*3.5. Fourier-Transform Infrared Spectroscopy (FTIR)*

The structural changes in synthesized liposomes with and without HS and the successful chitosan coating were confirmed by FTIR spectroscopy. From the IR spectrum of the HS (Figure 3a), a broadband at 3300 cm−<sup>1</sup> was attributed to O–H and N–H stretching and two bands at 2926 and 2853 cm−<sup>1</sup> were related to CH2 stretching vibrations of aliphatic chains. The amide region bands (1658 cm−<sup>1</sup> corresponding to protein amide I, 1550 cm−<sup>1</sup> corresponding to protein amide II, and 1247 cm−<sup>1</sup> related to protein amide III) were clearly visible in the IR spectrum of the HS [29].

**Figure 3.** FTIR spectra of (**a**) *Spirulina* hydrolysate (HS); (**b**) blank nanoliposomes stabilized with γ-oryzanol; (**c**) HS-loaded γ-oryzanol nanoliposomes; and (**d**) HS-loaded γ-oryzanol-chitosomes.

Blank nanoliposomes (Figure 3b) were observed at the following wavenumbers: 3438 cm−<sup>1</sup> related to hydroxyl stretch vibration, 2925 cm−<sup>1</sup> attributed to stretch vibrations of a methylene group, 1735 cm−<sup>1</sup> mainly related to the stretching vibration of the polar head ester groups of phospholipids, 1654 cm−<sup>1</sup> related to C=C stretching vibrations, 1246 and 1109 cm−<sup>1</sup> corresponding to symmetric and antisymmetric stretch vibrations of a phosphate group, and 958 cm−<sup>1</sup> related to asymmetrical stretch vibrations of N+/CH3.

Comparing the IR spectra of HS-loaded γ-oryzanol-liposomes (Figure 3c) and corresponding blank liposomes (Figure 3b), a great similarity between their spectra can be observed. The incorporation of hydrolysate into the liposomal carrier resulted in a shift in some frequencies. The most important of these changes were slight shifts at 1735 and 1654 cm−<sup>1</sup> to 1737 and 1658 cm−<sup>1</sup> for HS-loaded liposomes, which may correspond to the possible interaction of HS with carbonyl ester groups at the interfacial part of the liposomal bilayers [30].

For the HS-loaded γ-oryzanol-chitosomes (Figure 3d), a shift from 3379 to 3415 cm−<sup>1</sup> was detected, which may correspond to hydrogen bonding between hydroxyl or amino groups of chitosan and carboxylic acid or amino groups of hydrolysates. Moreover, after chitosan coating, significant changes in the absorption bands of acyl chains (3000–2800 cm–1) were detected. These peaks were converted into two narrow and intense peaks at 2862 and 2924 cm−<sup>1</sup> in the case of chitosan-coated liposomes. Further evidence for electrostatic conjugation of chitosan on liposome surface was a considerable shift to higher frequencies in the carbonyl group (from 1737 to 1739 cm–1). This indicates that the carbonyl groups are involved with cationic groups of chitosan, resulting in the destruction of some hydrogen bonds [31].
