Zein nanoparticles for oral folic acid delivery
Graphical abstract
Introduction
Zein, the major storage protein of maize, is located in the “zein-bodies”, of approximately 1 μm, that are distributed uniformly throughout the cytoplasm of the corn endosperm cells between starch granules of 5–35 μm [1]. From a physicochemical point of view, the key characteristic of zein is its insolubility in water except at extreme pH conditions (e.g., pH 11 or above) or in presence of high concentrations of urea, alcohol or anionic detergents [2]. This characteristic is directly related with its composition in amino acids. Thus, zein is particularly rich in glutamic acid (21–26%) and non-polar amino acids such as leucine (20%), proline (10%) and alanine (10%), but it is deficient in basic and acidic amino acids [3].
Actually, zein is not a single protein but a mixture of four main fractions (α-, β-, γ-, and δ-zein) that differentiated in their solubility and sequence [1], [4]. Alpha-zein is the most abundant (around 80% of total zein) and includes two prolamin groups with apparent molecular weights of 24 and 27 kDa. Beta-zein consists of a methionine-rich polypeptide of 17 kDa and constitutes up to 10% of the total zein; whereas γ-zein is also composed of two peptides of 27 and 18 kDa. Finally, δ-zein is a minor fraction and has a molecular weight of about 10 kDa [1], [4], [5].
Because of its hydrophobic character and deficiency in essential amino acids (e.g. lysine and tryptophan), the use of this corn protein in human food products is limited. However, zein has been proposed as material for the manufacture of a wide variety of products, including textile fibres for clothes [6], biodegradable films and plastics used for packaging [7], coatings for food and pharmaceutical dosage forms [8], [9] and scaffolds for tissue engineering [10].
In the last years, microparticles and nanoparticles from zein have also been studied as carriers of non-polar compounds including vitamin D3 [11], curcumin [12] or thymol [13]. Such devices were capable of protecting the loaded compounds from stomach harsh conditions and providing a mechanism for their controlled release [14], [15].
Folic acid (pteroyl-L-glutamic acid, vitamin B9) is a water soluble vitamin that is essential during periods of rapid cell division and growth. It is implicated in cell replication and has an important role in the one-carbon metabolic pathway, essential for cardiovascular and neurological functions [16]. During periods of inadequate folate intake or malabsorption, biochemical changes due to this lack of folic acid/folate may result in deleterious consequences, including increased risk for certain types of chronic diseases [17] and developmental disorders (e.g., neural tube defects) [18]. In this way, previous studies have shown that folate deficiency is associated with higher incidence of mental symptoms in general population and poor cognitive performance that may increase the risk of dementia in old age [18], [19]. Particularly in major depression, low folic acid levels are frequently described in clinical studies [20]. Corroborating these findings, a variety of controlled and open-label studies have shown that the efficacy of antidepressants is influenced by folate status and may be enhanced by folic acid supplementation [21]. On the other hand, low folate intake or low plasma folate concentration has also been associated with increased cardiovascular and cerebrovascular risks [22]. All of these effects would be related with high plasma levels of homocysteine, a cytotoxic sulfur-containing amino acid that can induce DNA strand breakage, oxidative stress and apoptosis [22].
Interestingly, folic acid supplementation might reduce the hyperhomocysteinaemia [23], [24]. However, the supply of folate coenzymes in vivo depends primarily on the quantity and bioavailability of ingested folic acid/folate and the rate of loss by urinary and fecal routes and through catabolism. Additionally, folate is highly susceptible to oxidative destruction. In fact, 50–95% of folate content in food is estimated to be lost during storage, preparation, or manufacturing processes [25].
The aim of this work was to design and evaluate zein nanoparticles as carriers capable of improving the bioavailability of folic acid when orally administered. For this purpose, these zein nanoparticles were prepared by an original procedure and their capability to improve the oral bioavailability of folic acid was evaluated and compared with a conventional aqueous solution of the vitamin in rats.
Section snippets
Materials
Zein, folic acid, lysine, arginine, pepsin, pancreatin, mannitol and sodium chloride were purchased from Sigma–Aldrich (Steinheim, Germany). Ethanol, acetonitrile and o-phosphoric acid (HPLC grade) were obtained from Merck (Darmstadt, Germany). Perylene-Red (BASF Lumogen® F Red 305; Lumogen red) was from Kremer Pigments Inc. (Aichstetten, Germany) and Tissue-Tek® OCT compound from Sakura Finetek Europe (Alphen, The Netherlands). 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Biotium
Folic acid loaded zein nanoparticles
Table 1 summarizes the main physicochemical properties of folic acid-loaded nanoparticles. When folic acid was encapsulated into zein nanoparticles, a moderate increase in the mean size of the resulting carriers was observed (about 164 nm for empty nanoparticles vs 193 nm for FA-NP-Z); whereas the negative zeta potential decreased from −46 mV (control nanoparticles) to −30 mV (folic acid-loaded nanoparticles). The folic acid loading into the zein nanoparticles (FA-NP-Z) was calculated to be
Discussion
Folic acid, as other weak acid compounds, possesses a pH-dependent aqueous solubility, being insoluble in aqueous media below pH 5 [28]. In vivo, the pH of the stomach contents may induce the precipitation of the vitamin in macroscopic aggregates that, once in the small intestine (pH around 5–6), would be (at least in part) re-dissolved. However, in these pH conditions, and because of the hydrophilic nature of the charged molecule, specific transporters are required for folic acid absorption.
Conclusions
Zein nanoparticles offer adequate properties for oral delivery purposes. Orally administered, these nanoparticles are localized within the gut in close contact with the gut mucosa. Regarding folic acid, its encapsulation in zein nanoparticles improved its relative oral bioavailability about 2-fold when compared with an aqueous solution of the vitamin. This fact would be related with the capability of these nanoparticles to reach the small intestine mucosa and develop mucoadhesive interactions.
Acknowledgements
This work was supported by the Regional Government of Navarra (Alimentos Funcionales, Euroinnova call) and the Spanish Ministry of Science and Innovation (ADICAP; ref. IPT-2011-1717-900000). Rebeca Penalva acknowledges the “Asociación de Amigos Universidad de Navarra” for the financial support.
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