Stability and bioactivity of nanocomplex of TNF-related apoptosis-inducing ligand

Abstract

The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has gained much attention due to its potent therapeutic effect for cancer and rheumatoid arthritis. In this study, we attempted to develop the injectable formulations which can stabilize TRAIL and thus show prolonged blood circulation in vivo. The positively charged TRAIL was mixed with hyaluronic acid (HA), resulting in the formation of nanocomplexes. The zeta-potentials of nanocomplexes and their mean diameters were significantly dependent on the feed ratio of HA to TRAIL. The increase in the feed ratio of HA reduced the particle size and decreased the value of the zeta-potential. The bioactivity of TRAIL in the complexes was comparable to that of native TRAIL, indicating that the complex formation did not affect the activity of TRAIL. Furthermore, the stability of TRAIL in the complexes was retained for 6 days, during which the bioactivity of native TRAIL disappeared. When native TRAIL was subcutaneously injected into the rats, its plasma concentration was not detectable after 12 h. In contrast, for HA/TRAIL nanocomplexes in 1% HA solution, substantial amount of TRAIL was circulated in blood for up to 5 days. These results imply that HA-based formulations of TRAIL hold the potential as the therapeutics.

Introduction

The tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), expressed as a type II transmembrane protein, has received much attention because of its potential as a therapeutic agent (Fesik, 2005, Bremer et al., 2006, Cretney et al., 2007). TRAIL has shown particular promise as an anti-cancer agent, capable of selectively inducing apoptosis through death receptor-mediated signaling pathways in cancer cells without significant cytotoxicity to normal cells (Walczak et al., 1999, Lawrence et al., 2001, Ray and Almasan, 2003). This selective and potent activity of TRAIL is due to its binding affinity to the death receptors that are over-expressed on the cancer cells and induce apoptosis. Although TRAIL is a membrane-bound ligand, its truncated version that contains the extracellular domain is water-soluble and exhibits potent anti-cancer activity both in vitro and in vivo (Ashkenazi et al., 1999). Therefore, recombinant TRAIL (rTRAIL) and its agonistic antibodies have been developed to target the death receptors, and a few of them are currently being evaluated as the anti-cancer drug in Phases I and II clinical trials (Fesik, 2005, Schaefer et al., 2007). The half-life of TRAIL, however, is reported to be less than 30 min, which may require frequent administration for preserving the therapeutic level in blood (Kelley et al., 2001, Xiang et al., 2004).

The clinical use of protein drugs often suffered from their short half-lives and susceptibility to proteolytic enzymes in biological fluids. In an attempt to surmount these problems, much effort has been made to develop effective delivery systems that can improve the stability of protein drugs and prolong their therapeutic effects. In particular, biodegradable microspheres using poly(lactic-co-glycolic acid) (PLGA) have been most widely used for the sustained protein delivery systems (Bartus et al., 1998, Wei et al., 2004, Tamber et al., 2005). However, these formulations have encountered some limitations on practical applications: (1) protein can be readily denatured by an acidic local environment, associated with degradation of PLGA microspheres; (2) current methods of preparing microspheres often involve exposure of protein drugs to harsh environments such as organic/aqueous interface, high temperature, and physical stresses; and (3) acidic exudates by degradation of PLGA may cause inflammation (Johnson et al., 1996, Putney and Burke, 1998, Fu et al., 2000). In recent years, a lot of approaches have provided the promising candidates for the protein delivery system. For example, the incorporation of basic exipients (e.g., Mg(OH)₂ and MgCO₃) into the PLGA microsphere could prevent a pH drop during degradation of microspheres, thus improving protein stability (Zhu et al., 2000). The solvent exchange method, implemented by the ultrasonic atomizer system, allowed producing reservoir-type microcapsules without protein aggregation and loss of its biological activity (Yeo and Park, 2004, Park et al., 2006). The complex formation of the protein drugs with the oppositely charged polymer has shown to improve protein stability in the microsphere (Lee et al., 2007b).

In this study, nano-sized complexes were prepared by mixing TRAIL with the negatively charged polysaccharide. Hyaluronic acid (HA), composed of d-glucuronic acid and N-acetyl-d-glucosamine, was chosen for ionic complexation of TRAIL because HA have unique and excellent physicochemical properties for drug delivery such as biocompatibility, biodegradability, and nonimmunogenicity (Fraser et al., 1997, Hahn et al., 2004, Liao et al., 2005). The effect of the complex formation between HA and TRAIL on protein stability and biological activities were determined. Furthermore, the injectable HA-based TRAIL formulation was subcutaneously injected into the rats to observe the pharmacokinetics of TRAIL.