer Nano instrument in the molecular weight module. The analysis was performed using glass cuvettes with square aperture. Toluene was used as a reference standard solvent and water as a common solvent for silk sericin. Zeta potential and isoelectric point were also determined by DLS using several protein solutions with pH ranging between acid and neutral (pH 1–7.45). The zeta potential value was considered at a neutral point. The isoelectric point value was considered for zeta potential.

#### 2.5.4. Conformational Analysis by Circular Dichroism (CD)

The secondary structure of silk sericin solution and sericin nanoparticles dispersion was evaluated by a Jasco J-1500 spectrophotometer, Japan (J-1500 Circular Dichroism Spectrophotometer) using a quartz cell of 1 mm path length. During this analysis, the samples were scanned three times at low concentrations in the range of 180–250 nm with a scan rate of 100 nm/min.
