*2.1. Shape and Size of CS-TPP Encapsulated CBLO Nanoparticles*

Refined cobia liver oil (CBLO) with an acid value of 0.15 mg KOH g−<sup>1</sup> was used as the core material for encapsulation. The fatty acid profile of the CBLO is presented in Figure 1. The CBLO contained 24.52% total ω-3 PUFAs (18.85% DHA, 4.25% EPA, and 1.42% α-linolenic acid).

**Figure 1.** GC analysis of (**a**) the fatty acid profile of CBLO, (**b**) EPA standard, and (**c**) DHA standard.

The two-step method used for the fabrication of CS@CBLO NPs is illustrated in Scheme 1. The first step was emulsification; the chitosan was treated with the surfactant reagent Tween 80 for entrapment. CBLO gained an oil-in-water micelle structure. The second step was the solidification of the micelles by the ionic gelation of chitosan with TPP to form CS@CBLO NPs. The morphology of the obtained particles was observed by SEM. As shown in Figure 2a,b, both CS NPs and CS@CBLO NPs exhibit a spherical shape and nanosized structure. The particle sizes of CS NPs and CS@CBLO NPs measured by SEM were 726 ± 136 nm and 347 ± 118 nm, respectively. The smaller particle size of CS@CBLO NPs was due to the homogenization dispersing the hydrophobic CBLO in the solution and forming micelles with the surfactant. While the CS NPs were formed by the electrostatic interaction of chitosan and TPP, the CS@CBLO NPs were formed by the ionic gelation of chitosan absorbed on the micelle. The hydrophobic CBLO in the micelle reduced the aggregation of chitosan on the micelle surface and thus produced smaller particles.

The z-average diameter, PDI, and Zeta potential of CS NPs and CS@CBLO NPs were examined by DLS. Figure 3 shows that the z-average diameter of CS NPs is ~658 nm, while the z-average diameter of CS@CBLO NPs is between 174 and 456 nm. The DLS analysis was carried out in the aqueous environment; thus, the particle size would depend on the extent of the aggregation or swelling of the chitosan. Since the CBLO was entrapped inside the CS@CBLO NPs, the hydrophobicity of CBLO decreased the aggregation and/or the swelling of chitosan, resulting in the formation of smaller particles, meaning that the z-average diameter of CS@CBLO NPs decreased with the increasing ratio of CBLO to chitosan. This phenomenon was similar to that seen in other studies [36,37].

**Scheme 1.** The two-step method used for fabrication of CS@CBLO NPs via emulsification and ionic gelation.

**Figure 2.** SEM images of (**a**) CS NPs and (**b**) CS@CBLO NPs (weight ratio of CBLO:chitosan at 1:1) at 2 kV. (**i**) and (**ii**) are the measured magnification at 5000× and 15,000×, respectively.

**Figure 3.** Z-average diameter, PDI and zeta potential of CS NPs and CS@CBLO NPs with different weight ratios of CBLO to chitosan. Data are the mean + standard deviation (*n* = 3).

PDI is a key parameter showing the quality of the size distribution of nanoparticles in suspensions. With a PDI lower than 0.3 and a single peak in the size distribution curve, it is considered to be monodisperse-sized dispersion [38]. In Figure 3, it can be seen that the highest PDI value was found to be 0.35 for CS NPs, while CS@CBLO NPs had a lower PDI value of 0.22–0.27. The results indicated that CS@CBLO NPs were a monodisperse dispersion with a low variability and no aggregation as compared to CS NPs. In addition, the zeta potential measurement is also shown in Figure 3. The zeta potential of CS NPs showed a positive charge of +36.9 mV contributed by the protonated amino group (NH3 +) of chitosan. On the contrary, the zeta potential of CS@CBLO NPs decreased as the ratio of CBLO to chitosan increased. It has been reported that the zeta potential is related to the number of TPP and chitosan charge groups [39]. With the increasing CBLO, the relative proportion of TPP and chitosan decreased. Moreover, the reason for the decreasing zeta potential may be due to the shielding effect of CBLO reducing the surface charge of chitosan. Several studies have pointed out that when the chitosan encapsulated carvacrol [40], krill oil [36], and eugenol [41], the positively charged surface was reduced with the increasing initial content of the loaded drugs.
