Preparation and characterization of zein/chitosan complex for encapsulation of α-tocopherol, and its in vitro controlled release study
Graphical abstract
Highlights
► Zein/CS nanocomplex could be considered as a polyelectrolyte complex based on our results. ► The hydrogen bonds and hydrophobic interactions could induce the formation of nanoparticles. ► The nanocomplex had spherical shape and uniform diameter about 200–300 nm. ► The complex could provide controlled release of α-tocopherol and protection from degrading in GI tract.
Introduction
Vitamin E is the main dietary fat-soluble antioxidant, playing important roles in the body. It is a family of four tocopherols (α, β, γ, and δ) and four corresponding tocotrienols (α, β, γ, and δ), of which α-tocopherol (TOC) has the highest biological activity. Vitamin E acts as a chain-breaking antioxidant preventing the propagation of free radical reactions, and thus consumption of vitamin E has been widely considered to help reduce risk of many chronic diseases, such as cardiovascular diseases [1], [2]. Although the overt deficiency of vitamin E is rare in humans, the marginal vitamin E deficiency could lead to increased susceptibility to free radical damage, especially in premature infants and hypercholesterolemic subjects, resulting in neuromuscular abnormalities, myophthies, and other neurological diseases [3], [4]. Therefore, supplementation of vitamin E is required in aforementioned cases. However, like other lipophilic nutraceuticals, TOC is poorly soluble in water and biologically unstable when exposed to environmental factors, such as light, temperature, and oxygen [5], [6].
In order to overcome the susceptibility and improve the stability of bioactive compounds during processing and storage, the emerging technology of nano-/micro-encapsulation has been recently applied in food and nutraceutical industries. Besides protecting them from the harsh processing conditions and adverse storage environment, the encapsulations of bioactive compounds can also achieve targeted delivery and controlled release of entrapped nutrients to the specific site. TOC has been early encapsulated into gliadin, a plant protein from wheat gluten, to form microparticles around 900 nm, however, the controlled release was only performed in organic medium (decane) at 25 °C, which cannot simulate release of TOC in vivo environment [7]. The controlled release of TOC in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) without enzymes could be achieved by microencapsulation of TOC in calcium-alginate gels [8] and calcium-pectinate microcapsules [9]. The in vivo study also revealed that encapsulation of TOC in calsium-pectinate microcapsules could provide sustained release of TOC in plasma and thus improve the bioavailability of TOC through oral administration. A new study showed that free TOC decomposed rapidly when it was incubated in simulated gastrointestinal tract (SGI) with presence of enzymes, however, its stability was dramatically improved after encapsulated into β-lactoglobulin emulsion gels [10]. Therefore, encapsulation of TOC by polymer-based delivery systems could not only improve its stability but also enhance the bioavailability and controlled release property of TOC in vivo. To the best of our knowledge, however, most delivery systems of TOC with controlled release property are at micro-scale beads or gels; the encapsulation and delivery systems at nano-scale have not been fully developed and characterized.
Zein is considered as generally recognized as safe (GRAS) and food-grade ingredient by the Food and Drug Administration (FDA). It contains three quarter of lipophilic and one quarter of hydrophilic amino acid residues. Because of its high hydrophobicity, zein has been successfully applied as a promising carrier for encapsulation and controlled release of fat-soluble compounds (e.g. gitoxin, fish oil, etc.) in the pharmacuetical and food areas [11], [12]. Chitosan (CS) is a linear polysaccharide consisting of randomly distributed N-acetyl-d-glucosamine and β-(1,4)-linked d-glucosamine units. CS has been widely considered as a versatile polymer used in pharmaceutical and nutraceutical areas as wall materials for development of delivery systems, due to its favorable biological properties such as biodegradability, biocompatibility, and low toxicity [13]. Compared with other delivery systems, nano- or micro-particles prepared by CS have a special feature of being able to adhere to mucosal surface and transiently open tight junctions between epithelial cells, due to its positive surface charge of CS molecules. In recent years, CS has been involved in delivery systems of TOC. For instance, CS has been used as a wall material to coat TOC encapsulated liposome nanoparticles, resulting in a great improvement of TOC stability, compared with non-coated liposome nanoparticles [14]. Also, it has recently been shown that TOC encapsulated CS nanoparticles can be successfully prepared by ultrasound [15]. However, it has been indicated that CS–tripolyphosphate (CS–TPP) nanoparticles might not be able to provide adequate protection and sustained release of small molecular drugs encapsulated in gastrointestinal environment, due to the high solubility of CS in aqueous acidic conditions resulting in the fast release of encapsulated drugs [16], [17]. Therefore, in order to overcome these obstacles, it would be helpful to introduce a second polymer to form stronger polymeric complex with CS.
Recently, many studies have been carried out to investigate the preparation, characterization, and application of micro-/nanocomplexes formed by two or more polymers [18], [19]. Zein and CS complexes have been newly developed to form antimicrobial ultrathin fiber structure by electrospinning [20], [21]. However, the investigation of polymeric complexes of zein and CS through their self-assembly properties is still lacking in the literature. A CS/zein nano-delivery system has been successfully developed in our lab to encapsulate hydrophilic nutrient with high bioactivities, and the release profile of hydrophilic nutrient from CS nanoparticles can be greatly improved after the nanoparticles are coated by zein protein [17]. In present study, a novel nano-scale delivery system of TOC using zein/CS complex was developed in order to provide protection of TOC against gastrointestinal conditions and enhance it releasing property. The various formulations with different zein concentrations, ratios of zein/CS, and loading percentages of TOC were investigated. Characterization was carried out by evaluating the particle size, zeta potential, and encapsulation efficiency of TOC/zein–CS complexes. The molecular interactions were investigated by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The controlled release properties of encapsulated TOC in different media with or without presence of enzymes was also studied.
Section snippets
Materials
Low molecular weight CS with 92% deacytelation degree (Batch No.: MKBB4232), vitamin E (TOC, Fluka >= 97%), Tween-20 (Tw), and pepsin were obtained from Sigma–Aldrich Chemical Co. Ltd. (St. Louis, MO). Zein sample with a minimum protein content of 97% was provided by Showa Sangyo (Tokyo, Japan). Phosphate buffer saline (PBS) was purchased from EMD Chemicals Incorporation (Gibbstown, NJ). Simulated gastric fluid without pepsin (SGF) and simulated intestinal fluid with pancreatin (SIF) were
FTIR results
FTIR was applied to characterize the intermolecular interactions of complex. The representative spectra of each component and their composites were shown in Fig. 1. In the original spectra of zein (Fig. 1A), CS (Fig. 1B), and TOC (Fig. 1C), the bands of hydrogen bonds were at 3312, 3368, and 3477 cm−1, respectively. However, after formation of complex, shift of hydrogen bands occurred, and the peaks were at 3312, 3309, 3315 cm−1 in the spectra of TOC/zein nanoparticles, zein–CS blank complex, and
Conclusions
In the present study, TOC/zein–CS complex was successfully prepared under mild conditions. Physicochemical analyses suggested that electrostatic interactions, hydrogen bonds, and hydrophobic interactions are the main forces in TOC/zein–CS complex. By coating TOC/zein nanoparticles with CS, particle size was dramatically reduced and zeta potential was increased to be highly positive, depending on different formulations. CS coating did not affect the encapsulation efficiency but greatly improved
Acknowledgements
This study was partially supported by Hatch fund of USDA. Authors acknowledge the support of Maryland NanoCenter and Center for Advanced Life Cycle Engineering in University of Maryland.
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