**1. Introduction**

Cobia (*Rachycentron canadum*) are a medium-sized migratory carnivorous fish that is widely distributed in tropical marine locations. Cobia has a high economic value and is a fish species that is popular for commercial aquaculture, with a global production of ~43,000 tons per year [1]. Cobia are mainly processed into fillets, with by-products of around 65% of the total weight generated [2,3]. Cobia liver is one of these waste products, accounting for 4% of the total weight and containing 46~48% fat; it is a potential raw material that could be used for producing fish oil. Cobia liver oil (CBLO) is rich in polyunsaturated fatty acids (PUFAs), such as DHA (Docosahexaenoicacid) and EPA (Eicosapentaenoic acid) [4]. DHA and EPA have been reported to possess anti-inflammatory

**Citation:** Chang, P.-K.; Tsai, M.-F.; Huang, C.-Y.; Lee, C.-L.; Lin, C.; Shieh, C.-J.; Kuo, C.-H. Chitosan-Based Anti-Oxidation Delivery Nano-Platform: Applications in the Encapsulation of DHA-Enriched Fish Oil. *Mar. Drugs* **2021**, *19*, 470. https://doi.org/ 10.3390/md19080470

Academic Editors: Maria do Rosário Domingues and Philippe Soudant

Received: 5 August 2021 Accepted: 20 August 2021 Published: 22 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and antidiabetic activities and can reduce the risk of cardiovascular disease, cancer, and Alzheimer's disease [5–7]. However, DHA and EPA are unstable due to the risk of oxidation, as the rate of oxidation increases with the number of double bonds in a fatty acid [8,9]. Oxidation exerts negative effects on flavor and nutritional value, thereby limiting the application of PUFA-enriched fish oil in nutritional supplements.

Reducing exposure to oxygen is one way to avoid the oxidation of fish oils. The encapsulation of lipophilic compounds in biocompatible materials with a nano-technique is a feasible means for achieving this purpose [10,11]. As a result, the encapsulation of CBLO can improve its stability, helping to avoid irradiation, oxidation, and thermal degradation while reducing the fishy smell. Chitosan, a cationic polysaccharide with an outstanding biocompatibility and biodegradability, is widely applied in the field of biomedicine [12,13]. The structure of chitosan comprises β(1,4)-linked D-glucosamine and *N*-acetyl-D-glucosamine; the pKa value of the primary amine is around 6.5, depending on the degree of *N*-deacetylation [14]. Chitosan is pH-sensitive due to the amino groups of Dglucosamine possessing a positive charge at pHs below 6, which cause chitosan to become a water-soluble cationic biopolymer. However, chitosan is insoluble in physiological conditions at neutral pH. Such characteristics make chitosan suitable for the encapsulation and delivery of lipophilic compounds [15,16]. In particular, chitosan exhibits many health benefits, helping one to resist ulcers, lowering cholesterol, reducing blood lipids, and helping in the prevention of coeliac disease [17,18]; based on the above benefits, chitosan is a suitable wall material that can be used for the encapsulation of drugs [19] and can help to achieving the purpose of controlled delivery [20].

Recently, several nanoencapsulation processes utilizing chitosan have been developed, such as ionic gelation [21], electrospinning [22], emulsion-homogenization [23], self-assembly [24], and antisolvent precipitation [25]. The ionic gelation method is the favorite among these, as it is non-toxic, non-solvent, and easily controllable. The ionic gelation technique is based on the electrostatic interaction between the positively charged amino groups of chitosan and the negatively charged groups of anions (such as sodium tripolyphosphate, TPP) to form a safety component, CS-TPP nanoparticles (CS NPs) [26,27]. CS NPs have been used for loading insulin and also applied in diabetes therapy [28]. Moreover, several food bioactive ingredients have been encapsulated in CS NPs, including curcumin [29], flavonoids [30], lutein [31], polyphenols [32], resveratrol [33], and vitamins (B9, B12, and C) [34]. Fish oils are rich in ω-3 PUFAs, which are highly prone to oxidation due to their higher content of unsaturated fatty acid undergoing lipid oxidation. The encapsulation of fish oil can protect unsaturated fatty acids against oxidation and help to avoid unwanted reactions [35]. However, as there is little literature available regarding CS NPs encapsulated fish oil, the effect of encapsulation on preventing the oxidation of ω-3 PUFAs is worthy of study.

In this study, the encapsulation of CBLO containing ω-3 PUFAs by chitosan at the nano-scale was explored. A two-step procedure (emulsification and ionic gelation) was performed to fabricate CS-TPP-encapsulated CBLO nanoparticles (CS@CBLO NPs). The characterizations of CS@CBLO NPs were investigated by scanning electron microscopy (SEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The effects of the initial CBLO content on the encapsulation efficiency (EE) and loading capacity (LC) were also investigated. Finally, the effect of CS@CBLO NPs on the oxidative stability of CBLO was evaluated.
