Glycyrrhetinic acid-modified chitosan/poly(ethylene glycol) nanoparticles for liver-targeted delivery

Abstract

A liver-targeted drug delivery carrier, composed of chitosan/poly(ethylene glycol)–glycyrrhetinic acid (CTS/PEG–GA) nanoparticles, was prepared by an ionic gelation process, in which glycyrrhetinic acid (GA) acted as the targeting ligand. The formation and characterization of these nanoparticles were confirmed by FT-IR, dynamic light scattering (DLS) and zeta potential measurements. The biodistribution of the nanoparticles was assessed by single-photon emission computed tomography (SPECT), and the cellular uptake was evaluated using human hepatic carcinoma cells (QGY-7703 cells). The anti-neoplastic effect of the doxorubicin·HCl-loaded nanoparticles (DOX-loaded nanoparticles) was also investigated in vitro and in vivo. The results showed that the CTS/PEG–GA nanoparticles were remarkably targeted to the liver, and keep at a high level during the experiment. The accumulation in the liver was 51.3% at 3 h after injection; this was nearly 2.6 times that obtained with the CTS/PEG nanoparticles. The DOX-loaded nanoparticles were greatly cytotoxic to QGY-7703 cells, and the IC₅₀ (50% inhibitory concentration) for the free doxorubicin·HCl (DOX·HCl) and the DOX-loaded CTS/PEG–GA nanoparticles were 47 and 79 ng/mL, respectively. Moreover, the DOX-loaded CTS/PEG–GA nanoparticles could effectively inhibit tumor growth in H22 cell-bearing mice.

Introduction

Liver cancer is one of the most prevalent cancers, with a high mortality. Up to now, chemotherapy has been the main treatment for liver cancer, except for surgical resection. However, most anticancer drugs have high toxicity, and the specificity of these drugs is poor, leading to systemic toxicity and adverse effects that are painful for the patient. Therefore, it is very important to seek new therapeutic strategies to treat liver cancer. Targeted drug delivery, that can ferry the drugs to specific organs or tissues, is a highly desirable strategy to improve the therapeutic outcome, with significantly decreased toxic side-effects compared to traditional chemotherapy [1], [2], [3].

In the past, most studies relied on conjugating carriers or drugs with targeting ligands [4], such as antibodies [5], [6] and sugars [7], [8], for targeted drug delivery to the liver or the treatment of hepatocellular carcinoma. Although successes such as Licartin [9], [10] have been achieved for antibody-mediated treatment of liver cancer, most antibodies are derived from mice, safety can become a major concern and the antibodies are expensive, thus limiting their applications. It had also been reported that asialoglycoprotein (ASGP) receptors are overexpressed on the surface of hepatoma cells [11], [12], and targeting could be accomplished through introduction of galactose residues, that can specifically bind to the ASGP receptors on hepatoma cells, into drug carriers. Kim et al. prepared ^99mTc hydrazinonicotinamide-galactosylated chitosan and showed that 16% of the injected dose could accumulate in the liver within 120 min of injection [13]. Wang et al. also prepared galactosylated liposomes (GalL) and showed a relatively high liver-targeting efficiency (64.6%) [14]. The density and the activity of the ASGP receptors, however, are lower in patients suffering from liver disease, because of the presence in serum of inhibitors that reduce the binding capacity of primary hepatocellular carcinomas by 95%. Thus, ASGP receptor-mediated targeting to the liver may not be so effective under pathological conditions [15]. Therefore, the discovery of new ligands for liver-targeting, instead of the use of the conventional ligands, is very important.

Glycyrrhizin (GL) and glycyrrhetinic acid (GA) (Fig. 1) are the main bioactive compounds of licorice, and are widely used in medicine for the treatment of many pathologies [16], [17], such as anti-inflammatory, anti-gastric, anti-hepatitis, anti-allergic and anti-hepatotoxic effects. In the 1990s, Negishi et al. [18] showed that there are specific binding sites for GL and GA on the cellular membrane of rat hepatocytes, and that the number of binding sites for GA is much more than that for GL. Recently, Lin et al. [19] prepared chitosan nanoparticles modified with glycyrrhizin (CTS-NPs-GL), and confirmed that CTS-NPs-GL preferentially accumulated in rat hepatocytes by a ligand–receptor interaction. Mao et al. [20] also found that the cellular uptake of liposomes modified with glycyrrhetinic acid by rat hepatocytes was 3.3-fold higher than that of unmodified ones. Although the liver cell-targeting ability of the GA-modified polymers has been confirmed in vitro, there are no reports on the distribution of GA-modified materials in vivo, and on the presence of GA receptors on human hepatic cells. Therefore, it is necessary to investigate the distribution of GA-modified carriers in vivo and their affinity for human liver cells. These studies would be of importance for the development of GA-mediated liver-targeted drug delivery.

Polymeric nanoparticles have been used widely as drug delivery vehicles. Chitosan (CTS) is a very important naturally occurring polysaccharide derived from the deacetylation of chitin, and has been used extensively in pharmaceutics due to its excellent biocompatibility and biodegradability. Poly(ethylene glycol) (PEG) is also a non-toxic and biodegradable polymer. PEG-coated nanoparticles have been found to be of great potential in therapeutic applications for controlled release of drugs [21], [22].

In this study, glycyrrhetinic acid-modified nanoparticles (CTS/PEG–GA) were prepared via an ionic gelation process. The aim of this work was to investigate the feasibility of using the CTS/PEG–GA nanoparticles as a drug carrier targeted to the liver. Thus, the biodistribution of these nanoparticles in vivo was investigated by single-photon emission computed tomography (SPECT), and the cellular uptake in vitro was also carried out using human hepatic carcinoma cells (QGY-7703 cells). In addition, the anti-neoplastic effect of doxorubicin·HCl-loaded nanoparticles(DOX-loaded nanoparticles) was studied in vitro and in vivo.