Physicochemical characterization of liposomes produced by ultrasonication and coated with pectin for the coencapsulation of vitamins D3 and B12

Abstract

Liposomes represent a versatile platform for the coencapsulation of hydrophilic and hydrophobic bioactive compounds, thereby increasing their potential for micronutrient delivery in food products. In this study, vitamins D3 (VD3) and B12 (VB12) were coencapsulated within liposomes produced via ultrasonication using hydrogenated or nonhydrogenated phospholipids. Additionally, pectin was investigated as a coating material for phospholipid vesicles, with the goal of potentially enhancing their stability during storage. The diameters of the uncoated liposomes ranged from 98 to 392 nm, depending on the type of phospholipid used, and all formulations exhibited stability over a 30-day period. Pectin-coated liposomes had diameters ranging from 177 to 236 nm, and their stability was largely influenced by the use of unsaturated phospholipids, with the polysaccharide concentration impacting long-term stability. Coencapsulation of VD3 and VB12 was successfully achieved in both uncoated and coated liposomes, with VD3 demonstrating significantly greater encapsulation efficiency than VB12. Notably, both vitamins were effectively protected from degradation during storage.

Introduction

Liposomes are phospholipid-based vesicles capable of encapsulating both hydrophilic and hydrophobic molecules within their structure. The amphiphilic nature of phospholipids enables liposomes to entrap lipophilic compounds within their phospholipid bilayers while simultaneously accommodating hydrophilic molecules in their aqueous internal core (Lasic, 1998). These intrinsic properties render liposomes highly versatile delivery systems, with coencapsulation emerging as an effective and widely adopted strategy in pharmaceutical applications (Chaves & Pinho, 2021; Gürsoy et al., 2004; Halwani et al., 2008).
Coencapsulation within liposomes also represents an efficient method for enhancing the delivery of micronutrients in food products. Vitamins, among the most commonly used micronutrients used for food fortification, have garnered significant attention in recent years for their encapsulation within liposomes (Chaves et al., 2023). This growing interest arises from the numerous advantages offered by liposomes as encapsulation and delivery systems, such as their high biocompatibility, versatility, potential for targeted delivery, controlled release, and availability of various production methods (Emami et al., 2016; Thompson et al., 2007). Vitamins D3 and B12 are essential micronutrients in human nutrition, with particular significance for elderly individuals, given the physiological changes associated with aging. Ensuring their adequate absorption is crucial in this context. Consequently, there is an increasing need to develop strategies that facilitate the delivery of these vitamins in sufficient quantities, potentially through coencapsulation of cholecalciferol and cobalamin. Such strategies are particularly vital, as elderly individuals may experience a reduced appetite, changes in eating patterns due to chewing difficulties, or conditions such as dysphagia, which can lead to micronutrient malabsorption and deficiencies in VD3 and VB12 (Barkoukis, 2016).
With respect to VD3 (cholecalciferol), there is a notable decline in the ability of the skin to synthesize this vitamin as a result of the aging process. VD3 functions as a steroid hormone and plays a pivotal role in maintaining calcium and phosphorus homeostasis, promoting bone health, and potentially mitigating the risks of conditions such as diabetes, cardiovascular diseases, and certain types of cancer (Chaves & Pinho, 2021; Tan et al., 2019). In contrast, VB12 is essential for red blood cell production and DNA synthesis, and its deficiency can lead to anemia, cognitive impairment, and neurological disorders (Chaves et al., 2023).
The coencapsulation of these two vitamins therefore has considerable potential for enhancing food formulations. However, the use of liposomes as delivery systems presents certain limitations, particularly their susceptibility to harsh conditions within the gastric environment, an issue that is especially relevant in the context of food as an oral delivery medium. A promising strategy to overcome this challenge involves coating phospholipid vesicles, thereby increasing their resilience to factors such as pH fluctuations, ionic strength, and enzymatic activity within the gastrointestinal tract (Pasarin et al., 2023). Polysaccharides, including chitosan and pectin, are frequently used as coating materials for orally administered liposomes (Caddeo et al., 2016; Chen et al., 2022; Li et al., 2022; Salehi et al., 2022; Tan et al., 2024; Nguyen et al., 2011; Li et al., 2014; Lopes et al., 2019; Feng et al., 2020; Šeremet et al., 2022), alongside proteins such as whey (Pan et al., 2020; Pan et al., 2022; Tamaddon et al., 2021; Yi et al., 2019). Studies have also explored the use of combinations of these materials and/or other polymers in coatings (De Leo et al., 2021), such as pectin-chitosan (Gibis et al., 2013; Hamadou et al., 2022; Karim et al., 2020), pectin-whey (Nazeer et al., 2019), chitosan-zein (Mehdizadeh et al., 2021), sodium alginate-chitosan (Tan et al., 2023), pectin-WPI (Gomaa et al., 2017), and Eudragit® S100/PEG-2000 (De Leo et al., 2023). Pectin was the polysaccharide selected for liposome coating in this study because of its favorable properties. It is an abundant polysaccharide widely used in the food industry as a gelling, thickening and emulsifying agent, in addition to its applications in edible coatings and films (Kalita et al., 2025; Roy et al., 2023). Pectin is classified as generally recognized as safe (GRAS) by the Food and Drug Administration (Espitia et al., 2014). Another emerging application of pectin is its use as a controlled-release agent in the pharmaceutical industry (Nguyen et al., 2011; Zhou et al., 2014), as it is resistant to low pH conditions and enzymatic digestion by proteases and amylases in the stomach, with degradation occurring only in the intestinal tract (Ribeiro et al., 2014). Given these characteristics, pectin is a promising polymer for liposome coating (Nguyen et al., 2011).
This study involved the synthesis of liposomes via ultrasonication using hydrogenated or nonhydrogenated phospholipids. Liposomes were produced with and without a pectin coating, enabling comparative analysis. These formulations were designed for the coencapsulation of vitamins D3 and B12. Ultrasonication parameters, including the number of cycles and amplitude, were optimized to achieve the desired vesicle characteristics. Following optimization, the liposomes were subjected to comprehensive characterization, including particle size analysis, zeta potential measurement, encapsulation efficiency evaluation, and an assessment of the stability of both liposomes and encapsulated vitamins during storage. The primary objective was to develop and thoroughly characterize a lipid-based carrier system aimed at the targeted delivery of essential vitamins, with a particular emphasis on addressing the dietary requirements of elderly populations in food products.

Materials

Liposomes were produced using two types of soy-derived purified lecithin: Phospholipon 90G (P90G, >94 % w/w phosphatidylcholine (PC), hydrogenated) and Lipoid S45 (PS45, >45 % w/w PC, 10–18 % phosphatidylethanolamine (PE), nonhydrogenated), both of which were sourced from Lipoid GmbH (Ludwigshafen, Germany). Apple pectin, with a degree of esterification ranging from 50 % to 75 %, was obtained from Sigma–Aldrich (St. Louis, MO, USA). Vitamin D3 (VD3, cholecalciferol, >98 % purity) and vitamin B12

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Letícia S. Ferreira, Beatriz B. da Silva, Matheus A. Chaves, Samantha C. Pinho, Physicochemical characterization of liposomes produced by ultrasonication and coated with pectin for the coencapsulation of vitamins D3 and B12, Food Chemistry, Volume 485, 2025, 144441, ISSN 0308-8146,
https://doi.org/10.1016/j.foodchem.2025.144441.