Encapsulation of curcumin in recombinant human H-chain ferritin increases its water-solubility and stability

Highlights

• Curcumin encapsulation within ferritin shell

• High water solubility of curcumin–ferritin nanocomposites

• Improved light and heat stability of encapsulated curcumin

Abstract

Curcumin is a natural bioactive agent found in turmeric (Curcuma longa) and has many health-promoting benefits. However, its poor water-solubility and thermal and photostability limit its application in foodstuffs. In the present study, recombinant human H-chain ferritin (rHuHF), a typical shell-like protein with an inner diameter as ~ 8 nm was used to encapsulate curcumin by taking advantage of the reversible dissociation and reassembly characteristic of apoferritin at different pH values. We demonstrated that curcumin molecules were successfully encapsulated within protein cages with a curcumin/protein molar ratio of 14.7 to 1. Upon such molecular encapsulation, curcumin molecules become greatly water-soluble, while their thermal and photostability were greatly improved due to protection by protein cages as compared to free analogs. This novel approach reported in this study has a potential to be applied in other poorly water-soluble bioactive compounds.

Introduction

Curcumin [(E,E)-1,7-bis(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-ione] (Fig. 1A) is a hydrophobic polyphenol derived from the rhizome of turmeric (Curcuma longa, an East Indian plant), along with demethoxy curcumin and bisdemethoxy curcumin (Anderson, Mitchell, & Mohan, 2000). It is a yellow pigment and has been extensively used in food and chemical industries as coloring, flavoring and preservative (Bhawana, Basniwal, Buttar, Jain, & Jain, 2011). Interests in this dietary polyphenol have grown in recent years due to its vast array of beneficial pharmacological effects such as antioxidant, anti-inflammatory (treatment of osteoarthritis), anticarcinogenic, antibacterial, wound healing, antispasmodic, and anticoagulant activities (Aggarwal et al., 2003, Pizzo et al., 2010). Furthermore, curcumin is nontoxic, even at relatively high dose (Strimpakos & Sharma, 2008). Though having multiple medicinal benefits and extremely high safety profile, wide applications of this promising molecule in food and chemical industries have been hindered by its poor water solubility (estimated to be 11 ng/mL), short biological half-life, and low bioavailability following oral administration (Anand, Kunnumakkara, Newman, & Aggarwal, 2007). Therefore, it is of great importance to improve the water solubility and stability of curcumin.

The water-insoluble bioactive compounds such as carotenoids, phytosterols and natural antioxidants can become water-soluble with higher dissolution velocity and saturation solubility, if they are in the form of nanodispersions with a particle size in nanometer range (Bhawana et al., 2011). Similarly, to overcome the problems of poor solubility and low bioavailability of curcumin, popular techniques that are used to manufacture nano-sized particles are solvent-based processes, which include emulsification–solvent evaporation, emulsification–solvent diffusion, and precipitation methods (Bhawana et al., 2011, Horn and Rieger, 2001). The disadvantage with these methods is that they require the addition of considerable amounts of surfactants to prevent coalescence during particle formation. Additionally, organic solvent used in these methods can lead to sample contamination and environmental pollution. Recently nanoparticle-based drug delivery approaches have been reported (Anand et al., 2010), in which curcumin is encapsulated in liposomes (Wang et al., 2008), protein-based microparticles, such as bovine serum albumin (Gupta, Aseh, Rios, Aggarwal, & Mathur, 2009) and chitosan (Das, Kasoju, & Bora, 2010), or complexed with phospholipids (Maiti, Mukherjee, Gantait, Saha, & Mukherjee, 2007) and cyclodextrin (Dhule et al., 2012). Additionally, the utilization of N-isopropylacrylamide with N-vinyl-2-pyrrolidone and poly(ethyleneglycol) monoacrylate to synthesize curcumin-encapsulated polymeric nanoparticles has been reported recently (Bisht et al., 2010). Another approach for increasing the rate of dissolution of curcumin is by increasing its surface area. This can be achieved by decreasing the particle size by milling or grinding (Bhawana et al., 2011).

Despite these significant developments, much work is needed to prepare monodispersed nanoparticles for transparent functional beverage applications using generally recognized as safe (GRAS) ingredients. Fortunately, the widespread occurrence of ferritins with a specific nanocage in nature offers a good opportunity for such purpose. Ferritins are a class of iron storage proteins that are widely distributed in animals, plants and bacteria except for yeast, which have an important function of iron storage and detoxification in all living organisms and play important roles in controlling cellar iron homeostasis (Arosio et al., 2008, Zhao, 2010). Ferritin is usually composed of 24 identical/different subunits that are arranged in an octahedral symmetry to form a hollow protein shell (outside diameter is about 12–13 nm, and inside diameter is about 7–8 nm) capable of storing up to 4500 iron atoms as a ferric inorganic complex. Each ferritin molecule has eight 3-fold channels and six 4-fold channels, through which the inner cavity of ferritin and outside solution are connected (Arosio et al., 2008, Zhao, 2010) (Fig. 1B). The native iron oxide particle can be easily removed from the protein cage by reduction of Fe(III) and subsequent chelation of Fe(II), resulting in the formation of apoferritin having an empty, intact protein cage. Apoferritin can be disassociated into subunits at extreme acid conditions (pH ≤ 2) but not denatured, and then reconstituted when pH is adjusted to pH ~ 7.0 (Kim et al., 2011). During this process, small molecules in solution could be trapped within its interior (Liu, Wang, Lea, & Lin, 2006). This interesting property has been used to prepare various inorganic nanoparticles. All obtained nanoparticles with ferritin were found to have the same and narrow size distribution (Iwahori et al., 2005, Li et al., 2007). Ferritin encapsulation has several advantages over the abovementioned methods. First, ferritin-encapsulated nanoparticles are homogeneous, and have nearly the same and narrow size as ~ 12 nm. Second, by using the reversible dissociation and reassembly characteristic of apoferritin at different pH values, small molecules can be easily and effectively encapsulated within the inner cavity of ferritin without the use of any organic solvents. Third, ferritin is a naturally occurring protein, which is edible and environment friendly.

The objective of this work is to encapsulate the lipophilic component, curcumin, within the apoferritin shell. More importantly, we demonstrated that associated with such encapsulation is an increase in the photo and thermal stability of curcumin as well as its solubility. These new properties of the curcumin molecules within ferritin nanocages may be favorable for their application in water-based food and pharmacological formulations.