3.2.2. Acidic Medium

FTIR was performed to investigate the drug–protein interaction. As previously mentioned, there are significant physical interactions that keep the drug inside the nanoparticles. Considering that the main interactions appeared between carboxyl and amino groups, a new in vitro test was performed in an acidic medium. The idea was to protonate the carboxyl negative conjugate form of COO− within sericin structure to disrupt the interaction with amino positive conjugate form NH3+ from doxorubicin. Thus, the entrapped drug could be released. This approach was based on the sericin zeta potential results, which had a considerable negative charge into a neutral medium (pH 7.45). The analysis showed a higher release efficiency within a shorter time period, as compared to the neutral medium. Figure 4 revealed a release efficiency of over 90% for all formulations SER 0.1%–SER 1% in a release time of 30–50 min. The release profile showed different behavior with a faster

release for SER 0.1% and SER 0.25% for the first 15 min, followed by a slower release for the next 15 min. SER 0.5% and SER 1% revealed a relatively constant release behavior for longer release time, as compared to SER 0.1% and SER 0.25%. This fact allowed them to reach a higher release.

**Figure 4.** Doxorubicin release profile from sericin nanoparticles in acidic medium (pH 3).

#### 3.2.3. Enzymatic Media

The drug–protein interaction was also investigated by enzymatic activity. Two specific enzymes, namely, protease type XIV from *Streptomyces griseus* and protease α-chymotrypsin from bovine pancreas were used. The enzymatic activity involved an enzymatic degradation of sericin protein with the easier release of entrapped drug molecules due to the disruption of physical interactions in drug–protein structure. The results showed an unexpected behavior considering the lower release with respect to the neutral medium. In the case of protease type XIV from *Streptomyces griseus*, the release efficiency was slightly over 20% (Figure 5a), while in the case of protease α-chymotrypsin from bovine pancreas, the release efficiency was slightly under 20% (Figure 5b). Both situations followed a similar profile release and a release time of 180 min. The profile showed a faster release within the first 50 min, followed by a slower release. The poor release efficiency can be explained by the strong physical interactions between the doxorubicin molecule and enzymes' chemical structure. In this case, the drug molecules released from the nanoparticles' mass were further embedded in the structure of the enzymes. These results are in good agreement with the FTIR analysis that showed stronger interactions within drug–enzyme association as compared to sericin nanoparticles.

#### *3.3. Morphological Characterization*

#### 3.3.1. SEM Analysis

SER 1% nanoparticles were obtained with a size distribution of 200–300 nanometers with round and specific fusiform shapes (Figure 6a). Higher magnification images revealed closer insights into nanoparticles' morphology with a bunch nanostructure and nanowaved surface (Figure 6b). The nanowaved morphology showed lower sizes of 15–20 nm winding the surface. Individual nanoparticles of 23 nm could be detected, suggesting that the bunch nanostructuring is formed of aggregates of smaller nanoparticles. Morphological characterization continued with formulations SER 0.5% and SER 0.25%. The results for SER 0.5% and SER 0.25% formulations revealed nanoparticle aggregates with a size range size of 100–200 nm (SER 0.5%) and 100–150 nm (SER 0.25%), as shown in Figure 6c,d. Aggregates for SER 0.5% had round and fusiform shapes (Figure 6c), while those for SER 0.25% exhibited only round shapes (Figure 6d). This could be explained by the size range differences since the size increase led to the deviation from the usual round shape. Formulation SER 0.1% was investigated in order to reveal a specific trend of

sericin formulations. The lower the sericin concentration was, the smaller the nanoparticle aggregates were obtained. Figure 6e showed aggregates with a size range of 80–130 nm. The aggregates had a round shape like SER 0.25%. The higher magnification image revealed the same bunch nanostructuring with a nanowaved surface (Figure 6f).

**Figure 5.** Doxorubicin release profiles from sericin nanoparticles in enzymatic media: protease XIV (**a**); chymotrypsin (**b**).

#### 3.3.2. TEM Analysis

TEM analysis was performed to confirm the SEM results regarding aggregates size, morphology, and nanostructuring. Formulation SER 1% exhibited individualized nanoparticles with a range size between 20 and 35 nm (Figure 7a, higher magnification). The overview image showed also individualized nanoparticles in a significant number (Figure 7b). The nanoparticle aggregates revealed the internal nanostructuring. The aggregates were composed of smaller nanoparticles of 20–35 nm (Figure 7c,d). Besides aggregates, one may notice individualized nanoparticles, suggesting that only a part of them associate with such structures. Formulation SER 0.1% had even smaller individualized nanoparticles with respect to other formulations (15–25 nm, Figure 7e,f). This result confirmed the formulation trend with smaller nanoparticles for lower sericin concentration. This fact can explain the size differences between the aggregates or their shape. The nanoprecipitation appeared as a suitable self-assembling method able to optimize the nanoparticles' features among other nanoformulation methods [30,48,49]. The nanoparticle low average dimension was directly related to some important sericin properties. The mechanism followed a nucleation step, which is typical for the nanoprecipitation method [37,50–52]. This mechanism assured the preparation of a high number of small nanoparticles.

**Figure 6.** SEM microphotographs for sericin nanoparticles from various sericin concentrations: (**a**,**b**) SER 1%; (**c**,**d**) SER 0.5% and SER 0.25%; (**e**,**f**) SER 0.1%.

**Figure 7.** TEM images for individualized nanoparticles obtained from 1% sericin concentration (**a**,**b**); TEM images for nanoparticles aggregates obtained from 1% sericin concentration (SER 1%) (**c**,**d**); TEM images for individualized nanoparticles obtained from 0.1% sericin concentration (SER 0.1%) (**e**,**f**).

#### *3.4. Dynamic Light Scattering, Zeta Potential, and Isoelectric Point*

DLS analysis showed more specifically nanoparticles aggregates size. The sericin nanoparticle formulations SER 0.1%–SER 1% revealed an increase of the mean diameter with the increase of the concentration of sericin solutions. All formulations showed a close size distribution profile and close mean size diameter. The size distribution of sericin nanoparticle aggregates and mean diameter are shown in Figure 8a. The results confirmed the morphological investigation by SEM and TEM on aggregates size increasing profile but with a higher size distribution due to the swelling effect.

**Figure 8.** DLS dimensional distribution of sericin nanoparticles with various concentrations (**a**); DLS zeta potential of sericin nanoparticles—SER 0.1% formulation (**b**).

The nanoparticles' surface zeta potential and isoelectric point were used to evaluate the protein surface charging at various pH values. The results revealed a negative surface charging with a zeta potential of −20.2 mV with a standard deviation ±0.9 mV (Figure 8b.) This value represents the zeta potential for neutral pH (7.45). The isoelectric point was established in the pH range 2–2.5. This value of zeta potential can influence the tumor cell line interaction or the mechanism of nanoparticle synthesis.

### Molecular Weight Evaluation by DLS

The average molecular weight of silk sericin was evaluated by the detection of light scattering based on the interactions of protein molecule–light. Therefore, the sericin solutions were exposed to a monochromatic wave of light and using multiple detectors. The analysis supposed the investigation of four diluted solutions with various concentrations between 0.2 and 2% (*w*/*v*) of silk sericin. An average molecular weight of about 11,700 ± 100 g/mole was determined.

#### *3.5. Conformational Analysis by Circular Dichroism (CD)*

CD analysis for sericin showed two positive peaks and one sharp negative peak. The positive peaks at 180 and 186 nm, together with the negative peak at 205 nm, suggest

a secondary conformational arrangement dominated by β-sheet and random coil. The sericin nanoparticles had a shifting of the negative peak to lower values and shifting of the positive peaks to higher values. The positive peak was also split into two peaks (Figure 9). This shows some conformational changes of random coil toward the β-sheet structure.

**Figure 9.** CD curves of native sericin (0.05 wt.% concentration) and sericin nanoparticles (SER-0.1%).

The sericin formulations led to the obtaining of sericin nanoparticles ranging between 15 and 40 nm depending on the solution concentration. These size values are below the usual sizes of polymeric nanoparticles. This fact can be attributed to several important factors including sericin chemistry, molecular weight, concentration, or preparation method. In the case of molecular weight (MW), there are various studies in the literature showing the influence or not of the MW on the size of the nanoparticles for different polymeric systems. Most probably, the molecular weight's influence on nanoparticles size is directly correlated to every studied system. In our study, the low molecular weight fitted the sericin in the oligomeric range (polypeptides), and it could induce such small nanoparticles. The sericin chemistry clearly influenced the hydrophilicity, water solubility, or behavior within the organic phase (acetone). The high water solubility could be also influenced by the low molecular weight. The high water solubility, together with the low molecular weight and chemistry, positively influenced the sericin behavior in acetone dispersion. In contact with acetone, the sericin molecules gather and induce a nucleation process. This is a more controlled process mechanism than instant precipitation. This approach is directly correlated to the preparation method. Nanoprecipitation follows a three-stage process: nucleation, growth, and aggregation [52–54]. Therefore, this approach allowed a supersaturation of sericin molecules per volume with a nucleating process, followed by a growth step. A high sericin concentration led to the generation of nanoparticles with a larger size distribution reaching 35–40 nm, while a low concentration led to the generation of nanoparticles with a narrow distribution. Both high (1% *w*/*v*) and low (0.1 *w*/*v*) concentrations led to the generation of a relatively high number of nanoparticles, suggesting no influence on the number of nanoparticles. Thus, in the initial stage, a high number of nanoparticles were formed, while the size differences appeared in the growth step due to the addition of new molecules on the nuclei surface. Another important issue to be addressed is the aggregation process. The dimensional and morphological investigation showed larger nanoparticle aggregates with a range size between 100 and 300 nm. These nanoparticle aggregates were formed

along with individual nanoparticles. Therefore, this process probably appears only in the case of supersaturation for nanoparticles' concentration per volume of dispersion media.
