Elsevier

Carbohydrate Polymers

Volume 99, 2 January 2014, Pages 499-507
Carbohydrate Polymers

Conjugation of curcumin onto alginate enhances aqueous solubility and stability of curcumin

https://doi.org/10.1016/j.carbpol.2013.08.067Get rights and content

Highlights

  • A novel Alginate-Curcumin (Alg-Ccm) conjugate was developed.

  • The conjugate self-assembled in aqueous medium forming nanosized micelles.

  • Alg-Ccm enhanced the aqueous solubility of curcumin compared to free curcumin.

  • The conjugate micelles augmented the stability of curcumin in water at pH 7.4.

  • Alg-Ccm showed excellent cytotoxic activity compared to free curcumin.

Abstract

Curcumin is a potential drug for various diseases including cancer. Prime limitations associated with curcumin are low water solubility, rapid hydrolytic degradation and poor bioavailability. In order to redress these issues we developed Alginate-Curcumin (Alg-Ccm) conjugate which was characterized by FTIR and 1H NMR spectroscopy. The conjugate self-assembled in aqueous solution forming micelles with an average hydrodynamic diameter of 459 ± 0.32 nm and negative zeta potential. The spherical micelles were visualized by TEM. The critical micelle concentration (CMC) of Alg-Ccm conjugate was determined. A significant enhancement in the aqueous solubility of curcumin was observed upon conjugation with alginate. Formation of micelles improved the stability of curcumin in water at physiological pH. The cytotoxic activity of Alg-Ccm was quantified by MTT assay using L-929 fibroblast cells and it was found to be potentially cytotoxic. Hence, Alg-Ccm could be a promising drug conjugate as well as a nanosized delivery vehicle.

Introduction

Curcumin (diferuloylmethane) is the principal active ingredient in the spice turmeric (curcuma longa). It is basically a low molecular weight natural polyphenol with versatile medicinal assets which are well documented in ancient Indian literatures (Goel, Kunnumakkara, & Aggarwal, 2008). Over the last couple of decades, there has been tremendous interest in curcumin because of its potent antioxidant, anti-inflammatory, antiproliferative and antiangiogenic activities (Aggarwal et al., 2003, Aggarwal and Sung, 2009, Chattopadhyay et al., 2004, Lantz et al., 2005, Ruby et al., 1995, Shi et al., 2006). Extensive research works have proved curcumin to be a highly pleiotropic molecule and it can interact with various molecular targets including cytokines, enzymes, genes, transcription factors, growth factors and their receptors etc. to control cell proliferation and apoptosis (Goel et al., 2008, Ravindran et al., 2009). Thus, the likelihood of developing resistance to curcumin is very less as it induces apoptosis through multiple cell signaling pathways. Besides, curcumin can play a vital role in controlling multidrug resistance (MDR) in cancer cells by down regulating the levels of Pgp, ABCG2 and MRP-1 (three major ABC drug transporters responsible for the development of MDR) (Bava et al., 2005, Manju and Sreenivasan, 2012). Curcumin has been documented to be highly effective against many cancer cells of human origin (Sandur et al., 2007), an efficient photodynamic therapeutic agent for the treatment of skin cancer (Park & Lee, 2007) and a competent curative agent against Alzheimer's and other incapacitating diseases (Mythri et al., 2011, Ringman et al., 2005). In spite of possessing striking anticancer properties, curcumin is not extensively used for cancer treatments due to its poor aqueous solubility and low bioavailability (Anand, Kunnumakkara, Newman, & Aggarwal, 2007). Hydrophobic nature of this polyphenolic compound along with its rapid metabolism, physicochemical and biological instability contribute to its poor bioavailability. To redress these problems several approaches have been proposed like encapsulation of curcumin in liposomes and polymeric micelles, inclusion complex formation with cyclodextrin, formation of polymer-curcumin conjugates etc. (Bisht et al., 2007, Manju and Sreenivasan, 2011a, Manju and Sreenivasan, 2011b, Manju and Sreenivasan, 2012, Thangapazham et al., 2008, Yallapu et al., 2010). Among the diverse drug delivery approaches, the “polymer-drug” conjugate is a very popular and positive approach for the delivery of hydrophobic drugs. The concept of covalently bound polymer-drug conjugates, proposed by Ringsdorf (1975), is widely accepted for improved aqueous solubility, controlled delivery of drugs in optimum dosages, enhanced therapeutic efficacy, reduced side effects and improved patient compliances (Alexis et al., 2008, Van et al., 2010, Zhou et al., 2010). Another important aspect associated with drug conjugates is that they can promote passive tumor targeting via the enhanced permeation and retention (EPR) effect (Maeda, 2010). Choosing the exact polymer in one hand can enhance the safer systemic circulation time of a conjugate and on the other hand, controlling parameters like size, functionality, charge etc. can amend the effect of biological half-life of a drug in a conjugate (Li & Huang, 2008). Various types of natural and synthetic polymers have been conjugated with hydrophobic drugs for the development of polymer-drug conjugates. Polyethylene glycol (PEG) with hydrophilic nature and ability to avoid nonspecific protein adsorption (stealth property) is the most widely studied polymer for the development of conjugates with longer blood circulation time. The main demerit associated with PEG is its non-biodegradability. Several hydrophilic, biodegradable, natural polymers have also been used for development of polymer drug conjugates. Alginate is an anionic, polysaccharide copolymer consisting of (1  4) linked β-d-mannuronic acid (M) and its C-5 epimer α-l-guluronic acid (G) residues. The natural block copolymer contains regions of sequential M-units (M blocks), G-units (G blocks) and atactically organized M and G units. Alginate is widely used in biomedical applications owing to its several advantages such as biodegradability, high biocompatibility, non-toxicity, non-immunogenicity and the scope of chemical modification. The two secondary C-2 and C-3 hydroxyl groups and the C-6 carboxylate group are the attractive sites in alginate for the desired chemical modifications to develop derivatives with unique characteristics.

In this study, hydrophobic drug curcumin was covalently conjugated to the C-6 carboxylate functionality of hydrophilic sodium alginate via an ester linkage to produce Alg-Ccm conjugate. The covalent conjugation was confirmed by FTIR, 1H NMR, fluorescence spectroscopy etc.

The conjugate was developed with the aim to enhance the solubility and stability of curcumin in aqueous medium. It was hypothesized that Alg-Ccm could stabilize curcumin in aqueous solution by self-aggregation to generate micelles with hydrophobic curcumin in the inner core and hydrophilic alginate in the outer shell. The Alg-Ccm conjugate micelles were thoroughly characterized by dynamic light scattering, TEM and the critical micelle concentration was also determined. The in vitro cytotoxicity of Alg-Ccm was also assessed using L-929 mouse fibroblast cells and was quantified by MTT assay.

Section snippets

Materials

Curcumin (95% total curcuminoid content) from turmeric rhizome was obtained from Alfa Aesar (Bangalore, India). Sodium alginate (average molecular weight 4 × 105) was purchased from Sd fine chemicals (Mumbai, India). 3-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP) and 1-Pyrenecarboxaldehyde (1-PyCHO) were purchased from Sigma-Aldrich (Bangalore, India). Dimethyl sulfoxide (DMSO) and ethanol were obtained from Merck (Mumbai, India). Ultra pure water (18.2 mΩ resistivity) was

Synthesis and structural characterization of Alg-Ccm conjugate

As a natural copolymer, alginate is highly useful for biomedical applications due to its striking characteristics like biodegradability, non-toxicity, non-immunogenicity, chelating ability etc. Hydrophobically modified alginate derivatives have also been studied extensively. In order to obtain a potential curcumin conjugate for therapeutic applications with improved aqueous solubility and stability of curcumin, the Alg-Ccm conjugate was synthesized. In order to synthesize Alg-Ccm, extremely

Conclusions

In the present study, we have developed a polymer-drug conjugate by direct covalent conjugation of hydrophobic drug curcumin with the C-6 carboxylate group of hydrophilic, biodegradable and biocompatible natural polymer sodium alginate via esterification reaction. Alg-Ccm conjugate was found to be extensively water soluble and thereby the aqueous solubility of curcumin (in conjugated form) was enhanced by several folds than that of free curcumin. The conjugate self-aggregated in aqueous

Acknowledgements

The Council for Scientific and Industrial Research (CSIR), India, is gratefully acknowledged for financial assistance. The authors also thank the Director and the Head of Biomedical Technology Wing, SCTIMST for providing necessary facilities.

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