*3.1. Materials*

Crude cobia liver oil was extracted from cobia liver using homogenization in addition to the sonication method [53]. Briefly, 100 g of cobia liver was homogenized with 1 L hexane using a Polytron PT2100 homogenizer (Kinematica, Littau, Switzerland) equipped with a Polytron PT-DA 2120/2EC probe at 15,000 rpm for 2 min at room temperature, followed by treatment in an ultrasonic bath (40 kHz, Delta D400H, Taipei, Taiwan) for 1 h. The mixture was centrifuged at 3000 rpm for 5 min to remove the solid cobia liver. The supernatant was transferred to a rotary evaporator at 70 ◦C in order to remove the hexane and recover the oil. The refined cobia liver oil (CBLO) was obtained via degumming, neutralization, and bleaching, according to the procedures described previously [4]. The CBLO containing 24.52% total ω-3 PUFAs (18.85% DHA, 4.25% EPA, and 1.42% α-linolenic acid) was stored at −20 ◦C until further use. Fatty acid methyl esters of the standards of DHA and EPA and CBLO were prepared via saponification and methylation. The fatty acid composition was analyzed using the GC method by the Center for Aquatic Products Inspection Service, NKUST. cis-4,7,10,13,16,19-Docosahexaenoic acid was purchased from Acros (Fair Lawn, NJ, USA). cis-5,8,11,14,17-Eicosapentaenoic acid was purchased from TCI Co., LT. (Tokyo, Japan). Fatty acid methyl ester standards (Supelco 37 Component FAME Mix, Catalog No. 47885) and BF3-methanol reagent (14% BF3 in CH3OH, *w/v*) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chitosan (degree of deacetylation of 94.42%) with an average molecular weight of 350 kDa was obtained from Charming & Beauty Co., Ltd. (Taipei, Taiwan). Tween 80 was purchased from Scharlau Chemical Reagent Co., Ltd. (Barcelona, Spain). Acetic acid was purchased from Riedel-de Haën (Seelze, Germany). The sodium tripolyphosphate (TPP) and sodium hydroxide were purchased from Wako Pure Chemical Industries (Osaka, Japan). Unless otherwise specified, all the reagents and chemicals used in this study were of analytical grade. All of the experiments were carried out using double-deionized water (18.2 Ω) using a machine from Merck Millipore.

#### *3.2. Fabrication of CS-TPP Encapsulated CBLO Nanoparticles*

The CS@CBLO NPs was prepared by a two-step method—i.e., via emulsification and ionic gelation [54]—with some modification. Briefly, chitosan powder was added to 1% (*v/v*) aqueous acetic acid solution at room temperature and stirred gently overnight to prepare the 1.5% (*w/v*) chitosan solution. The chitosan solution was centrifuged at 8000 rpm for 20 min, and the supernatant was collected for further use. Afterwards, 40 mL of chitosan solution was added to 0.5 g of Tween 80 and stirred at 45 ◦C for 2 h to obtain a transparent solution. The CBLO was gradually dropped into the transparent chitosan solution (40 mL) during homogenization (Polytron PT2100 with PT-DA 2112/2EC probe, Kinematica, Littau, Switzerland) at a speed of 13,000 rpm for 1 min and 16,500 rpm for 2 min to obtain an oil-in-water emulsion. Emulsions with different weight ratios of CBLO to chitosan (0:1, 0.25:1, 0.5:1, 0.75:1, 1:1, and 1.25:1, respectively) were prepared. After emulsification, 40 mL of TPP solution (0.5%, *v/v*) was gradually dropped into the emulsion under continuous stirring for 40 min. The formed particles were harvested using centrifugation at 10,000 rpm for 30 min, then subsequently washed several times with deionized water. Finally, the wet particles were dispersed in 25 mL of water by Q700 sonicator (Qsonica, CT, USA) to produce a homogeneous suspension. The ultrasonication was carried out in an ice bath operated at 30% amplitude for 2 min. The suspensions were immediately freeze-dried at −40 ◦C for 72 h to obtain the final product, CS@CBLO NPs, and were stored in dry conditions at room temperature.

#### *3.3. Morphology of Nanoparticles*

SEM was used to observe the morphology of the CS NPs and CS@CBLO NPs. One drop of the sample (50 μg/mL) was placed on a glass plate and dried at room temperature. The dried sample was sputter-coated with gold and then observed through an environmental scanning electron microscope (ESEM; FEI Quanta-200, Brno-Cernovice, Czech Republic) ˇ under an accelerated voltage of 20 keV. The average particle size was measured from SEM images using an image analyzer (SigmaScan Pro 5, Chicago, IL, USA); in total, about 50 particles were measured.
