Abstract
Lipophilic bioactive compounds often exhibit poor aqueous solubility, chemical instability and low intestinal absorption, limiting their effectiveness in nutraceutical and pharmaceutical formulations. This study aimed to develop gastro-resistant spray-dried microparticles (MPs) capable of encapsulating and modulating the release of selected liposoluble molecules (fisetin, quercetin, β-carotene, vitamin A acetate and vitamin E). Five formulations (MILC-Fis, MILC-Q, MILC-β-car, MILC-Vit.A and MILC-Vit.E) were prepared within the innovative MILC® matrix (Micro Intelligent Lite Carrier), composed of milk and whey proteins (WPs), to obtain microparticles prepared exclusively with food-based materials. Scanning electron microscopy (SEM) analysis confirmed the formation of predominantly spherical particles with a mean diameter of approximately 15 ± 5 μm. MPs showed high encapsulation efficiencies, up to 79.6% for MILC-Q and 75.7% for MILC-Fis, exhibited pH-dependent release profiles with minimal release under gastric conditions (pH 1–2) and enhanced release at intestinal pH (6.8). In vitro studies demonstrated a significant increase in bioaccessibility, while in vivo pharmacokinetic evaluation revealed an improvement in bioavailability, with MILC-Q and MILC-Fis increasing the AUC by 8.2-fold and 2.47-fold, respectively, compared with the non-encapsulated compounds. These results highlight the potential of the MILC® protein matrix to protect lipophilic bioactives during gastric transit and enhance their intestinal availability and systemic exposure, supporting its applicability as a food-grade delivery platform for poorly soluble nutraceuticals and functional food formulations.
Introduction
Lipophilic bioactive compounds, such as fat-soluble vitamins and selected polyphenols, are increasingly studied for their broad spectrum of antioxidant, anti-inflammatory and cytoprotective activities. Despite their biological relevance, their incorporation into food, nutraceutical and pharmaceutical formulations is often compromised by intrinsic physicochemical limitations, including poor aqueous solubility, susceptibility to oxidative and thermal degradation and low intestinal absorption. These challenges underline the need for oral delivery systems capable of improving their stability, protecting them during gastrointestinal transit and enhancing their bioavailability.
Vitamins represent essential micronutrients required to sustain normal physiological functions and overall human health, acting as key regulators of growth, metabolism and tissue maintenance [1,2]. Because humans cannot synthesize them endogenously, the recommended intake levels established by the RDA (Recommended Daily Allowance) must be achieved primarily through dietary sources [3]. Situations involving insufficient intake, impaired absorption, or increased metabolic demand can lead to states of hypovitaminosis, which are associated with a wide range of clinical disorders [4,5]. Under such conditions, the use of oral vitamin supplements in addition to the regular diet is commonly advised [6].
Vitamins are generally categorized into water-soluble and fat-soluble classes. Water-soluble vitamins contain ionizable or polar functional groups and dissolve readily in aqueous environments; they are not extensively stored in the body and are rapidly excreted, which necessitates continuous dietary replenishment. Fat-soluble vitamins, including β-carotene, vitamin A and vitamin E, are essential nutrients involved in key physiological processes [7–9]. However, they are prone to oxidation, sensitive to light and heat, and generally characterized by variable absorption. Their predominantly hydrophobic nature requires incorporation into mixed micelles to allow intestinal absorption, and their stability is strongly influenced by formulation parameters and exposure to environmental stressors.
Similarly, flavonoids such as fisetin and quercetin exhibit high antioxidant, neuroprotective and anti-inflammatory potential [10–15], but suffer from limited water solubility and rapid degradation under physiological conditions, resulting in poor intestinal uptake. In fact, despite their importance, the efficacy of lipophilic vitamins and flavonoids as oral supplements is often compromised. Their hydrophobic nature, susceptibility to degradation processes and limited permeability across biological membranes frequently result in low bioavailability [16]. Consequently, encapsulation strategies that improve their dispersibility, protect them from degradation and modulate their release profiles have become critical to support their efficient oral delivery [17]. Microparticulate formulations, in particular, are promising tools for stabilizing lipophilic compounds, improving dissolution, permeability, and gastrointestinal stability while reducing the risk of hypervitaminosis, minimizing adverse effects, and permitting less frequent administration [18,19].
Microencapsulation using biocompatible, food-grade materials has emerged as a promising approach to address these limitations, offering improved dissolution and gastrointestinal stability while reducing degradation and enabling controlled release [20,21]. In this context, milk and whey proteins have gained attention due to their biocompatibility, food-grade status, favourable emulsifying properties, ability to interact with hydrophobic molecules and capacity to form protective, structured matrices around bioactive compounds [22,23]. These proteins can stabilize emulsions, limit oxidation and improve the gastrointestinal resistance of sensitive ingredients, thereby contributing to improved intestinal availability and controlled release [24].
Among the various encapsulation technologies, spray drying is one of the most widely used due to its efficiency, rapid solvent removal, scalability, cost-effectiveness and compatibility with thermosensitive compounds when properly optimized [25]. The technique involves atomizing a feed liquid, such as an emulsion or dispersion, in a stream of hot air, resulting in rapid evaporation of the solvent and the formation of dry microparticles and stable powders [26] and is widely used in both the food and pharmaceutical industries to protect bioactive substances from degradation [27]. Moreover, when combined with appropriate formulation strategies, spray drying can generate gastro-resistant systems capable of preserving bioactive compounds in acidic environments while enabling their selective release under intestinal pH conditions [30].
Despite the widespread application of spray drying, a protein-based gastro-resistant encapsulation platform exclusively based on food-grade milk and whey proteins, capable of simultaneously improving the stability, intestinal release and in vivo bioavailability of lipophilic bioactives, remains insufficiently explored. Existing studies typically employ synthetic polymers, gums, polysaccharides and mixed excipient systems, while protein-only matrices have been primarily investigated for emulsification rather than for targeted protection during gastric transit. This represents a relevant gap in the development of natural, food-compliant delivery systems capable of simultaneously improving stability, in vitro bioaccessibility and in vivo bioavailability of hydrophobic compounds [31].
Given these premises, the present study aimed to develop and characterize gastro-resistant microparticles by spray drying loaded with fisetin, quercetin, β-carotene, vitamin A and vitamin E, employing an innovative milk and whey protein matrix MILC® (Micro Intelligent Lite Carrier) as the encapsulating system. The resulting formulations were investigated for their morphological features, pH-responsive release profiles and in vivo bioavailability to assess the potential of the MILC® matrix as a natural, food-grade platform for improving the protection, stability and intestinal uptake of lipophilic bioactive compounds.
Materials
Ethanol, Dulbecco’s Phosphate Buffer Saline (DPBS), methanol (MeOH), trifluoroacetic acid (TFA), octanol, EDTA, were purchased from Sigma-Aldrich, Italy. Retinyl acetate, β-carotene, α-tocopherol and quercetin 95% were purchased from A.C.E.F., Italy; Fisetin 98% was purchased from Hansen Supplements; HCl and NaOH were purchased from VWR, Italy; gum arabic and soy lecithin were purchased from Galeno, Italy; WPs (purity ≥90%) were purchased from Bulk Powders, England. Phytosomal quercetin (Quercetin Phytosome®; Indena S.p.A., Milan, Italy, declared drug loading 34-42%) was purchased by Farmalabor Srl.
For in vivo studies, Wistar rats (weighing 250-400 g), purchased from ENVIGO Srl (San Pietro al Natisone UD, Italy), were employed throughout the study. The animals were housed in temperature-controlled rooms with a 12-h light cycle at 22–24 °C and 50–60% humidity. They were fed standard laboratory chow and tap water ad libitum. Animals were allowed to acclimate to the housing conditions for 1 week before starting the experiment. Procedures involving animals and their care were conducted in conformity with the Italian D.Lgs 26/2014 and following the provisions of the European Community Council Directive 210/63/UE, recognized and adopted by the Italian Government. The experiments were approved by the Ethical Committee for Animal Experimentation of the University of Palermo and by the Italian Ministry of Health (Authorization n. 56/2023-PR). All animal experiments comply with the ARRIVE guidelines.
Download the full article as PDF here Controlled release and enhanced bioavailability of spray-dried microparticles loaded with liposoluble vitamins and natural flavonoids quercetin and fisetin
or continue reading here
Maria Grazia Zizzo, Francesca Terracina, Carla Buzzanca, Sergio Scirè, Rosa Maria Dina, Noemi Aloi, Francesco Montalbano, Riccardo Messina, Gaetano Caldara, Mariano Licciardi, Controlled release and enhanced bioavailability of spray-dried microparticles loaded with liposoluble vitamins and natural flavonoids quercetin and fisetin, Journal of Drug Delivery Science and Technology 121 (2026) 108287. https://doi.org/10.1016/j.jddst.2026.108287
