Gallic acid loaded PEO-core/zein-shell nanofibers for chemopreventive action on gallbladder cancer cells
Graphical abstract
Introduction
Natural products containing polyphenolic compounds are useful for the prevention and treatment of diseases caused by oxidative damage such as cancer (Nijveldt et al., 2001). Among polyphenols, gallic acid (GA) is potentially beneficial to human health as a strong antioxidant (Kim et al., 2002), anti-inflammatory (Kroes et al., 1992), and antimutagenic (Gichner et al., 1987) agent. GA also exhibits anti-tumor activity in hepatocellular carcinoma cells (Lima et al., 2016), leukemia, and prostate, lung, gastric, colon, breast, cervical and esophageal cancer (Park, 2017). However, GA is poorly stable at high temperature (Snow Boles et al., 1988), in the presence of oxygen or light (Jacques et al., 2010; Aytac et al., 2016) and at alkaline pH (Friedman and Jürgens, 2000). Once orally administered, GA shows limited absorption and rapid clearance (Bhattacharyya et al., 2013; Ferruzzi et al., 2009), which in turn leads to low, inefficient plasma levels (Siranoush et al., 2001). The major metabolites 4-omethylgallic acid and pyrogallol possess lower antioxidant activity compared with GA (Yasuda et al., 2000).
Encapsulation in nanometric structures may improve stability and bioavailability, and consequently the therapeutic efficiency of GA. In this context, electrospinning is gaining an increasing attention as it combines the advantages of additive manufacturing (mainly 100% yield encapsulation) with versatility of materials to be processed and of drug release performances (Dalton et al., 2013; Hu et al., 2014). Only recently, electrospun fibers have been tested for oral administration of poorly soluble or unstable drugs (Ignatious et al., 2010). For example, formulation of diosmin or flubendazole in electrospun nanofibers leads to complete amorphization of the drug and thus very rapid release, which in turn increases bioavailability rate and extent, compared to the micronized drug (Vrbata et al., 2014; Vigha et al., 2017). Gellan/poly vinyl alcohol (PVA) nanofibers have been shown to enhance oral bioavailability and in vivo antimicrobial efficacy of ofloxacin because of their mucoadhesion and gastro-retention properties (Vashisth et al., 2017). Moreover, grinded fibers can be tableted to prepare easy-to-handle immediate release formulations (Vigha et al., 2017; Poller et al., 2017).
The encapsulating polymer plays a crucial role in controlling drug release. Compared to synthetic polymers, biopolymers, such as globular proteins (e.g., collagen, silk, soy protein) and linear/branched polysaccharides (e.g., chitosan, alginate, starch) (Khandare et al., 2012; Rošic et al., 2011), resemble better biomacromolecules present in the human body minimizing foreign-body reactions (Cui et al., 2010; Pakravan et al., 2012). Plant proteins are particularly attractive because they are non-toxic, biodegradable, abundantly available from renewable sources, and versatile in terms of processability in different formats such as films, fibers, and gels (Lelkes et al., 2008). In addition, proteins have been known to interact strongly with active substances (e.g., polyphenols) through hydrogen bonding, van der Waals and/or hydrophobic interactions, enhancing stability (Maiti et al., 2006). For instance, electrospun fibers based on zein (major storage protein of corn) have been shown suitable to stabilize catechin (Li et al., 2009). Zein is considered as suitable carrier for oral delivery of drugs due to its hydrophobicity, reduced susceptibility to proteolytic degradation, and ability to withstand gastric pH (Karthikeyana et al., 2012). In previous studies, GA was successfully encapsulated in zein nanofibers by uniaxial electrospinning, preserving its antioxidant and antibacterial activities for food packaging purposes (Neo et al., 2013a; Neo et al., 2013b). However, an important burst release was detected in the first 20 min corresponding to 67–88% of the total loaded GA (Neo et al., 2013b). This rapid release might be due to GA presents on or near the uniaxial nanofiber surface. To slow down the release, zein-based nanofibers were heat-cured (150 °C, 24 h) to denature the protein and also to promote interactions of GA carboxylic groups with zein amino acids, which increased fibers hydrophobicity and morphological stability after immersion in water (Neo et al., 2014). Nevertheless, maintenance of the chemotherapeutic potential of GA after encapsulation in the nanofibers was not evaluated.
So far, encapsulation of GA in core-shell electrospun fibers using a coaxial spinneret has not been tested yet. Coaxial electrospinning is a useful technology for reducing the burst release phenomena, without the need of post-processing that may compromise drug stability (Meinel et al., 2012; Vysloužilová et al., 2017). The polymer at the shell contributes to the protection of the drug from deleterious environmental and biologic conditions. The release behavior is controlled by diffusion through the core and the shell layers and/or erosion of the shell material. For instance, ethyl cellulose/poly-(N-isopropylacrylamide) core/shell nanofibers have been shown to prolong ketoprofen release (Lv et al., 2017), and poly(butylene adipate)/poly(vinylpirrolidone) core/shell nanofibers have been successfully developed for efficient artemisinin delivery for malaria and prostate cancer treatments (Bonadies et al., 2017).
Core/shell nanofibers that combine natural proteins such as gelatin or collagen and synthetic polymers such as poly(ε-caprolactone) (PCL) or poly(ethylene oxide) (PEO) allow for an easy tuning of drug release patterns (Zhang et al., 2004; Huang et al., 2006), while preserving adequate biocompatibility for tissue engineering and drug delivery applications. For example, silk fibroin/PEO core/shell nanofibers can provide sustained release of dexamethasone and protect artery endothelial cells against inflammatory damage and apoptosis (Chen et al., 2015). Globular proteins are particularly attractive for the design of fibers intended for oral drug administration (Scheibel, 2005) but the high viscosity of their solutions may hinder the electrospinning process (Rošic et al., 2012). For example, soy protein form gel networks when aggregates instead of interconnected chains. To overcome this drawback, synthetic polymers, such as PEO or poly(vinyl alcohol) (PVOH), with good electrospinnability performance are usually added (Vega Lugo and Lim, 2009). The addition of PEO facilitates chain entanglements in solution and enables the formation of continuous fibers (Shenoy et al., 2005). Moreover, PEO allows for the processing using water as solvent (Pakravan et al., 2012), which may be also suitable for dissolving GA.
The aim of this work was to implement an electrospinning procedure for the formulation of GA in core/shell nanofibers that can be orally administered for chemopreventive action in human gallbladder cancer. Coaxial electrospinning was performed using PEO as core and zein as shell with the purpose of achieving GA sustained release. A Plackett Burman design was followed to study the effect of PEO and zein concentration and applied voltage on diameter and morphology index of nanofibers. Then, coaxial nanofibers were characterized applying a variety of complementary techniques, and GA release pattern was recorded in acid and neutral pH medium. Finally, cytotoxicity and reactive oxygen species (ROS) production on gallbladder cancer GB-d1 and NOZ cells in the presence of GA-nanofibers were assessed (for the first time to the best of our knowledge) in order to elucidate the chemopreventive potential of encapsulated GA.
Section snippets
Materials
Zein from corn, poly(ethylene oxide) (PEO, Mv~300,000) and gallic acid monohydrate (GA) were purchased from Sigma Aldrich (Chile). Briefly, zein solutions were prepared at different concentration by dissolving zein powder in ethanol:water 70:30% v/v under stirring at 25 °C (Li et al., 2009). PEO solutions (1–10% w/v) were prepared in distilled water. GA solution (2.5% w/v) was prepared by dissolving GA in the PEO solution at 25 °C and stored in the dark at 4 °C until use.
Definition of working range of independent variables
An electrospinning
Core-shell nanofiber production
Fiber diameter and morphology are influenced by a number of variables interrelated each other, such as solution parameters (e.g., polymer concentration, viscosity, conductivity, dielectric constant, surface tension), process parameters (e.g., electric potential, flow rate, distance between the spinneret and collector) and ambient conditions (e.g., temperature, humidity) (Pham et al., 2006; Yu et al., 2009; Meinel et al., 2012). By varying one or more of the above variables, morphology of
Conclusions
Core-shell nanofibers can be successfully obtained using PEO and zein as main components. The Plackett-Burman experimental design allowed identifying the more relevant variables to be considered for tuning the structural characteristics of the nanofibers. Optimized composition provided sustained GA release profiles both at acid and neutral pH medium. GA-nanofibers were more cytotoxic than free GA against human gallbladder cancer cells lines. NOZ was more susceptible than GB-d1 probably due to
Acknowledgements
Work financially supported by FONDECYT (Project 11140127) Chile, MICINN (Grant SAF2014-52632-R) Spain, and FEDER.
Conflict of interest
The authors have no conflict of interests to declare.
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