*3.1. FTIR–ATR Analysis*

FTIR investigation showed the main characteristic peaks for doxorubicin, enzymes, sericin, and their interactions. The spectrum of doxorubicin had the following peaks: peak at 3517 cm−<sup>1</sup> was assigned to water molecules bonded within the drug structure; peak at 3323 cm−<sup>1</sup> was attributed to hydroxyl stretching vibration; peak at 3128 cm−<sup>1</sup> was attributed to N-H stretching vibration; peaks at 2977 cm−<sup>1</sup> and 2931 cm−<sup>1</sup> were assigned to C-H stretching vibration within the ring; peak at 1728 cm−<sup>1</sup> was assigned to C=O stretching in carbonyl group within vibrating in quinone and ketone; peak at 1584 cm−<sup>1</sup> was attributed to N-H bending vibration and C-N stretching vibration; peak at 1525 cm−<sup>1</sup> was assigned to hydroxyl group bending vibration; peak at 1410 cm−<sup>1</sup> was attributed to methyl bending vibration; peaks at 1285 cm−1, 1207 cm−1, 1115 cm−1, and 1075 cm−<sup>1</sup> were attributed to C-N and C-O-C stretching vibrations, C-O stretching vibration within the tertiary, and secondary and primary alcohols; peaks at 999 cm−<sup>1</sup> and 865 cm−<sup>1</sup> were assigned to the skeletal ring (Figure 1). The sericin spectrum revealed a specific protein spectrum with the following characteristic peaks: peak at 3291 cm−<sup>1</sup> was attributed to hydroxyl stretching vibration; peak at 3070 cm−<sup>1</sup> was attributed to N-H stretching vibration; peak at 2972 cm−<sup>1</sup> was assigned to C-H stretching vibration; peak at 1649 cm−<sup>1</sup> was assigned to C=O stretching in carbonyl group within amidic backbone (amide I); peak at 1529 cm−<sup>1</sup> was attributed to N-H bending vibration and C-N stretching vibration (amide II); peak at 1395 cm−<sup>1</sup> was attributed to methyl, methylene, and methine groups bending vibration; peak at 1242 cm−<sup>1</sup> was attributed to C-N (amide III); peak at 1068 cm−<sup>1</sup> was attributed to C-O-C and C-O stretching vibrations (Figure 1).

**Figure 1.** FTIR–ATR spectra of sericin, doxorubicin, and doxorubicin-loaded sericin nanoparticles.

Both enzymes (protease IV and chymotrypsin) showed a typical protein-specific spectrum with amide I, amide II, and amide III. The main peaks are shown in Figure 2a,b. Sericin–doxorubicin interaction revealed a drug-specific peak at 3510 cm<sup>−</sup>1, assigned to the water molecules bonded within the drug molecules. Another important peak appeared at 3070 cm−1, attributed to unsaturated =C-H stretching vibration characteristic for the aromatic structures similar to doxorubicin structure. Therefore, FTIR analysis showed the presence of doxorubicin within the sericin structure. Furthermore, the analysis revealed no chemical bonds between the sericin and drug, suggesting that the association was induced only by physical interactions (Figure 1).

**Figure 2.** FTIR–ATR spectra of enzymes (protease IV and chymotrypsin), doxorubicin, and association of chymotrypsin/doxorubicin (**a**) and protease IV/doxorubicin (**b**).

Protease IV and chymotrypsin revealed an association with doxorubicin even to a greater extent than sericin. Thus, chymotrypsin–drug association showed two new peaks at 3176 cm−<sup>1</sup> and 3113 cm<sup>−</sup>1, attributed to N-H stretching vibration. This fact may suggest that the amidic groups are more visible, as compared to the singular drug or enzyme. A higher contribution to the spectrum can be explained by the fact that these groups manifested supplementary physical interactions. Another important peak at 860 cm−1, attributed to the drug skeletal ring, appeared into the association spectrum (Figure 2a). This approach revealed that the presence of the drug molecules within the chymotrypsin structure was also governed by physical interactions. Protease IV had also some association with doxorubicin. The spectrum (Figure 2b) showed a different overview regarding drug association, as compared to the chymotrypsin approach. Therefore, the spectrum showed the absence of amide I and the presence of a broad peak centered at 1558 cm−<sup>1</sup> with a significant visible shoulder around 1600 cm−1. This peak probably represents the contributions of amide I and amide II from the protease and peak contribution assigned to hydroxyl group bending vibration (1584 cm−1). The peak at 1419 cm−<sup>1</sup> increased its intensity, and a new peak appeared at 1345 cm<sup>−</sup>1. This fact can be attributed to the contribution of the bending vibration for methyl, methylene, and methine groups. Furthermore, the presence of the peak at 850 cm−1, attributed to the skeletal drug structure, confirmed the presence of doxorubicin within the protease XIV structure (physical interactions). One last aspect that can be concluded from protease investigation with respect to sericin highlights the apparent stronger association interactions for the enzymes. This fact can be explained in the case of enzymes by the amide, hydroxyl, and skeletal contributions.
