International Journal of Pharmaceutics 486 (2015) 59–68
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Casein/pectin nanocomplexes as potential oral delivery vehicles Yangchao Luo a,b, * , Kang Pan a , Qixin Zhong a, ** a b
Department of Food Science and Technology, University of Tennessee, 2510 River Drive, Knoxville, TN 37996, United States Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 January 2015 Received in revised form 18 March 2015 Accepted 19 March 2015 Available online 20 March 2015
Delivery systems prepared with natural biopolymers are of particular interests for applications in food, pharmaceutics and biomedicine. In this study, nanocomplex particles of sodium caseinate (NaCas) and pectin were fabricated and investigated as potential oral delivery vehicles. Nanocomplexes were prepared with three mass ratios of NaCas/pectin by acidification using glucono-d-lactone and thermal treatment. NaCas/pectin at 1:1 mass ratio resulted in dispersions with the lowest turbidity and the smallest and most uniform nanocomplexes. Thermal treatment at 85 C for 30 min facilitated the formation of stable, compact, and spherical nanocomplexes. Heating not only greatly increased the yield of nanocomplexes but also significantly improved the encapsulation capability of rutin studied as a model compound. Pectin in nanocomplexes delayed the hydrolysis of NaCas by pepsin at gastric conditions and enabled the controlled release of most rutin in simulated intestinal conditions. The nanocomplexes based on food-sourced biopolymers have promising features for oral delivery of nutrients and medicines. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Casein Pectin Nanocomplexes Delayed gastric digestion Oral delivery vehicles
1. Introduction Drug delivery systems based on biopolymer polyelectrolyte complexes (PECs) have attracted increasing attention in recent years (Jones and McClements, 2011; Luo and Wang, 2014a,b). The PECs are formed by electrostatic interactions between two or more oppositely charged polyelectrolytes. The dimension and morphology of PECs are largely determined by the structure of biopolymers and fabrication conditions. Recently, protein–polysaccharide PECs have drawn interest for food and pharmaceutical applications, due to their encapsulation capability and tunable drug delivery properties (Schmitt and Turgeon, 2011; Turgeon et al., 2007). PECs formed with proteins and polysaccharides have unique advantages when compared to those formed with one type of polyelectrolytes. Proteins contribute to encapsulation efficiency by providing affinity to bind bioactive compounds via hydrogen bonding and hydrophobic interactions, while polysaccharides work as a barrier to protect the enzymatic degradation of protein in gastric conditions and thus provide sustained release of encapsulated compounds in the intestine. For instance, zein/
* Corresponding author at: Department of Nutritional Sciences, University of Connecticut, 3624 Horsebarn Road Extension, U-4017, Storrs, CT 06269-4017, United States. Tel.: +1 860 486 2186; fax: +1 860 486 3674. ** Corresponding author. Tel.: +1 865 974 6196; fax: +1 865 974 7332. E-mail addresses: [email protected] (Y. Luo), [email protected] (Q. Zhong). http://dx.doi.org/10.1016/j.ijpharm.2015.03.043 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
chitosan PECs have been studied to form nanoparticles (Luo et al., 2013b, 2011) and microspheres (Müller et al., 2011) to encapsulate various drugs and nutrients and provide sustained release in gastrointestinal conditions. Sodium caseinate (NaCas) is a commercially available water soluble protein ingredient, produced by acid precipitation of casein micelles from bovine milk followed by neutralization using sodium hydroxide for spray-drying (Walstra et al., 2006). The nutritional and functional properties of NaCas have been extensively studied (Aimutis, 2004; Kinsella, 1984; Luo et al., 2014). Although NaCas and casein micelles share similar casein compositions, they differ in amounts of calcium and phosphate, and NaCas has a smaller mass and are more soluble than casein micelles (Walstra et al., 2006). NaCas is comprised of both hydrophobic and hydrophilic segments, and the amphiphilicity enables the self-assembly as nanoparticles in aqueous solutions. As a group of natural biopolymers, NaCas has excellent biocompatibility, great digestibility, and low toxicity, and is a promising candidate for the development of novel drug delivery systems (Elzoghby et al., 2011). Encapsulation of hydrophobic curcumin was recently studied by spray-drying a warm ethanol solution with NaCas and curcumin (Pan et al., 2013). In our another recent study, a novel pH-driven technique was developed to encapsulate curcumin (Pan et al., 2014b). The technique is based on the dissociation of NaCas and deprotonation/dissolving of curcumin at pH 12, followed by neutralization to reassemble NaCas and simultaneous encapsulation of precipitated curcumin. Another recent study suggested that
60
Y. Luo et al. / International Journal of Pharmaceutics 486 (2015) 59–68
NaCas could facilitate the cellular uptake of hydrophobic nutrients by interacting with intestinal epithelial cells (Luo et al., 2013a). Nevertheless, when developing oral drug delivery systems, protection of protein nanoparticles from gastric digestion is needed so as to release drugs at the absorption sites in intestines. Pectin is an anionic polysaccharide, and a great number of studies have shown that pectin is capable of stabilizing casein micelles in acidified milk (Laurent and Boulenguer, 2003; Maroziene and de Kruif, 2000; Pereyra et al., 1997; Tuinier et al., 2002). The adsorption of pectin onto casein micelles at pH 3.5– 5.0 is driven by electrostatic attraction between carboxylate groups of pectin and cationic amino acid residues of casein (Tuinier et al., 2002). Another study suggested that NaCas has similar properties as casein micelles in forming PECs with pectin (Rediguieri et al., 2007). The study also revealed that a slow rate of acidification (1 drop/min of 1 M citric acid) facilitated the formation of soluble pectin/casein PECs. Due to high concentrations of both biopolymers, the as-prepared NaCas/pectin PECs are spherical microparticles with a dimension of 3 mm. We hypothesize that soluble nano-scale PECs can be formed from solutions with low concentrations of NaCas/pectin and the fabricated nanoPECs can be used to deliver bioactive compounds for food and pharmaceutic applications. The first objective of the present work was to fabricate and characterize NaCas/pectin nanoPECs prepared by the controlled acidification enabled by glucono-d-lactone (GDL). GDL lowers solution pH after gradual hydrolysis and has been previously used for acid-induced gelation of casein and other biopolymers such as xanthan gum (Braga and Cunha, 2004), pectin (Matia-Merino and Singh, 2007), carrageenan (Ribeiro et al., 2004), gellan gum (Nag et al., 2011), and whey protein (Vasbinder et al., 2004). The physicochemical properties and in vitro digestion stability were studied for PECs prepared at various conditions. The second objective was to explore the drug delivery potential of NaCas/ pectin nanoPECs using rutin as a model drug. Rutin (30 ,40 , 5,7-tetrahydroxyflavone-3-rutinoside) is a plant-derived dietary flavonoid and is an important phytochemical with significant pharmacological activities (Chua, 2013). Because the phenolic group of rutin can be deprotonated at alkaline conditions, we hypothesize rutin can be encapsulated in nanoPECs using our recently developed pH-driven encapsulation technique (Pan et al., 2014b). 2. Materials and methods
on a magnetic stir plate and the pH was monitored until reaching a constant value (in less than 1 h). After stirring for 1 h, the mixture was heated at 85 C for 30 min in a water bath. Both the samples before and after heating were characterized. The optical density of samples was measured at 500 nm using a UV/vis spectrophotometer (Evolution 201, Thermo Scientific, Waltham, MA, USA) for comparison of turbidity. Samples were also photographed for comparison of visual appearance. Dispersions were ultra-centrifuged at 125,000 g for 40 min to separate PECs from free polyelectrolytes which remained in the supernatant after centrifugation. The NaCas and pectin concentrations in the supernatant were determined using the bicinchoninic acid (Walker, 1994) and phenol–sulfuric acid (Kochert, 1978) assays, respectively, with reference to standard curves established from NaCas and pectin. 2.3. Particle size and zeta potential determination The particle size was determined using dynamic light scattering (DLS, model Delsa Nano analyzer, Beckman Coulter, Atlanta, GA, USA) with a scattering angle of 165 . Samples were diluted to fit the instrument signal requirement. In addition to the hydrodynamic diameter and polydispersity index, the intensity, which is relevant to particle count rate, was reported after multiplying by dilution factors. Zeta potential was calculated from electrophoretic mobility determined using a laser Doppler velocimeter (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). 2.4. Morphology of complexes The morphology of nanoPECs was first examined with scanning electron microscopy (SEM). Briefly, one drop of a freshly prepared sample was cast-dried on a 5 5 mm silicon wafer, which was then sputter-coated by a thin layer (
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Casein/pectin nanocomplexes as potential oral delivery vehicles Yangchao Luo a,b, * , Kang Pan a , Qixin Zhong a, ** a b
Department of Food Science and Technology, University of Tennessee, 2510 River Drive, Knoxville, TN 37996, United States Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 January 2015 Received in revised form 18 March 2015 Accepted 19 March 2015 Available online 20 March 2015
Delivery systems prepared with natural biopolymers are of particular interests for applications in food, pharmaceutics and biomedicine. In this study, nanocomplex particles of sodium caseinate (NaCas) and pectin were fabricated and investigated as potential oral delivery vehicles. Nanocomplexes were prepared with three mass ratios of NaCas/pectin by acidification using glucono-d-lactone and thermal treatment. NaCas/pectin at 1:1 mass ratio resulted in dispersions with the lowest turbidity and the smallest and most uniform nanocomplexes. Thermal treatment at 85 C for 30 min facilitated the formation of stable, compact, and spherical nanocomplexes. Heating not only greatly increased the yield of nanocomplexes but also significantly improved the encapsulation capability of rutin studied as a model compound. Pectin in nanocomplexes delayed the hydrolysis of NaCas by pepsin at gastric conditions and enabled the controlled release of most rutin in simulated intestinal conditions. The nanocomplexes based on food-sourced biopolymers have promising features for oral delivery of nutrients and medicines. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Casein Pectin Nanocomplexes Delayed gastric digestion Oral delivery vehicles
1. Introduction Drug delivery systems based on biopolymer polyelectrolyte complexes (PECs) have attracted increasing attention in recent years (Jones and McClements, 2011; Luo and Wang, 2014a,b). The PECs are formed by electrostatic interactions between two or more oppositely charged polyelectrolytes. The dimension and morphology of PECs are largely determined by the structure of biopolymers and fabrication conditions. Recently, protein–polysaccharide PECs have drawn interest for food and pharmaceutical applications, due to their encapsulation capability and tunable drug delivery properties (Schmitt and Turgeon, 2011; Turgeon et al., 2007). PECs formed with proteins and polysaccharides have unique advantages when compared to those formed with one type of polyelectrolytes. Proteins contribute to encapsulation efficiency by providing affinity to bind bioactive compounds via hydrogen bonding and hydrophobic interactions, while polysaccharides work as a barrier to protect the enzymatic degradation of protein in gastric conditions and thus provide sustained release of encapsulated compounds in the intestine. For instance, zein/
* Corresponding author at: Department of Nutritional Sciences, University of Connecticut, 3624 Horsebarn Road Extension, U-4017, Storrs, CT 06269-4017, United States. Tel.: +1 860 486 2186; fax: +1 860 486 3674. ** Corresponding author. Tel.: +1 865 974 6196; fax: +1 865 974 7332. E-mail addresses: [email protected] (Y. Luo), [email protected] (Q. Zhong). http://dx.doi.org/10.1016/j.ijpharm.2015.03.043 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
chitosan PECs have been studied to form nanoparticles (Luo et al., 2013b, 2011) and microspheres (Müller et al., 2011) to encapsulate various drugs and nutrients and provide sustained release in gastrointestinal conditions. Sodium caseinate (NaCas) is a commercially available water soluble protein ingredient, produced by acid precipitation of casein micelles from bovine milk followed by neutralization using sodium hydroxide for spray-drying (Walstra et al., 2006). The nutritional and functional properties of NaCas have been extensively studied (Aimutis, 2004; Kinsella, 1984; Luo et al., 2014). Although NaCas and casein micelles share similar casein compositions, they differ in amounts of calcium and phosphate, and NaCas has a smaller mass and are more soluble than casein micelles (Walstra et al., 2006). NaCas is comprised of both hydrophobic and hydrophilic segments, and the amphiphilicity enables the self-assembly as nanoparticles in aqueous solutions. As a group of natural biopolymers, NaCas has excellent biocompatibility, great digestibility, and low toxicity, and is a promising candidate for the development of novel drug delivery systems (Elzoghby et al., 2011). Encapsulation of hydrophobic curcumin was recently studied by spray-drying a warm ethanol solution with NaCas and curcumin (Pan et al., 2013). In our another recent study, a novel pH-driven technique was developed to encapsulate curcumin (Pan et al., 2014b). The technique is based on the dissociation of NaCas and deprotonation/dissolving of curcumin at pH 12, followed by neutralization to reassemble NaCas and simultaneous encapsulation of precipitated curcumin. Another recent study suggested that
60
Y. Luo et al. / International Journal of Pharmaceutics 486 (2015) 59–68
NaCas could facilitate the cellular uptake of hydrophobic nutrients by interacting with intestinal epithelial cells (Luo et al., 2013a). Nevertheless, when developing oral drug delivery systems, protection of protein nanoparticles from gastric digestion is needed so as to release drugs at the absorption sites in intestines. Pectin is an anionic polysaccharide, and a great number of studies have shown that pectin is capable of stabilizing casein micelles in acidified milk (Laurent and Boulenguer, 2003; Maroziene and de Kruif, 2000; Pereyra et al., 1997; Tuinier et al., 2002). The adsorption of pectin onto casein micelles at pH 3.5– 5.0 is driven by electrostatic attraction between carboxylate groups of pectin and cationic amino acid residues of casein (Tuinier et al., 2002). Another study suggested that NaCas has similar properties as casein micelles in forming PECs with pectin (Rediguieri et al., 2007). The study also revealed that a slow rate of acidification (1 drop/min of 1 M citric acid) facilitated the formation of soluble pectin/casein PECs. Due to high concentrations of both biopolymers, the as-prepared NaCas/pectin PECs are spherical microparticles with a dimension of 3 mm. We hypothesize that soluble nano-scale PECs can be formed from solutions with low concentrations of NaCas/pectin and the fabricated nanoPECs can be used to deliver bioactive compounds for food and pharmaceutic applications. The first objective of the present work was to fabricate and characterize NaCas/pectin nanoPECs prepared by the controlled acidification enabled by glucono-d-lactone (GDL). GDL lowers solution pH after gradual hydrolysis and has been previously used for acid-induced gelation of casein and other biopolymers such as xanthan gum (Braga and Cunha, 2004), pectin (Matia-Merino and Singh, 2007), carrageenan (Ribeiro et al., 2004), gellan gum (Nag et al., 2011), and whey protein (Vasbinder et al., 2004). The physicochemical properties and in vitro digestion stability were studied for PECs prepared at various conditions. The second objective was to explore the drug delivery potential of NaCas/ pectin nanoPECs using rutin as a model drug. Rutin (30 ,40 , 5,7-tetrahydroxyflavone-3-rutinoside) is a plant-derived dietary flavonoid and is an important phytochemical with significant pharmacological activities (Chua, 2013). Because the phenolic group of rutin can be deprotonated at alkaline conditions, we hypothesize rutin can be encapsulated in nanoPECs using our recently developed pH-driven encapsulation technique (Pan et al., 2014b). 2. Materials and methods
on a magnetic stir plate and the pH was monitored until reaching a constant value (in less than 1 h). After stirring for 1 h, the mixture was heated at 85 C for 30 min in a water bath. Both the samples before and after heating were characterized. The optical density of samples was measured at 500 nm using a UV/vis spectrophotometer (Evolution 201, Thermo Scientific, Waltham, MA, USA) for comparison of turbidity. Samples were also photographed for comparison of visual appearance. Dispersions were ultra-centrifuged at 125,000 g for 40 min to separate PECs from free polyelectrolytes which remained in the supernatant after centrifugation. The NaCas and pectin concentrations in the supernatant were determined using the bicinchoninic acid (Walker, 1994) and phenol–sulfuric acid (Kochert, 1978) assays, respectively, with reference to standard curves established from NaCas and pectin. 2.3. Particle size and zeta potential determination The particle size was determined using dynamic light scattering (DLS, model Delsa Nano analyzer, Beckman Coulter, Atlanta, GA, USA) with a scattering angle of 165 . Samples were diluted to fit the instrument signal requirement. In addition to the hydrodynamic diameter and polydispersity index, the intensity, which is relevant to particle count rate, was reported after multiplying by dilution factors. Zeta potential was calculated from electrophoretic mobility determined using a laser Doppler velocimeter (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). 2.4. Morphology of complexes The morphology of nanoPECs was first examined with scanning electron microscopy (SEM). Briefly, one drop of a freshly prepared sample was cast-dried on a 5 5 mm silicon wafer, which was then sputter-coated by a thin layer (
