Hydroxytyrosol encapsulated in biocompatible water-in-oil microemulsions: How the structure affects in vitro absorption

Highlights

• W/O microemulsions were formulated using non-ionic surfactants and edible oils.

• Nanoencapsulation of hydroxytyrosol (HT) was achieved in the microemulsions.

• DLS and EPR revealed that the bioactive molecule was localized at the interface.

• Intestinal cell coculture Caco-2/TC7 and HT29-MTX viability was not affected.

• Microemulsion’s composition strongly affected HT permeation in cells.

Abstract

Over the last years, the incorporation of natural antioxidants in food and pharmaceutical formulations has gained attention, delaying or preventing oxidation phenomena in the final products. In order to take full advantage of their properties, protection in special microenvironments is of great importance. The unique features of the natural phenolic compound hydroxytyrosol (HT) - including antioxidant, anti-inflammatory, antiproliferative and cardioprotective properties - have been studied to clarify its mechanism of action. In the present study novel biocompatible water-in-oil (W/O) microemulsions were developed as hosts for HT and subsequently examined for their absorption profile following their oral uptake. The absorption of HT in solution was compared with the encapsulated one in vitro, using a coculture model (Caco-2/TC7 and HT29-MTX cell lines). The systems were structurally characterized by means of Dynamic Light Scattering (DLS) and Electron Paramagnetic Resonance (EPR) techniques. The diameter of the micelles remained unaltered after the incorporation of 678 ppm of HT but the interfacial properties were slightly affected, indicating the involvement of the HT molecules in the surfactant monolayer. EPR was used towards a lipophilic stable free radial, namely galvinoxyl, indicating a high scavenging activity of the systems and encapsulated HT. Finally, after the biocompatibility study of the microemulsions the intestinal absorption of the encapsulated HT was compared with its aqueous solution in vitro. The higher the surfactants’ concentration in the system the lower the HT concentration that penetrated the constructed epithelium, indicating the involvement of the amphiphiles in the antioxidant’s absorption and its entrapment in the mucus layer.

Introduction

Through the last years, both food and pharmaceutical industries have shifted their focus to natural products by exploiting their beneficial properties. This trend is motivated by the avoidance of the high-cost procedures and the harmful side effects that synthetic bioactive molecules may exhibit. Recent studies have revealed many of the health-benefit properties of natural compounds including antioxidant [1], anti-proliferative [2] and anti-depressive [3] activities. Interestingly, in the pharmaceutical sector there is a growing interest for natural substances serving as lead compounds. Numerous drugs in the market are based on natural compounds from plants, whereas others are in clinical trials. Moreover, in the food industry, in order to obviate the oxidation of products during storage and to design high added-value products, companies tend to replace synthetic compounds by naturally occurring ones. Antioxidants may be defined as substances that, when present in foods, delay, control, or inhibit oxidation and deterioration of food quality [4]. The replacement of synthetic antioxidants, with natural ones, limits the necessary high-cost and time-consuming testing procedures.

Hydroxytyrosol (HT, 3,4-dihydroxyphenylethanol) is a secoiridoid of amphiphilic nature, deriving from the hydrolysis of oleuropein, with a great antioxidant capacity [5]. HT is naturally present in high concentrations in the leaves of the Olea europea tree and is recognized as one of the principal bioactive compounds of Extra Virgin Olive Oil (EVOO). Owing to its structural and molecular features, HT provides many beneficial effects after consumption. HT may act as a controller of lipid oxidation of natural oils [6] and as an antioxidant in films used for active food packaging [7]. Many studies have evidenced health benefits including neuroprotective [8], antiproliferative [9] and antimicrobial [10] properties. European Food and Safety Authority (EFSA) has claimed that HT acts as a protector of the cardiovascular system avoiding oxidation of LDL cholesterol by free radicals, maintaining normal blood HDL cholesterol concentrations and preventing atherosclerosis [11].

In order to take full advantage of the health benefits of molecules such as HT, both pharmaceutical and food industries are trying to develop strategies to incorporate them in products so that they are more soluble, stable against oxidation and degradation phenomena, with extended shelf-life, while possessing an increased bioavailability profile. To undertake these challenges, a variety of non-toxic systems such as nanoparticles [12], nanodispersions [13,14] and gelled emulsions [15], has been proposed for the incorporation of the functional compounds. Much attention has been paid to liquid-in-liquid colloidal systems, and especially nanoemulsions [16] and microemulsions [17]. Both oil-in-water (O/W) and water-in-oil (W/O) nanodispersions have been studied: the first type for the delivery of poorly water-soluble bioactive compounds and the second for antioxidants, peptides, etc. These colloidal formulations allow overcoming hurdles related to unpleasant taste, product instability and poor bioavailability. Especially, microemulsions, which are thermodynamically stable and isotropic mixtures of two immiscible phases stabilized by surfactants (and potentially co-surfactants), have been extensively studied for their ability to improve the bioavailability of peptides, drugs and other compounds [18,19]. In addition, microemulsions represent an interesting and potentially powerful alternative system for drug delivery because of their high solubilization capacity, transparency, ease of preparation, and high diffusion and absorption rates when compared with solvents without surfactant molecules. W/O microemulsions have been tested for the delivery and absorption of highly water-soluble compounds in the treatment of pathologies, such as diabetes, revealing increased intestinal bioavailability of the active molecules after their intake [20]. Nanoencapsulation ensures protection of antioxidants, controlled release and increased bioavailability. As part of the daily diet, a nutraceutical and potentially therapeutic agent, HT and its encapsulation in nanocarriers has attracted increasing attention over the last years [21]. As far as our knowledge is concerned, few references have focused on the intestinal absorption of polyphenolic molecules encapsulated in nanosystems after their consumption. Trying to elucidate the processes concerning the fate of novel formulations in the gastrointestinal tract (GIT), our group focuses on the digestion and absorption of oil-based nanodispersions and their encapsulated compounds. The gastric and intestinal digestion of W/O emulsions and microemulsions composed of medium chain triglycerides (MCT) and a variety of surfactants with encapsulated HT has been tested with a two-step digestion model using gastric and pancreatic lipases [22]. As a subsequent step, the present study aims at characterizing the intestinal absorption profile of HT encapsulated in W/O microemulsions.

Following the oral uptake of lipid-based formulations with encapsulated bioactive compounds, absorption studies allow to evaluate their bioavailability and their fate in the intestine. During the development of new products, epithelium permeability studies are required to assess the efficacy of the final product. In vitro cell culture models provide an approach to predict permeability by utilizing cell monolayers cultivated in permeable filter inserts to mimic the transport of a compound from the intestinal lumen. The cell culture models are widely used as they provide simplicity and reproducibility allowing inter-laboratory comparison of results. For the described reasons, different well-characterized cancer cell lines are used. Cancer cell lines are an auto-replication source of high homogeneity which can be easily manipulated while simultaneously providing reproducible results for drug delivery studies. Although, isolation of primary enterocytes from the human small intestine has been intended, their application remain limited due to obstacles such as poor viability and short life span [23]. Caco-2 cell line, one the most widely used and studied culture model, is able to construct a monolayer with morphological and functional characteristics similar to enterocytes. The permeability of drugs across Caco-2 monolayers has been shown to correlate well with the percentage of drug absorbed by humans for both passively absorbed and actively transported compounds [24].

However, many studies propose the use of coculture models in order to mimic the human intestinal epithelium more realistically by combining absorptive cells and mucus secreting goblet ones. The coculture of Caco-2/TC7 and HT29-MTX cell lines in a 9:1 proportion is the model closest to the physiological conditions. The mucus layer mimics the physiological barrier for the absorption of molecules and of high molecular weight compounds. Their culture on specific filters leads to the formation of a monolayer which permits rapid evaluation of bioactive absorption [25]. The above described co-culture model was chosen as it represents the epithelium of small intestine, the mainly absorption site of bioactives into systemic circulation after oral administration. As the present study deals with the HT absorption and the microemulsion’s effect on it, we focused on the intestinal epithelium without studying the fate of the system along the whole GIT.

In the present study we designed biocompatible non-ionic W/O microemulsions composed of Isopropyl myristate (IPM) and/or Extra Virgin Olive Oil (EVOO) as the oil phase and Polysorbate 80 (Tween 80™) in combination with distilled monoglycerides (DMG) as surfactants. As the formulated microemulsions are intended to be used either to enrich food products or to stand alone as functional formulations, the choice of the ingredients was made based on their safety [26] as approved ingredients in the food industry. For that reason, EVOO was used as one edible natural oil with beneficial health properties. Nevertheless, due to its complex nature, and its endogenous humidity, high surfactant amounts are needed for microemulsion formation [27,28]. Therefore, a less complex oil should be used in order to increase the dispersed aqueous phase. The natural occurring IPM [29], the isopropyl ester of myristic acid, is widely used in the pharmaceutical industry due to its penetration enhancing properties in topical drug delivery [30] but is also used as flavoring agent in food products. In addition, in the present work the food-grade Tween 80 and DMG were used as surfactants. The formulated W/O microemulsions were used as hosts for the encapsulation of HT. The colloidal nanodispersions were studied structurally before and after the encapsulation of HT with the use of Dynamic Light Scattering (DLS) and Electron Paramagnetic Resonance (EPR) technique. EPR technique was also used for the investigation of the antioxidant scavenging effect of HT after its encapsulation in the reverse micelles. Additionally, the aim of the present study was to evaluate the intestinal absorption of free HT in comparison with the encapsulated in biocompatible microemulsion systems. As far as we know, only few studies have reported the use of W/O systems for oral bioavailability of natural hydrophilic compounds and none of them has used natural oils such as EVOO in combination with absorption in vitro studies across the intestinal epithelium.