Several naturally occurring nanofoods and their modified products serve as excellent carriers for the nanoencapsulation of pharmaceuticals, nutrients, and supplements. These carriers offer improved stability and bioavailability (Pushparaj et al., 2022). They primarily include biological macromolecules such as polysaccharide (Guo et al., 2021, Hua et al., 2021), and proteins (Zou, et al., 2016), as well as small lipid molecules like phospholipids (Semenova et al., 2021). For example, soybean phospholipid nanoliposomes have been shown to enhance the bioavailability of flavonoids (Ayala-Fuentes & Chavez-Santoscoy, 2021). Cyclodextrin (CD), a product of starch hydrolysis, can be utilized to optimize the physicochemical properties of essential oils (Cid-Samamed et al., 2022). Moreover, the incorporation of low concentrations of CD in the aqueous core of liposomes preserves the integrity of the membrane without impacting the characteristics of the liposomes (Lim et al., 2021). Therefore, the combination of drug-in-cyclodextrin-in-liposome (DCL) represents an intriguing carrier that harnesses the advantages of both CD and liposome carriers (Baldim et al., 2022).

CDs belong to the family of cage molecules due to their structure, which features a hydrophobic cavity capable of encapsulating hydrophobic molecules through host–guest complexation (Braga, 2022). The most commonly encountered CDs consist of six (α-CD), seven (β-CD), and eight (γ-CD) glycosyl units (Kurkov & Loftsson, 2013). To address the limitations of native cyclodextrins, their properties can be enhanced through the synthesis of derivatives. These derivatives aim to increase water solubility, enhance inclusion capacity, or reduce toxicity (Przybyla et al., 2020). One widely utilized derivative is hydroxypropyl-β-CD (HP-β-CD), an etherified derivative of β-CD. It finds extensive application in the food and pharmaceutical industries due to its high solubility and safety profile.

Limonene (Lim) is a monoterpene essential oil with a pleasant lemon-like odor. It is typically obtained through the cold pressing of citrus peel wastes, where it can be found at concentrations exceeding 90 % (Siddiqui et al., 2022). Due to its strong citrus flavor and abundant availability in nature, Lim finds widespread use in the food industry. Additionally, Lim offers a diverse range of health benefits, including antioxidant, antiarrhythmic, anti-cancer, anti-inflammatory, and antibacterial activities (Marinho et al., 2022, Ravichandran et al., 2018). However, the utilization of Lim is subject to certain limitations due to its instability, volatile nature, and low solubility in water (Ibanez et al., 2020).

We employed a drug-in-cyclodextrin-in-liposome (DCL) formulation to encapsulate Lim, aiming to address its limitations. The formulation consisted of two main segments: the Lim/HP-β-CD inclusion complex (IC) was first prepared and then encapsulated into liposome using the ethanol-injection method. Although previous studies have utilized DCL for encapsulating essential oil ingredients (Hammoud et al., 2020, Sundar and Parikh, 2023), none have specifically encapsulated Lim using this approach. Moreover, the characterization methods for DCL formulations during preparation have been limited. The complexation mechanism of Lim-IC was analyzed X-ray Diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and Thermogravimetry (TGA) technologies. Therefore, this study aimed to extensively elucidate the structural characteristics of the DCL formulation using multiple techniques and highlight its advantages as a nanocarrier for encapsulating Lim.