Design of the interface of edible nanoemulsions to modulate the bioaccessibility of neuroprotective antioxidants.

International Journal of Pharmaceutics 490 (2015) 209–218

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Design of the interface of edible nanoemulsions to modulate the bioaccessibility of neuroprotective antioxidants M. Plaza-Oliver a,b,1, J.Fernández Sainz de Baranda a,b,1, V. Rodríguez Robledo a,b , L. Castro-Vázquez a,b , J. Gonzalez-Fuentes a,b , P. Marcos a,b , M.V. Lozano a,b , M.J. Santander-Ortega a,b, * , M.M. Arroyo-Jimenez a,b, ** a b

Cellular Neurobiology and Molecular Chemistry of the Central Nervous System Group, Faculty of Pharmacy, University of Castilla-La Mancha, Albacete, Spain Regional Centre of Biomedical Research (CRIB),University of Castilla-La Mancha, Albacete, Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 31 March 2015 Received in revised form 5 May 2015 Accepted 11 May 2015 Available online 18 May 2015

Most frequently the use of bioactive molecules for the supplementation of food and beverages is hampered by stability limitations or inadequate intestinal absorption. This work evaluates in vitro the role that the interface of the nanoemulsion has on the physicochemical properties, the stability behavior and the enzymatic degradation after oral intake. For that purpose three soybean oil (SB) formulations were studied. These formulations were based on the emulsifier lecithin but modified with two non-ionic surfactants Pluronic1 F68 (PF68) or Pluronic1 F127 (PF127) yielding (i) SB-NE (only lecithin on the interface), (ii) SB-NE PF68 (lecithin plus PF68) and 9 (iii) SB-NE PF127 (lecithin plus PF127). All the formulations tested were low polydispersed and showed a size of about 200 nm and z-potential of 50 mV. The in vitro colloidal stability assay showed that lecithin itself was able to promote that formulations reach unaltered to the small intestine and facilitate the absorption of the antioxidant payload on a tunable fashion there (with in vitro bioaccessibility values from around 40% up to a 70%). PF68 was able to sterically stabilize the formulation against the aggregation induced by the pH and electrolytes of the simulated gastrointestinal track; however, this surfactant was easily displaced by the lipases of the simulated intestinal milieu being unable to modulate the digestion pattern of the oil droplets in the small intestine. Finally, PF127 displayed a strong steric potential that dramatically reduced the interaction of the oil droplets with lipases in vitro, which will compromise the capacity of the formulation to improve the bioaccessibility of the loaded antioxidant. ã2015 Elsevier B.V. All rights reserved.

Keywords: Nanoemulsions Soybean oil Lecithin Pluronic1 a-tocopherol Bioaccessibility

1. Introduction Currently, the food industry is trying to implement the valuable knowledge acquired in the pharmaceutical field for designing new nanostructures able to overcome biological barriers. These nanostructures would be used to encapsulate active therapeutic molecules for food and beverage products supplementation in order to increase and homogenise the bioavailability of the active molecule (Acosta, 2012; McClements and Xiao, 2012; Porter et al., 2007). In this case the concept would not be the treatment of a disease but the diet-based prevention of chronic diseases such as

* Corresponding author at: University of Castilla-La Mancha, Medical Sciences, Albacete, Spain. Tel. :+ 967 59 92 00x2239. ** Corresponding author at: Tel. :+ 967 59 92 00x8249. E-mail addresses: [email protected] (M.J. Santander-Ortega), [email protected] (M.M. Arroyo-Jimenez). 1 These authors have equally contributed to this work. http://dx.doi.org/10.1016/j.ijpharm.2015.05.031 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

central nervous system pathological aging diseases or cardiovascular diseases among others (Acosta, 2012; Yang and McClements, 2013). Nevertheless, the direct incorporation of bioactive molecules such as antioxidants to food or beverage matrices is mainly hampered by (i) the modification of the product organoleptic properties; (ii) the degradation of the labile bioactive due to oxidative processes, pH, temperature changes or during the manufacturing and storage of the food or beverage products; (iii) the premature degradation of the bioactive after oral intake; (iv) the limited intestinal absorption (Acosta, 2012; Fang and Bhandari, 2010). All these factors can result in the refusal of the product by the consumer or in low bioavailability of the bioactive after ingestion, clearly hampering its efficacy (Scalbert and Williamson, 2000). As introduced before, the need to overcome these limitations has brought the food and beverage industry to consider the use of nanotechnology. Nanoparticles for the encapsulation of food supplements would allow not only to

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protect the food/beverage product from the modification of its organoleptic properties by the bioactive molecules, but also to preserve the nutritional properties of those molecules against degradation processes during the manufacturing and storage phases, as well as to protect the cargo up to its release at the target site (Acosta, 2012; McClements and Xiao, 2012; Porter et al., 2007). One of these nanoplatforms that potentially may improve this landscape are lipid nanostructures. These structures are used as food and beverage supplements for the encapsulation of lipophilic antioxidants to protect its cargo before releasing in the target site. In this sense, one key feature of the system is the maintenance of their nanometric entity up to its arrival to the small intestine (Acosta, 2012; Golding et al., 2011; Li et al., 2012; McClements and Xiao, 2012; Porter et al., 2007; Salvia-Trujillo et al., 2013). Size will affect the interaction of the nanostructure with the enzymes and others macromolecules present in the intestinal milieu. This interaction will promote the digestion of the nanostructure, and then, the enzyme triggered partition of the therapeutic antioxidant from the nanostructure matrix towards the mixed micelles and vesicles formed by bile salts, phospholipids and degradation products of the lipid nanostructure (Acosta, 2012; McClements and Xiao, 2012; Porter et al., 2007). Afterwards, these mixed micelles and vesicles with a therapeutic antioxidant enriched inner core will be absorbed by the enterocytes resulting in the release of the antioxidant to the systemic circulation in a reproducible and efficient fashion (Acosta, 2012; McClements and Xiao, 2012; Porter et al., 2007). In the specific case of lipophilic therapeutic antioxidants, such as vitamin E, marketed food supplements products have shown low and very variable bioavailability results (Acosta, 2012; Eitenmiller and Lee, 2005; McClements and Xiao, 2012). Hence, the entrapment of the lipophilic molecule in lipid nanostructures, already incorporated into the food or beverage matrix during the manufacturing process, have improved the magnitude and reduced the variability of the antioxidant intestinal absorption after oral intake (Acosta, 2012; Gong et al., 2012; Hatanaka et al., 2010; Li et al., 2012; McClements and Xiao, 2012; Salvia-Trujillo et al., 2013; Scalbert and Williamson, 2000; Yang and McClements, 2013). Similar results were obtained with other hydrophobic antioxidants such as b-carotene and curcumin (Acosta, 2012; Anand et al., 2010; Salvia-Trujillo et al., 2013; Takahashi et al., 2009). Under this scenario, the success of the establishment of nanocarriers loaded with specific bioactive molecules as food or beverage supplements depends on: 1. Our capacity to tune up the surface of the nanostructure to avoid the premature degradation/aggregation of the formulation and to selectively trigger the release of the cargo at the desired target (small intestine in our case). Among others factors, nanostructures are specially characterized by a high area/volume ratio (Israelachvili, 2010), this fact means that the design of the interface composition of the formulation is a key point to control not only the formulation process but also the colloidal behavior of the nanostructure once formulated (Israelachvili, 2010; Santander-Ortega et al., 2006, 2012). The interaction of the lipid nanostructures with the surrounding medium can be controlled by the superficial charge of the particles through a repulsion electrostatic potential of interaction, or by the incorporation of non-ionic polymers that will avoid the aggregation of the particles by the presence of electrolytes through a steric repulsion potential (Israelachvili, 2010). The proper coating of the lipid nanostructure with non-ionic polymers will also modulate the interaction of the particle with the macromolecules present in the physiological fluids, such as the lipases responsible of the system digestion in the

small intestine (Santander-Ortega et al., 2006, 2009; Tobio et al., 2000). 2. Our capacity to design lipid nanostructured matrices able to properly accommodate the bioactive molecule (hydrophobic antioxidants in our case). It is already reported that the lipid matrix of nanostructures will affect the loading capacity of the formulation, the hydrodynamic mean size of the system and the bioavailability of the antioxidant (Li et al., 2012; Saberi et al., 2013; Salvia-Trujillo et al., 2013; Yang and McClements, 2013). In this line, compared to medium chain triglyceride oils (MCT), long chain triglyceride oils (LCT) have shown excellent properties to encapsulate and improve the bioavailability of a-tocopherol a lipophilic antioxidant with proved neuroprotective properties (Eitenmiller and Lee 2005; Yang and McClements, 2013). 3. Additionally, as food supplements, all the raw materials should be of food grade and the incorporation of the nanostructures to the food product should not significantly increase the final market price. Bearing this in mind, the in vitro analysis of the effect of the interface composition of a lipid nanostructure was proposed as an improvement of the in vivo bioavailability of therapeutic antioxidants with already demonstrated neuroprotective properties. In order to achieve this aim three different oil nanoemulsions that share the same LCT oil matrix composed by soybean oil, which could help to improve the bioavailability of hydrophobic antioxidants were designed (Yang and McClements, 2013). The first oil nanoemulsion (SB-NE) has been formulated with an interface composed only by lecithin, a well-known emulsifier able to facilitate the formation of colloidally stable nanometric oil droplets thanks to an electrostatic repulsion potential (Israelachvili, 2010; Klang and Valenta, 2011; Santander-Ortega et al., 2010; van Nieuwenhuyzen and Szuhaj, 1998). It is important to highlight that this emulsifier can also modulate the interaction of pancreatic enzymes with the inner oil core of the nanoemulsion and then control the digestion of the oil droplets in the small intestine (Mun et al., 2007; TorcelloGomez et al., 2011a; Wulff-Perez et al., 2010). The other two types of soybean nanoemulsions have been formulated using lecithin plus PF68 (SB-NE PF68) or PF127 (SB-NE PF127). These two non-ionic surfactants share the same poly(oxyethylene)– poly(oxypropylene)-poly(oxyethylene) (POEa–POPb–POEa) architecture but differ on the POE/POP block length. These differences will have an effect on both, the adsorption of Pluronic1 onto the O/W interface of the nanostructure and its capacity to modulate the interaction of the nanostructure with the surrounding medium (Santander-Ortega et al., 2009; Torcello-Gomez et al., 2013; Wulff-Perez et al., 2012). This effect was analyzed when the role of the interface composition of the formulation on the colloidal behavior of the systems after oral intake was evaluated, thus achieving the first statement. In order to address the second one, the encapsulation capacity of the nanoemulsions initially for three model hydrophobic antioxidants, i.e., gentisic acid, p-hydroxybenzoic acid and p-coumaric acid, with interesting neuroprotective properties was analyzed (Garrido et al., 2012; Joshi et al., 2006). Next, the concept bioaccessibility was introduced, which is the fraction of antioxidant in the mixed micelles and vesicles after the digestion of the lipid nanostructure in the small intestine (Li et al., 2012; McClements and Xiao, 2012; Mun et al., 2007; Saberi et al., 2013; Salvia-Trujillo et al., 2013; Yang and McClements, 2013)). Therefore, the capacity of the soybean nanoemulsion to improve the bioaccessibility of hydrophobic antioxidants with neuroprotective capacity as a function of the interface composition was studied.


2. Materials and methods 2.1. Systems formulation Soybean nanoemulsions were formulated by the solvent displacement technique (Lozano et al., 2008, 2013). Briefly, 63 ml of soybean oil (Sigma, Spain) was emulsified using 20 mg of lecithin (Epikuron 145V1) kindly donated by Cargill (Spain), 0.25 ml of ethanol and 4.75 ml of acetone (Sigma, Spain) under magnetic stirring. As a function of the nanoemulsion coating aqueous phase was composed only by 5 ml MilliQ water (SB-NE) or by PF68 (SB-NE PF68) or PF127 (SB-NE PF127) both from Sigma (Spain) at a concentration of 5 mg/ml. After emulsification, the formulations were rota evaporated to a final volume of 5 ml at 37 C.

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concentration of each antioxidant was determined using an optimized high pressure liquid chromatography (HPLC) method with UV detector. All of chromatographic parameters were studied in order to get the best resolutions between peaks (Rs), high efficacies (N) and shorter total analysis time. The chromatography conditions using C18 column as reverse stationary phase (5 mm particular size and 4,6 150 mm), are the following: an isocratic mobile phase consisting of acetic acid 0,01 M as aqueous phase and methanol as organic phase 60:40 v/v respectively. A flow of mobile phase of 1 ml/min, injection volume of 20 ml and a wavelength of 230 nm as a compromise for the simultaneous detection of each compound were used. In all separations the total analysis time was of 5 min and values of Rs between peaks higher than 1.5 for each compounds. 2.5. Bioaccessibility assay

2.2. Physicochemical characterization and colloidal stability Determination of the hydrodynamic mean diameter and the z-potential of the formulations was carried out by dynamic light scattering technique using a Z-Sizer NanoZS from Malvern (UK) dispersing the nanoemulsions in phosphate buffer 2 mM. Simulated gastrointestinal fluids (GIF), i.e., simulated saliva, gastric and intestinal fluids, used in this work were prepared according to the USP (XXIX). Briefly, colloidal stability was carried out by the incubation of the formulations in plain (prepared without enzymes) and normal GIF during 4 h at 37 C. Mucin, pepsin and pancreatin used to prepare GIF were supplied by Sigma (Spain), USP XXIX (Yang and McClements, 2013). 2.3. Analysis of the enzymatic degradation in simulated intestinal fluids Lipolysis of the soybean nanoemulsions as a function of their surface composition was studied by the titration of the free fatty acids released to the aqueous phase as result of the digestion of the oil matrix triglycerides by the lipases present in the intestinal pancreatic milieu. Namely, 1 ml of formulation was diluted up to 5 ml with plain simulated intestinal fluid. A volume of 5 ml of SIF supplement with 2X fold of pancreatin was mixed with the nanoemulsion leading to a final volume of 10 ml (1% w/v pancreatin, USP XXIX). Lipases present in pancreatin will degrade each triglyceride molecule in two free fatty acids and one monoglyceride. Acidification of the medium was monitored with a pH electrode and titrated with NaOH 0.1 M during 2 h at 37 C under magnetic stirring (Mun et al., 2007; Salvia-Trujillo et al., 2013; Yang and McClements, 2013).

The bioaccessibility of the encapsulated antioxidant, defined as the fraction of antioxidant released from the nanostructure matrix and available for intestinal absorption through the mixed micelles and vesicles formed by bile salts, phospholipids and degradation products of the lipid nanostructure was determined after the in vitro digestion of the nanoemulsions in SIF using a method described previously (Mun et al., 2007; Salvia-Trujillo et al., 2013; Yang and McClements, 2013). After the incubation of the formulation in SIF for 2 h at 37 C under magnetic stirring, an aliquot of the raw digesta medium was centrifuged (Hettich 320R) at 5000 rpm for 60 min at 15 C and the supernatant was collected, this “micelle fraction” has the bioactive compound solubilised and available for intestinal absorption (Mun et al., 2007; Salvia-Trujillo et al., 2013; Yang and McClements, 2013). In some samples, a top layer of tiny droplets of non-digested oil was observed and was completely removed from the micelle fraction, prior to analysis. The micelle phase was then collected using another glass pipette. Aliquots of 1 ml of the micelle fraction or the raw digesta medium were mixed with 4 ml of hexane, vortexed and centrifuged at 5000 rpm for 10 min at 15 C. The bottom layer, with the solubilised antioxidant, was collected while the top layer was mixed with 4 ml of chloroform and the same procedure was followed. Both hexane fractions were mixed together and analyzed by HPLC. Bioaccessibility was calculated as follows: Bioaccessibilityð%Þ ¼ 100X

Cm CT

where Cm is the antioxidant concentration on the micelle phase and CT is the antioxidant concentration in the raw digesta medium. 3. Results

2.4. Antioxidants encapsulation and release In order to assess the potential use of these nanoemulsions as food supplements to improve the bioavailability of antioxidants with neuroprotective capacity, three model antioxidants were used. The three co-encapsulated antioxidants were gentisic acid (Mw: 154 Da; Log P: 1.56), p-hydroxybenzoic acid (Mw: 138 Da; Log P: 1.42) and p-coumaric acid (Mw: 164 Da; Log P: 1.88) all of them supplied by Sigma (Spain). The preparation method of the antioxidants co-loaded nanoemulsions was not modified with respect to the un-loaded formulations. Following the same procedure described above, plain ethanol was substituted by an ethanoic solution of the three antioxidants containing 1000 ppm of each antioxidant. The encapsulation efficiency (EE) was calculated by a dialysis method introducing the specific nanoemulsion on a dialysis bag (12 KDa cut off) under sink conditions. The release of the antioxidants from the nanoemulsions was evaluated using the same setup for a period of time of 24 h. The determination of the

The main objective of this work was the design of novel lipid nanostructures able to improve the bioavailability of antioxidants with neuroprotective properties by properly modifying their interfacial properties. Initially the formulation and the physicochemical characterization of the nanostructures were studied, followed by the evaluation of their colloidal behavior after oral intake as a function of their interface composition. The analysis of the potential of the lipid nanostructures to encapsulate and release different therapeutic antioxidants with neuroprotective properties was a key piece of work that finally led us to the evaluation of the bioaccessibility of a-tocopherol as a function of the interfacial composition of the prototypes. 3.1. Systems formulation As shown in Table 1, the use of LC alone or in parallel with PF68 or PF127 during the formulation of the nanoemulsions led to

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Table 1 Hydrodynamic mean size, polydispersity index (PDI) and z-potential of SB-NE, SBNE PF68 and SB-NE PF127. Encapsulation efficacy of gentisic acid (gen), phydroxybenzoic acid (p-OH-B) and p-coumaric acid (p) n 3.

0.15 56 2 0.16 53 2 0.18 35 5

88 3 81 1 79 2

p-OH-B

p-Cou

96 2 93 2 92 2

92 2 86 2 86 1

the formation of oily droplets with a hydrodynamic mean size (zaverage diameter) of around 200 nm and low polydispersity (PDI < 0.2). Although both Pluronics1 are widely known as excellent emulsifiers (Olbrich and Muller, 1999; Santander-Ortega et al., 2006, 2010), the results obtained indicate that LC itself seems to be good enough to allow the formulation of the systems with a narrow nanometric size distribution (Santander-Ortega et al., 2010). These results suggest that these systems should present a good potential to enhance the bioaccessibility of the encapsulated antioxidant (Gong et al., 2012; Hatanaka et al., 2010; Li et al., 2012; Salvia-Trujillo et al., 2013). Despite the similar hydrodynamic mean particle size of the systems formulated with the different surfactants, their z-potential magnitude displayed a clear dependence with respect to the surfactant used in the formulation, where PF68 coated system presented similar superficial charge that the non-coated nanoemulsion and the PF127 coated system had a clear reduction on its z-potential magnitude. 3.2. Colloidal stability after oral intake The next set of experiments was focused on the in vitro analysis of the colloidal stability of the three nanoemulsions through a three-step simulated GIT track model including mouth, stomach and small intestine compartments. Considering the complex composition of these media and in order to achieve a better understanding of the effect of each interface component on the colloidal behavior of the formulations, the size evolution of the systems in plain (to analyze the effect of pH and electrolytes) and normal (to analyze the effect of mucin and enzymes) simulated saliva (pH 6.8), gastric (pH 1.2) and intestinal (pH 6.8) fluids (SSF, SGF and SIF respectively) were studied for up to 4 h, which are the normal stomach and small intestine residence times (Acosta, 2012; McClements and Xiao, 2012; Yang and McClements, 2013). Monitoring of the hydrodynamic mean size evolution of the three systems either in plain or normal SSF showed that all of them maintained its z-average diameter during the incubation time at 37 C (data not shown). These results are in line to the ones recently published by Yang and McClements (2013). SB-NE PF68 and SB-NE PF127 systems were totally stable during the incubation time in SGF while SB-NE showed a slight size increase (from 200 nm up to 350 nm), see Fig. 1. It is important to highlight that, even with a hydrodynamic mean size around 350 nm, these systems would be able to improve the bioaccessibility of antioxidants after oral intake (Li et al., 2012). Interestingly the presence of gastric enzymes led to the colloidal stabilization of SB-NE formulation while both Pluronic1 coated systems presented barely the same hydrodynamic size that in plain (prepared without enzymes) SGF. The results obtained for the plain simulated intestinal fluid show that the three formulations were totally stable during the incubation time (see Fig. 2). Contrary to the colloidal stabilization observed in SGF, the incorporation of enzymes (pancreatin) to plain SIF led to the instant aggregation of the SB-NE formulation, see Fig. 2. The comparison between the behavior observed for

Hydrodynamic Mean Size (nm)

195 3 SB-NE SB-NE PF68 194 5 SB-NE PF 127 202 4

z-potential (mV) Gen

SB-NE SB-NE PF68 SB-NE PF127 SB-NE SB-NE PF68 SB-NE PF127

450 400 350 300 250 200 150 100 0

1

2

3

4

Time (h) Fig. 1. Hydrodynamic mean size of SB-NE (square), SB-NE PF68 (circle) and SB-NE PF127 (triangle) incubated either in simulated gastric fluid (SGF) without enzymes (solid lines, closed symbols) or in pepsin supplemented medium, (USP XXIX) (dashed lines, open symbols), n 3.

500

SB-NE SB-NE PF68 SB-NE PF127 SB-NE SB-NE PF68 SB-NE PF127

450

Hydrodynamic Mean Size (nm)

Encapsulation efficacy (%) Size (nm) PDI

500

400 350 300 250 200 150 100 0

1

2

3

4

Time (h) Fig. 2. Hydrodynamic mean size of plain (square), PF68 (circle) and PF127 (triangle) coated soybean nanoemulsions (SB-NE) incubated either in simulated intestinal fluid without enzymes (solid lines, closed symbols) or in pancreatin supplemented medium, (USP XXIX) (dashed lines, open symbols), n 3.

SB-NE in plain and in normal SIF shows that the aggregation of the formulation is due to the interaction of the oil droplets with the enzymes present in the medium but not to the reduction of the repulsive electrostatic potential by the electrolytes present in the SIF medium. Fig. 3 allows us to understand at a glance the GIT colloidal stability of all of the systems studied in the three-steps simulated GIT model used. As stated in the introduction, the enhanced antioxidants bioavailability at the central nervous system would require the protection and transport of the cargo through the GIT up to the small intestine. Once in the small intestine, the carrier should interact with intestinal enzymes and promote the absorption of its payload to the systemic circulation through the mixed micelles formed by bile salts, phospholipids and lipid digestion products (Acosta, 2012; McClements and Xiao, 2012). Bearing this in mind SB-NE seems to be the most promising formulation as it is able to maintain intact physicochemical properties through the mouth and stomach whiles once it reaches


3.3. Enzymatic degradation: lipolysis assay

800

SB-NE SB-NE PF 68 SB-NE PF 127

700

Size (nm)

600 500 400 300 200 100 0 Original

Mouth

Stomach

Small Intestine

Fig. 3. Hydrodynamic mean size of SB-NE, SB-NE PF68 and SB-NE PF127 formulations after its incubation in simulated saliva fluid (SSF), simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) during 4 h, n 3.

the small intestine aggregates by, the presence of the intestinal enzymes (see Fig. 3). On the other hand, the coating of the oil droplets, either with PF68 or PF127, resulted in the total stabilization of the systems in the three physiological environments, i.e., mouth, stomach and small intestine. Contrary to what it was observed in the colloidal stability assays, recent studies aimed to analyze the effect of the molecular differences between PF68 and PF127 on its capacity to protect lipid systems from pancreatic enzymes have concluded that, although both surfactants are able to form a protecting layer around the lipid surface, PF127 is much more efficient protecting the system from the enzymatic degradation (Torcello-Gomez et al., 2011a, 2013; Wulff-Perez et al., 2010, 2012). The reason behind this different behavior relies on the larger hydrophobic section of PF127 in comparison to PF68 (Torcello-Gomez et al., 2011c, 2013; WulffPerez et al., 2012). This bigger hydrophobic poly-PPO central block allows to achieve not only higher accumulation of the surfactant molecules onto the O/W interface (see z-potential value of SB-NE PF127 vs SB-NE PF68), but also avoids their displacement from the O/W interface by enzymes and bile salts (Torcello-Gómez et al., 2013, 2014). The similar behavior observed in the data obtained for PF68 and PF127 cannot be clearly due to the steric effect; indeed, we believe that the protein corona plays an important role on PF68 stabilization. Considering the similar physicochemical properties of SB-NE and SB-NE PF68 (see Table 1), it could be feasible that the colloidal stability observed for the PF68 coated system would not be due to the steric protection of the oil droplets but to a homogenous coating of the pancreatic macromolecules around the systems (Santander-Ortega et al., 2014; Tirado-Miranda et al., 2003). The properties of pancreatic lipase as interfacial enzyme would support this hypothesis, as once bound to the surface of the oil droplets the surface-to-core erosion of the lipid matrix would explain why the SB-NE PF68 presented constant size values during its incubation in SIF (Devraj et al., 2013; Landry et al., 1996). Bearing this in mind we consider that the study of the nanoemulsion-enzymes interaction on the colloidal stability should be supported by the analysis of the lipid matrix integrity after the incubation of the systems with the enzymes secreted by the pancreas. This set of knowledge would help us to get a better insight of the potential of these lipid nanostructures to improve the bioavailability of antioxidants with neuroprotective properties.

The basis of the lipolysis assay is the fact that when lipases reach the oil core of the nanoemulsions they will degrade the triglycerides (TG) of the lipid matrix releasing two free fatty acids (FFA) and one monoglyceride per each TG molecule to the SIF milieu (Mun et al., 2007; Salvia-Trujillo et al., 2013). Then, by the titration of the FFA molecules released with NaOH it is possible to quantify the percentage of digestion of the nanoemulsions by the lipases (Li et al., 2012; McClements and Xiao, 2012; Mun et al., 2007; Salvia-Trujillo et al., 2013). The digestion percentage of the three nanoemulsions in SIF as a function of their interface composition is represented in Fig. 4. As can be observed, SB-NE matrix was rapidly digested by pancreatic lipase, resulting in a degradation of 80% of the nanoemulsions just in 20 min. Despite to the lower digestion values, SB-NE PF68 presented a similar pattern, with a degradation of 70% during the first 20 min of the assay. On the opposite side, SB-NE PF127 had a negligible degradation (5%) during the first 20 min of incubation. This surfactant maintained its protective effect during the whole assay, leading to a final digestion of just of 15% of the lipid matrix of the nanoemulsions after 2 h of incubation in SIF. 3.4. Encapsulation and release of gentisic, p-hydroxy benzoic, p-coumaric acid The design of lipid nanostructure matrices able to properly accommodate the bioactive molecule was one of the two items exposed in the introduction as key features of an effective nanotechnology based nutraceutical. Hence, the second main section of this work was focused on the effect of the formulation interface composition on its encapsulation/release capacity of antioxidant with therapeutic effects. Lipid nanostructures are being widely used for the encapsulation of hydrophobic low molecular weight drugs (Lozano et al., 2008, 2013). Considering the promising results obtained for this type of nanostructures, the co-encapsulation of three therapeutic hydrophobic antioxidants (with low molecular weight) that have shown excellent properties as preventive agents against cardiovascular diseases, stroke and cancer (Nevado et al., 2009) as well as neuroprotective properties (Garrido et al., 2012; Joshi et al., 2006) was proposed. The three co-encapsulated antioxidants were gentisic acid (Mw:154 Da; Log P: 1.56), p-hydroxybenzoic acid

SB-NE SB-NE PF68 SB-NE PF127

100

80

Degradation (%)

1000 900

213

60

40

20

0 0

20

40

60

80

100

120

140

Time (min) Fig. 4. Percentage of lipolysis of the plain (black columns), PF68 (grey columns) and PF127 (white columns) soybean nanoemulsions incubated in SIF (USP XXIX). Degradation of the plain SB-NE after 5 h of incubation in SIF was set as 100% of degradation, n 3.

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(Mw:138 Da; Log P: 1.42) and p-coumaric acid (Mw: 164 Da; Log P: 1.88). As expected, the three antioxidants presented high affinity for the oil matrix of the SB-NE, SB-NE PF68 and SB-PF127 formulations showing EE values close to 90%, see Table 1. Once encapsulated, the co-release of the three antioxidants from the nanoemulsions was analysed. The release profiles of the three antioxidants from SB-NE were included in Fig. 5 as representative of what was observed for SB-NE PF68 and SB-NE PF127 formulations (data not shown). Interestingly, the encapsulation of the antioxidants into the soybean matrix resulted in their sustained release in a controlled fashion from the oil droplets avoiding the typical initial burst effect deeply described for this type of nanostructures (Lozano et al., 2008, 2013). The release pattern achieved for the model antioxidants were about 60% for gentisic acid, 70% for phydroxybenzoic acid and 80% of p-coumaric acid from the lipid nanostructure after 5 h of incubation. Nanostructures for food supplementation should encapsulate and protect the active molecule not only from the GIT environment after oral intake, but also during the manufacture and store process (Acosta, 2012; McClements and Xiao, 2012). This requisite would not be feasible when the active molecule follows a release pattern as the one represented in Fig. 5. Interestingly, the likeness observed in the encapsulation capacity and release profile of the three model antioxidants from SB-NE, SB-NE PF68 and SB-NE-PF127 indicates that the interface composition did not exert any clear effect on the capacity of the lipid nanostructures to modulate their interaction with the encapsulated active molecule. The design of a successful formulation in nanotechnology relies on the proper selection of the carrier and the cargo molecule. The summary of the results previously exposed for the three prototypes clearly shows that SB-NE formulation is the one with the highest possibilities for increasing the bioavailability of antioxidants with neuroprotective properties used as food supplements. On the other side, the proper cargo selection would require us to consider both, the physicochemical properties of the soybean oil matrix as well as the information from the encapsulation/ release profile obtained for the model antioxidants. Soybean oil is mainly composed by long chain triglycerides (LCT), in this sense it has been recently published that (in comparison to medium chain triglycerides) these triglycerides are especially efficient improving the bioavailability of a-tocopherol (Yang and McClements, 2013). The molecule a-tocopherol is one of the main isomers of vitamin E, a well-known antioxidant that has demonstrated, among many

100

Gentisic p-OH Benz p-Coum

90

Antioxidant Release (%)

80

benefits to health, promising neuroprotective properties (Eitenmiller and Lee, 2005). Unfortunately, actual marketed products supplemented with vitamin E suffer from low and highly variable bioavailability of the molecule after oral intake (Eitenmiller and Lee, 2005). In addition, the null solubility in water of a-tocopherol (Log P: 9.60) suggests that once encapsulated, it will be retained in the oil matrix until its digestion on the small intestine (Yang and McClements, 2013). The benefits that would involve the encapsulation of a-tocopherol into a lipid nanostructure would not only be the improvement of its bioaccessibility compared with actual marketed products, but also would lower the high absorption variability observed after the oral intake of vitamin E marketed products (Eitenmiller and Lee 2005; Gong et al., 2012; Hatanaka et al., 2010). 3.5. Formulation and bioaccessibility of a-tocopherol loaded nanoemulsions The encapsulation of a-tocopherol, with a theoretical loading of 33% or 66%, led to hydrodynamic mean size reduction from 195 nm (SB-NE) down to 160 nm (a-tocopherol loaded SB-NE), while the z-potential of the loaded nanoemulsions was similar to that observed for the empty system (around 60 mV). The encapsulation studies showed that a-tocopherol had high affinity for the soybean oil matrix with an EE close to 90% and a negligible leakage of the antioxidant under store conditions during a 2 weeks period of time (data not shown). As referred previously, the bioaccessibility of the SB-NE a-tocopherol loaded formulations was evaluated as the fraction of antioxidant present in the mixed micelles, which are directly available for intestinal absorption after the digestion of the systems in SIF for 2 h (Li et al., 2012; McClements and Xiao, 2012; Salvia-Trujillo et al., 2013; Yang and McClements, 2013). The analysis of a-tocopherol fraction retained in the mixed micelles after the incubation of the SB-NE in SIF gave a bioaccessibility value of 38 7.4 % for the nanoemulsion with a theoretical loading of 33% and 38 0.4 % for the systems with a loading of 66%. These results correlate to the ones recently reported by Yang and McClements using corn oil nanoemulsions (Yang and McClements, 2013), and mean that the SB-NE with a 33% loading of a-tocopherol was able to solubilize 1.7 0.3 mg of a-tocopherol per ml of formulation, while the SB-NE with a loading of 66% of a-tocopherol reached a value of 2.9 0.1 mg per ml of formulation into the mixed micelles. These excellent in vitro results would mean a promising in vivo performance, as previous studies have shown good correlation between bioaccessibility and in vivo conditions (Golding et al., 2011; Gong et al., 2012; Hatanaka et al., 2010; Li et al., 2012; McClements and Xiao, 2012; Mun et al., 2007). 3.6. Reformulation and bioavailability

70 60 50 40 30 20 10 0 0

1

2

3

4

5

Time (hours) Fig. 5. Release profile of gentisic acid (Squares), p-hydroxybenzoic acid (circles) and p-coumaric acid (triangles) from SB-NE, n 3.

The excellent emulsifying properties of LC allowed the formation of the soybean droplets by the solvent displacement method in the absence of any other surfactant. This fact might suggest that the high affinity of the LC for the O/W interface during the formation of the oil droplets could also affect the lipase/colipase access to the inner oil core of the system after its adsorption onto the oil droplets interface. We believe that the modification of the LC amount would have an effect on the formulation of the system and subsequently on the digestibility of the SB-NE most probably affecting the bioaccessibility of a-tocopherol (Mun et al., 2007; Torcello-Gomez et al., 2011a; Wulff-Perez et al., 2010). The amount of LC added to the organic phase of the formulation was reduced from 20 mg (original SB-NE) down to 10 mg (SB-NE10) or 5 mg (SB-NE5); lower amount of LC resulted in heterogeneous


4. Discussion As commented above, lipid nanostructures can be used for encapsulating antioxidants and improve their absorption (Acosta, 2012; McClements and Xiao, 2012). Moreover, the selection of the adequate surfactant placed at the oil/water interface can modulate the enzymatic degradation rate of the system in the intestine and subsequently the bioaccessibility of the desired antioxidant (Mun et al., 2007; Torcello-Gomez et al., 2011a; Wulff-Perez et al., 2010, 2012). Table 1 shows the main physicochemical properties of SB-NE, SB-NE PF68 and SB-NE PF127 systems. As commented above, despite to their similar hydrodynamic mean size, the superficial charge (z-potential) presented a clear dependence on the surface composition. The results gathered in Table 1 show that SB-NE presented highly negative superficial charge that ensures its colloidal stability under storage conditions (Santander-Ortega et al., 2010). It is important to highlight that the lecithin used to formulate these nanoemulsions (Epikuron 145V1) is a mixture of phospholipids that contains impurities with negatively charged groups and phosphatidylcholine as main component. It has been previously demonstrated that those impurities with carboxylic head groups present in Epikuron 145V1 are mainly responsible of the negative superficial charge displayed by the systems (Goycoolea et al., 2012; Santander-Ortega et al., 2010). On the other hand, the incorporation of the non-ionic Pluronics1 to the nanoemulsion surface should lead to a displacement of the shear plane (where the z-potential is defined) towards the aqueous phase, and then to a reduction on z-potential magnitude (Santander-Ortega et al., 2006). Similar z-potential obtained of SB-NE and SB-NE PF68 could be due to low incorporation of PF68 to the nanostructure (Santander-Ortega et al., 2010). This low incorporation can be attributed to low affinity of PF68 for the nanoemulsion surface, most probably due to the presence of hydrophilic LC polar heads at the O/W interface of the nanoemulsion (Santander-Ortega et al., 2010). The evident z-potential Table 2 Physicochemical properties of soybean nanoemulsions (SB-NE) with a theoretical loading of 66% of a-tocopherol formulated with decreasing amounts of lecithin, n 3. System

Lecithin (mg)

Size (nm)

PDI

z-potential (mV)

SB-NE SB-NE10 SB-NE5

20 10 5

160 3 166 6 171 11

0.14 0.12 0.12

62 2 53 1 58 2

80

Tocopherol bioaccessibility (%)

formulations with multiple populations. The main physicochemical characteristics of a-tocopherol loaded soybean nanoemulsions are gathered in Table 2 showing that the formulation with the 66% of a-tocopherol has quite similar physicochemical properties compared to SB-NE (hydrodynamic mean size, PDI and z-potential). These results may indicate that SB-NE10 and SB-NE5 should present the same colloidal behavior that the original SB-NE after oral intake. Although the physicochemical characterization cannot shed light on the LC composition of the droplet interface, we believe that lower LC amount will affect the lipase/co-lipase access to the inner oil core and subsequently improve the bioaccessibility of a-tocopherol after oral intake. This is confirmed by the results exposed in Fig. 6, which shows the percentage of bioaccessibility reached with the new prototypes in comparison to the original SB-NE formulation. As can be observed, the reduction in the LC content clearly improved the bioaccessibility of a-tocopherol, reaching an excellent maximum bioaccessibility of 70% with the nanoemulsion formulated with 5 mg of LC.

215

70 60 50 40 30 20 10 0

SB-NE

SB-NE10

SB-NE5

Fig. 6. Percentage of a-tocopherol bioaccessibility of soybean nanoemulsions as a function of the amount of LC used in the formulation: SB-NE (20 mg), SB-NE10 (10 mg) and SB-NE5 (5 mg), n 3.

magnitude reduction when PF127 was used, instead of PF68, can be explained by a higher incorporation of this surfactant onto the oily core surface due to its longer hydrophobic central block of PF127. Besides, this stronger interface-surfactant interaction will increase the capacity of PF127 to successfully compete with other macromolecules for the oil/water surface (Olbrich and Muller, 1999; Torcello-Gomez et al., 2013; Wulff-Perez et al., 2012). The success on the use of lipid nanostructures as nutraceuticals for food supplementation depends on our capacity to design nanostructures able to reach undamaged the small intestine and once there to promote the intestinal absorption of the cargo through the mixed micelles formed during the digestion of the nanostructure (Acosta, 2012; McClements and Xiao, 2012). Under this scenario the first objective of the studies described in this manuscript has been to analyse the effect of the interface composition of the soybean nanoemulsion on its capacity to reach unaltered the small intestine and be digested there by intestinal enzymes, which should help to improve the bioaccessibility of the associated antioxidant. For that purpose the colloidal stability under simulated physiological conditions and lipolysis for the three different lipid nanostructures were studied which main results are discussed as follows for the sake of understanding: SB-NE The formulation with the O/W interface composed only by LC (Epikuron 145V1) has shown a great potential to be used as food supplement platform to increase the bioavailability of antioxidants. This is mainly due to its capacity to preserve its physicochemical properties across the mouth and the gastric compartments. Colloidal stability in the SSF (pH 6.8) can be attributed to the electrostatic repulsion between the oil droplets (Israelachvili, 2010; Santander-Ortega et al., 2009, 2010), while the homogeneous coating of the SB-NE oil droplets by the gastric enzymes led to the formation of an homogeneous corona able to stabilize the system in the simulated gastric enviroment (see Table 1 and Fig. 3) (Nel et al., 2009; Santander-Ortega et al., 2014; Tirado-Miranda et al., 2003). Once the system reaches the small intestine without modifications in its nanometric size, the interaction with the pancreatic enzymes leads to the digestion of the system in a short period of time (see Fig. 4). The design of nanostructures able to reach the small intestine as a nanometric entity and then be digested there by the pancreatic enzymes has been recently described as a key factor in the success of

216


nanoplatforms for increasing the bioavailability of antioxidants used as nutraceuticals in food supplementation (Acosta, 2012; Golding et al., 2011; McClements and Xiao, 2012; Salvia-Trujillo et al., 2013). SB-NE PF68 The incorporation of PF68 to the O/W interface was aimed to preserve the physicochemical properties of the formulation in the mouth and the stomach. However, the surprisingly high stability showed by the bare SB-NE system in these media (based on the DLVO potentials of interaction) as well as the similar digestion pattern displayed by both formulations makes necessary to reconsider the role of the PF68 excipient in the formulation of the system (see Figures 3 and 4). Another issue in the use of PF68 as coating agent is its low incorporation to the nanostructure interface (see Table 1). This means that the surfactant of the formulation will mainly be as PF68 micelles instead of as a coating layer around the oil matrix (Santander-Ortega et al., 2006). This is a potential problem during the encapsulation of antioxidants as the active molecules would be either distributed in the edible oil matrix or in the non-digestible polymeric micelles reducing their bioavailability (McClements and Xiao, 2012; Torcello-Gomez et al., 2011a; Wulff-Perez et al., 2010). SB-NE PF127 The aim pursued by the incorporation of PF127 to the oil nanostructure was to preserve it from a premature degradation on the GIT, as was with PF68. All the results presented in this work clearly show the protective role of the PF127 coating layer around the oil core through a steric hindrance mechanism (see Table 1 and Fig. 3) (Torcello-Gomez et al., 2011a). Unfortunately, the strong adsorption of PF127 onto the interface of the oil droplets also avoided the digestion of the nanostructure in the small intestine, which, as will be commented below, would end up in the low bioaccessibility of the encapsulated antioxidant (Li et al., 2012). It can be anticipated that the low accessibility of the lipases to the inner oil matrix of the SB-NE PF127 will compromise the capacity of the formulation to improve the bioavailability of the encapsulated antioxidant (Li et al., 2012). Regarding to the PF68 coated nanoemulsions (SB-NE PF68), it has been recently demonstrated that PF68 can efficiently protect nanoparticles made of polylactic-co-glycolic acid (PLGA) from the enzymatic degradation in SIF (Santander-Ortega et al., 2006, 2009). However, as shown in Fig. 4, the incorporation of PF68 did not have a sensible effect on the degradation pattern of the nanoemulsion. These differences in the protective role of PF68 can be attributed to the different interaction of the surfactant with both substrates. PLGA nanoparticles with a hydrophobic character, similar to that of polystyrene nanoparticles, will favor the adsorption of PF68 and will prevent its desorption by the enzymes presented in the surrounding medium (Santander-Ortega et al., 2009). However, in the case of the oil nanoemulsion the presence of lecithin polar heads on its surface will reduce its hydrophobic character (Santander-Ortega et al., 2010). Therefore, although PF68 can be efficiently incorporated to the oily droplets (Torcello-Gomez et al., 2011a,b; Wulff-Perez et al., 2012), the weaker oily core-surfactant hydrophobic interactions will facilitate its desorption by the enzymes and proteins (Torcello-Gomez et al., 2013; Wulff-Perez et al., 2010) reducing its protective effect (Torcello-Gomez et al., 2011c). The analysis of the loading capacity of the three nanoemulsions revealed that all of them were highly efficient co-encapsulating gentisic, p-hydroxybenzoic and p-coumaric acids (EE > 80%). The nanoemulsion was able to release the antioxidant in a controlled

fashion avoiding the typical burst effect observed with other molecules (Lozano et al., 2008, 2013). Burst effect is related to the heterogeneous core-shell arrangement of the encapsulated drug in the oil matrix where the shell-associated drug molecules are rapidly released from the nanostructure while the core-associated drug molecules show a delayed/controlled release with respect to the former. However, release profiles depicted in Fig. 5 clearly indicated an excellent distribution of the three model antioxidants inside the soybean oil core. Unfortunately, more than 50% of the encapsulated antioxidants were release from the nanoemulsions in 5 h. Nanoemulsions for food supplementation should protect the cargo not only after oral intake but also along the manufacturing and distributing process. This requisite would not be achieved with the release pattern of these antioxidants. Based on the LCT nature of the soybean matrix, a-tocopherol, a therapeutic antioxidant with already demonstrated neuroprotective properties (Eitenmiller and Lee 2005), would be efficiently incorporated to the nanoemulsion matrix (Yang and McClements, 2013). Moreover, the high lipophilic character of this antioxidant (Log P: 9.60), indicates that, once encapsulated, the antioxidant would be retained in the lipid nanoemulsion matrix until the digestion of the formulation in the small intestine (Yang and McClements, 2013). In addition, inclusion of a-tocopherol into the nanoemulsion would not only improve the absorption of this antioxidant, but also would reduce the high absorption variability observed in the actual marketed products (Eitenmiller and Lee 2005; Gong et al., 2012; Hatanaka et al., 2010) As shown in Table 2, the encapsulation of a-tocopherol resulted in a reduction of the hydrodynamic mean size of the oily droplets from 195 nm down to 160 nm. Although the physicochemical mechanisms governing the size of the droplets produced by solvent displacement technique are still not clearly understood (Mora-Huertas et al., 2011), the reason behind the size reduction of the formulation could be related to the increase of the oil viscosity and the interfacial tension of the O/W interface by the addition of a-tocopherol to the soybean oil (Saberi et al., 2013). Regardless of the hydrodynamic mean size reduction, the similar physicochemical properties of the empty and the a-tocopherol loaded systems indicate that the encapsulation of the antioxidant should not affect its promising colloidal behavior after its oral intake. The success of the formulation for improving the bioaccessibility of a-tocopherol was confirmed by the quantification of the antioxidant fraction in the mixed micelles produced during the digestion of the formulation by the pancreatic enzymes, which will be internalized through the intestinal epithelial cells either by active or passive transport (Acosta, 2012; McClements and Xiao, 2012). Then, the quantification of the fraction of a-tocopherol present in the mixed micelles which are available for intestinal absorption, brought values of bioaccessibility of 38 7% (for the SB-NE with a theoretical loading of 33%) and 38 1% (for the SB-NE with a theoretical loading of 66%). These promising results clearly show the successful formulation of a lipid nanometric platform, through a low energy method, able not only to overcome the biological barriers of the gastrointestinal track but also to promote high and reproducible percentages of bioaccessibility of the cargo. Finally, our aims were focused on the optimization of the formulation to increase even more the bioaccessibility of the cargo. The selection of LC as emulsifier was found to be the optimal for a nutraceutical formulation. Our results of the lipolysis assay (see Fig. 4) were in line with other recently published studies which proved that LC presents lower capacity than non-ionic surfactants, such as PF68, PF127, Tweens or PEG-stearate derivatives to hamper the adsorption of lipase/co-lipase onto O/W interfaces (Mun et al., 2007; Torcello-Gomez et al., 2011a, 2013; Wulff-Perez et al., 2010,


2012). On the other side, Mun and co-authors have recently shown that LC presents higher protection capacity against lipase action than casein or whey proteins, two proteins widely used in the food industry as emulsifiers (Mun et al., 2007). This intermediate capacity of LC to control the lipolysis of lipid matrix suggest that LC emulsifying properties can be used for tuning the lipase/oil core interaction up and then for the modulation of the nanoemulsion digestibility (Mun et al., 2007; Torcello-Gomez et al., 2011a; WulffPerez et al., 2010). The modulation of the amount of LC present in the formulation (and then in the O/W interface) from 20 mg down to 5 mg resulted in improved bioaccessibility, reaching bioaccessibility values of a-tocopherol of around 70% thanks to the modulation of the lipase access to the inner oil core, meanwhile the original physicochemical properties of the nanoemulsion were unmodified, which guaranteed the colloidal behavior of the system after oral intake. 5. Conclusions There are two main features that a nanostructure should fulfil in order to success as a food supplement, i.e., adequate interface and matrix composition. This work has addressed both items for a soybean lipid nanostructure and has determined that for this formulation the best interface composition was the one composed only by lecithin, as it maintains the integrity of the system until it reaches the small intestine where it enables its digestion. This conclusion was achieved by performing a thorough physicochemical characterization that allowed us to understand the different mechanisms underneath the stability of three systems with different interface composition: SB-NE, SB-NE PF68 and SB-NE PF127, followed by a lipolysis assays. By the evaluation of the matrix composition effect on the encapsulation of antioxidants with neuroprotective activity we learned that the interface composition had no an appreciable role on the controlled release of the molecules tested from the nanostructure. Nevertheless, it led us to understand that an adequate selection of the nature of the cargo molecule and the composition of the matrix are needed for designing a promising nanoplatform for food supplementation. Indeed the results show the development of an optimised a-tocopherol lipid nanostructure with great potential for future in vivo studies, as shown by the high and consistent a-tocopherol bioaccessibility achieved. Acknowledgement Authors thank the financial support given by the PEII-2014040-P research project from the Junta de Comunidades de CastillaLa Mancha, as well as by the GI20153002 research project from the Vicerrectorado de Investigación, University of Castilla-La Mancha. Authors are also grateful to Dr. M. Rocío Fernández Santos for the support given. References Acosta, E.J., 2012. Association colloids as delivery systems: principles and applications in the food and nutraceutical industries. In: Huang, Q. (Ed.), Nanotechnology in The Food, Beverage and Nutraceutical Industries. Woodhead Publishing, Amsterdam. Anand, P., Nair, H.B., Sung, B.K., Kunnumakkara, A.B., Yadav, V.R., Tekmal, R.R., Aggarwal, B.B., 2010. Design of curcumin-loaded PLGA nanoparticles formulation with enhanced cellular uptake, and increased bioactivity in vitro and superior bioavailability in vivo. Biochem. Pharmacol. 79, 330–338. Devraj, R., Williams, H.D., Warren, D.B., Mullertz, A., Porter, C.J., Pouton, C.W., 2013. In vitro digestion testing of lipid-based delivery systems: calcium ions combine with fatty acids liberated from triglyceride rich lipid solutions to form soaps and reduce the solubilization capacity of colloidal digestion products. Int. J. Pharm. 441, 323–333. Eitenmiller, R.R., Lee, J., 2005. Vitamin E: Food Chemistry, Composition, and Analysis. CRC Press, London.

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Design and usability study of an iconic user interface to ease information retrieval of medical guidelines.

Design and performance of a novel electrospray interface.

Facet-Engineered Surface and Interface Design of Photocatalytic Materials.

Does boiling affect the bioaccessibility of selenium from cabbage?

Atomic scale interface design and characterisation.

Interface of Linguistic and Visual Information During Audience Design.

Design of radiation tolerant materials via interface engineering.