Polyethylene sebacate–doxorubicin nanoparticles for hepatic targeting

Abstract

The present study discusses polyethylene sebacate (PES)–doxorubicin (DOX) nanoparticles (PES–DOX NP) using pullulan as asialoglycoprotein receptor (ASGPR) ligand for hepatic targeting. Pullulan, a hydrophilic polymer served as ligand and as stealth agent. PES–DOX NP were prepared by modified nanoprecipitation using PES and Gantrez AN 119 (Gantrez), as complexing agent in the organic phase, while DOX was dissolved in the aqueous phase. Pullulan was adsorbed on the formed nanoparticles (PES–DOX–PUL). Intimate association of PES and Gantrez, and ionic complexation of DOX with Gantrez (confirmed by FTIR), coupled with rapidity of nanoprecipitation resulted in nanoparticles with high entrapment efficiency and high drug loading. Nanoparticles were successfully freeze dried. Drug release from PES NP followed zero order kinetics. PES–DOX NP and PES–DOX–PUL exhibited low hemolytic potential and good serum stability. Comparative biodistribution study in rats using ^99mTc labeled formulations revealed higher blood concentration and lower liver concentration of PES–DOX–PUL, confirming the long circulating nature of PES–DOX–PUL, and thereby the possibility of improved targeting to hepatocytes. Nanoparticles revealed lower DOX concentration in the heart suggestive of low cardiotoxicity. Our study presents a radically different yet simple approach for the design of PES–DOX nanoparticles with high drug loading for improved therapy in hepatic cancer.

Introduction

Hepatic cancer (HC) is one of the most widespread malignancies accounting for 4–5% of all human cancers, with nearly 5 million deaths occurring every year (Llovet et al., 2003, Schwartz and Llovet, 2008). Doxorubicin hydrochloride (DOX) is a drug of choice in the treatment of HC. Insufficient concentration at the tumor site coupled with cardiotoxicity, nephrotoxicity, myelosuppression and multiple drug resistance due to P-glycoprotein efflux and topoisomerase II resistance are serious limitations of current DOX therapy (Patil et al., 2008b). Enabled by their size and targetability, nanocarriers have been extensively studied to overcome the challenges in effective delivery of DOX. However conventional nanocarriers are rapidly cleared from circulation following intravenous injection mainly by kupffer cells of the liver and other macrophages (Brigger et al., 2002). Thus for efficient treatment of HC, a drug delivery strategy to enhance preferential uptake by hepatocytes with minimum kupffer cell uptake is essential (Xu et al., 2009). Long circulating targeted DOX nanocarriers could therefore provide important advantage in the treatment of HC.

Asialoglycoprotein receptor (ASGPR), also known as hepatic lectin represents a promising target for hepatocyte-specific delivery. ASGPR is predominantly present in large numbers on the sinusoidal cell membrane of hepatocyte and internalizes sugars such as galactose or lactose and glycoproteins with terminal galactose or N-acetylgalactosamine by endocytosis (Wu et al., 2002). ASGPR is reported to be over expressed on the surface of hepatocytes in patients with HC (Trouet and Jolles, 1984). Hence ASGPR targeting could provide a viable strategy for delivery of DOX in HC.

Several ligands such as asialo-feutin (Arangoa et al., 2003), asialo-transferrin, asialo-ceruloplasmin, asialo-lactoferrin, asialo-orosomucoid (Pathak et al., 2008), galactose and galactosylated or lactosylated residues including galactosylated cholesterol (Kawakami et al., 1998, Kawakami et al., 2000), galactosylated lipid (Wang et al., 2006), glycolipids (Sliedregt et al., 1999) and galactosylated polymers (Zanta et al., 1997) have been explored for selective targeting to hepatocytes. One such water soluble polysaccharide polymer pullulan, comprising of three α-1, 4-linked glucose molecules that are repeatedly polymerized at α-1, 6-linkages on terminals glucose is reported to be internalized by hepatocytes via ASGPR mediated endocytosis (Kaneo et al., 2001). Enhanced targeting to hepatocytes using pullulan–drug conjugates prepared by covalent bonding is reported (Hosseinkhani et al., 2002).

Polyethylene sebacte (PES), a new biodegradable polymer (Fig. 1) recently reported by our group has the advantages of ease of synthesis, good hydrolytic stability and low cost. Toxicity studies including genotoxicity and mutagenicity have confirmed safety of PES for biomedical and pharmaceutical applications (More et al., 2009). The objective of the present study is preparation and evaluation of DOX loaded PES nanoparticles (PES–DOX NP) for hepatic targeting using pullulan as ASGPR ligand. Pullulan, a hydrophilic polymer, when anchored on to nanoparticles could provide the dual advantage of stealth property and targeting to ASGPR by receptor mediated endocytosis. PES–DOX NP were prepared by modified nanoprecipitation using PES and Gantrez AN 119 (copolymer of methyl vinyl ether and maleic anhydride) as complexing agent in the organic phase, while DOX was dissolved in the aqueous phase.