**1. Introduction**

Naringenin (5,7,4-trihydroxyflavanone) and naringin (5,7,4-trihydroxyflavone 7- rhamno-glucoside) are flavones abundantly existent in fruits, including grapefruit, tomato skin and oranges [1]. Naringin can be formed by naringenin at the position of the 7th carbon atom with neohesperidose (Figure 1), which promotes its solubility in water. Their health-promoting features have been explored in vivo and in vitro models, such as antioxidant activity [2,3], anti-inflammatory activity [4,5], anticancer activity [6,7], anti-diabetic activity [8] and protective effects on the central nervous system [9] and the cardiovascular system [10]. However, naringenin and naringin are hydrophobic and low-soluble polyphenols with low water solubility of 38 and 500 μg/mL at room temperature, respectively [11,12], and taste bitter, which greatly limits their application in clinical and functional foods. To overcome these limits, the solubility and bioavailability of naringenin and naringin have been improved by encapsulation in various kinds of carefully designed

**Citation:** Chen, M.; Li, R.; Gao, Y.; Zheng, Y.; Liao, L.; Cao, Y.; Li, J.; Zhou, W. Encapsulation of Hydrophobic and Low-Soluble Polyphenols into Nanoliposomes by pH-Driven Method: Naringenin and Naringin as Model Compounds. *Foods* **2021**, *10*, 963. https://doi.org/ 10.3390/foods10050963

Academic Editors: Hong Wu and Hui Zhang

Received: 26 March 2021 Accepted: 27 April 2021 Published: 28 April 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/).

colloidal delivery systems, including cyclodextrins complexation [12], nanovehicles [13,14], liposomal [15] and ternary complex particles [16].

**Figure 1.** Three dimensions conformer of naringenin and naringin and their hydroxyl groups are non-ionized in acidic condition and negatively charged in alkaline condition.

The pH-driven method is a new method for constructing a delivery system, which has received extensive attention due to its easy-to-handle, cost-saving, energy-saving and organic solvent-free properties during operation. The process of this method includes dissolving the bioactivator under strongly alkaline conditions and then adjusting the pH to neutral or acidic after blending with the carrier system. Hydrophobic polyphenols like curcumin have poor solubility in neutral acidity, while they can be dissolved due to deprotonation at alkaline pH. According to this property of some polyphenols, Pan et al. firstly loaded curcumin into casein nanoparticles by the pH-driven method [17]. Subsequently, a series of studies on the colloidal delivery systems based on the pH-driven method began to emerge, especially for curcumin encapsulation. The successful preparation of a series of colloidal carrier systems loaded with curcumin enhanced its water solubility and bioavailability, which proved the feasibility of preparing the delivery system with the pH-driven method [18–25]. However, the pH-driven method has rarely been applied for the encapsulation of other hydrophobic polyphenols. A potential disadvantage of this method is that the encapsulation of polyphenols needs to be carried out under a highly alkaline conditions, which could facilitate the chemical degradation of polyphenols [26]. Peng and co-workers have demonstrated that the pH-driven method can be used to load certain kinds of lipophilic polyphenols such as curcumin and resveratrol but cannot encapsulate quercetin because the degradation rate of quercetin was extremely fast at alkaline pH values [26].

Liposomes are microcosmic phospholipid vesicles with a bilayered membrane structure. The hydrophilic segments of the phospholipids are located on the inside and outside surface of liposomes, while the hydrophobic tail forming lipid bilayers are separated from aqueous phase [27]. Nanoliposome is a nanometric versions of liposome, which can load bioactive substances with different lipophilicities into the various parts of liposomes, such as phospholipid bilayers, hydrophilic core or bilayer interface [28]. Therefore, nanoliposomes are an extremely promising encapsulation technology in the field of nutraceutical products. The superiority of nanoliposome technology is embodied in the protection of sensitive bioactivators, storage stability, excellent loading capacity, improved bioavailability and sustained-release ability [29]. The utilization of high-pressure homogenizer to prepare liposomes can overcome the traditional liposome preparation method that uses detergents or organic solvents that are either undesirable or not allowed ingredients in foods [25].

Naringenin and naringin, with hydrogen donor count as 3 and 8, respectively, whose hydroxyl groups are non-ionized under acidic conditions (Figure 1). When the pH changes to alkaline, they are easy to be negatively charged due to the deprotonation of the hydroxyl group, which extremely enhances their hydrophilic ability. We hypothesized that naringenin and naringin lose their charges due to the protonation of the same group under neutral and acidic conditions, which would reduce their water solubility. Therefore, when the naringenin/naringin alkaline solution and nanoliposome solutions are mixed, the pH is adjusted to an acidic condition, which causes the naringenin or naringin to be embedded into the hydrophobic region within nanoliposomes. In this study, we first determined the stability of naringenin and naringin under alkaline conditions and the change in solubility of naringenin and naringin in the transition from pH 12.0 to neutral or acidic were also measured, which ensured that they can be encapsulated into liposomes by the pHdriven method. Subsequently, nanoliposomes with different concentrations of naringenin or naringin and different contents of lecithin were prepared by microfluidazition and pH-driven methods, and their encapsulation efficiency and maximum loading capacity were determined. The structures of bioactivator-loaded nanoliposomes, particle size and ζ-potentials of nanoliposomes were determined to understand the bioactivator-loaded procedure using the pH-driven method. Finally, the stability of the naringenin-loaded nanoliposomes was studied. This study may lead to develop novel food-grade colloidal delivery systems and apply to introducing naringenin and naringin into foods, supplements, or drugs.
