Elsevier

Carbohydrate Research

Volume 367, 15 February 2013, Pages 41-47
Carbohydrate Research

Synthesis, characterization, and in vitro evaluation of palmitoylated arabinogalactan with potential for liver targeting

https://doi.org/10.1016/j.carres.2012.11.025Get rights and content

Abstract

Arabinogalactan (AG), a water soluble polysaccharide with more than 80 mol % galactose units, was hydrophobized by covalent attachment of palmitoyl chains using a base-catalyzed esterification reaction with the objective of effective amalgamation of arabinogalactan in liposomes for targeting asialoglycoprotein receptors (ASGPR) on liver parenchymal cells. Palmitoylated AG (PAG) was characterized by physico-chemical parameters, IR, 1H NMR, and 13C NMR and molecular weight determination by gel permeation chromatography. PAG was incorporated in liposomes and the liposomes were characterized by dynamic light scattering, optical microscopy, zeta potential, and transmission electron microscopic (TEM) techniques. The liposomal system was evaluated for acute toxicity in swiss albino mice and was found to be safe. Targeting ability of PAG was confirmed by in vitro binding affinity to Ricinus communis agglutinin (RCA120), a lectin specific for galactose. The liposomal system with PAG was evaluated for cytotoxicity on HepG2, MCF7, and A549 cancer cell lines. Cytotoxicity study revealed enhanced activity on ASGPR-expressive HepG2 cells as compared to MCF7.

Highlights

Arabinogalactan, asialoglycoprotein receptor specific polysaccharide, was hydrophobically modified using palmitoyl chloride. ► The safety profile of the targeting ligand, hydrophobic derivative of arabinogalactan, was established in vivo. ► The receptor binding ability of the targeting ligand (palmitoylated arabinogalactan) was established by agglutination study. ► A liposomal delivery system modified with the targeting ligand was successfully fabricated and characterized.

Introduction

Delivering drugs or bio-active molecules in various macromolecular carriers like liposomes, nanoparticles etc. have gained substantial importance due to the targeting potential of these carriers. Liposomes are the widely used macromolecular carriers for delivery of lipophilic drugs1 as well as hydrophilic drugs.2 Upon intravenous administration, conventional liposomes are cleared by the reticuloendothelial system limiting its use to passive targeting based on its size3 and lipid composition.4 Various ligands that have been incorporated in liposomes and investigated include anionic and cationic agents for charge interaction,5 monoclonal antibodies to specific antigens,6 and various carbohydrates like pullulan,7 dextran,8 amylopectin,9 and scleroglucan10 for actively targeting carbohydrate receptors on various cell types. Carbohydrates, owing to their natural origin, have better ability to bind to receptors on various cell types and can also provide a stealth protection11 to the macromolecular carrier like liposome.

Hepatic targeting is a challenging area to the formulation scientist. Liver is the major site of accumulation for macromolecular/particulate carriers;12 yet these carriers lack the desired therapeutic effect in diseases/disorders of the liver like hepatocellular carcinoma, liver cirrhosis, hepatitis, hepatic tuberculosis etc. The macromolecular/particulate carriers accumulate in the non-parenchymal cells (Kupffer cells) of the liver13 whereas the disorders/diseases of the liver are confined to the parenchymal cell. Parenchymal cells have carbohydrate sensitive receptors called ASGPR that recognize N-acetylgalactosamine and β-d-galactose units14, 15 as the major signaling unit.16 Specificity and selectivity of monosaccharides and disaccharides containing galactose as a ligand targeting ASGPR are reported.17, 18 Larch arabinogalactan obtained from Larix occidentalis (AG) is a polysaccharide with high content ∼80 mol % of galactose unit.19 AG has been reported for its anti-metastatic activity by stimulation of production of natural killer cells.20 Amalgamation of AG with high galactose content on the surface of liposomes could lead to a better receptor ligand interaction and in conjunction with an anti-cancer agent, can help in better management of hepatocellular carcinoma wherein an over-expression of ASGPR is reported.21

This work was aimed at the synthesis and characterization of a palmitoyl derivative of AG with potential to target the ASGPR on the parenchymal cells of the liver. PAG was characterized by advanced analytical techniques and incorporated into liposomes which were characterized by dynamic light scattering, optical microscopy and TEM. Cytotoxicity of PAG liposomes was studied on HepG2, MCF7 and A549 cancer cell lines. Receptor binding affinity of PAG liposomes was assessed by its binding with Ricinus communis agglutinin (RCA120).

Section snippets

Synthesis of PAG

AG is a US FDA approved dietary fiber. AG with its high galactose content has liver specificity,22, 23, 24 but its potential as a liver directing ligand for macromolecular carriers like liposomes has not been reported. In this study AG was hydrophobized by reacting palmitoyl chloride with AG in a base catalyzed esterification reaction. Physical properties of AG and PAG are shown in Table 1. The % yield of PAG was found out to be 40% with respect to the total weight of palmitoyl chloride and AG

Chemicals

Larch arabinogalactan, palmitoyl chloride, RCA120 were purchased from Sigma–Aldrich® (Germany). Phospholipon 90 (PL90) and Phospholipon 90H (PL90H) were obtained as gift samples from Lipoid® (Germany). Cholesterol was obtained from Fischer Scientific (India). TLC Silica plates were purchased from Merck (Germany). All the other chemicals and reagents used were of analytical grade unless otherwise specified.

Synthesis of PAG

PAG was synthesized from AG by esterification reaction between the hydroxyl group of the

Acknowledgments

S.M.S. is thankful to Indian Council of Medical Research, New Delhi, India; P.O.P. is thankful to Council of Scientific and Industrial Research, New Delhi, India and A.S.J. is thankful to Amrut Mody Research Foundation, Mumbai, India for financial support. Authors acknowledge the help provided by Sophisticated Analytical Instrument Facility, Indian Institute of Technology Bombay, Mumbai, India for TEM images.

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