Polymeric micelles of poly(2-ethyl-2-oxazoline)-block-poly(ε-caprolactone) copolymer as a carrier for paclitaxel

Abstract

Polymeric micelles based on amphiphilic block copolymers of poly(2-ethyl-2-oxazoline) (PEtOz) and poly(ε-caprolactone) (PCL) were prepared in an aqueous phase. The loading of paclitaxel into PEtOz–PCL micelles was confirmed by ¹H-NMR spectra. Paclitaxel was efficiently loaded into PEtOz–PCL micelles using dialysis method, and the loading content of paclitaxel in micelles was in the range 0.5–7.6 wt.% depending on the block composition of block copolymers, organic solvent used in the dialysis, and feed weight ratio of paclitaxel to block copolymer. The higher the content of hydrophobic block in the block copolymers, the higher the loading efficiency of micelles for paclitaxel. When acetonitrile was used as solvent, a higher drug loading efficiency was obtained than with THF. The loading efficiency decreased with increasing feed weight ratio of paclitaxel to block copolymer from 0.1:1 to 0.2:1. The hydrodynamic diameters of paclitaxel-loaded micelles were in the range 18.3–23.4 nm with narrow size distribution. The hemolysis test of PEtOz–PCL performed in vitro indicated that the toxicity of PEtOz–PCLs to lipid membrane was not significant compared with Tween 80, and was comparable to that observed with Cremophore EL. The proliferation inhibition activity of paclitaxel-loaded micelles for KB human epidermoid carcinoma cells was also evaluated in vitro. Paclitaxel-entrapped polymeric micelles exhibited comparable activity to that observed with Cremophore EL-based paclitaxel formulations in inhibiting the growth of KB cells.

Introduction

Paclitaxel (Fig. 1a), an anticancer agent extracted from the bark of the Pacific yew (Taxus brevifolia), has shown significant activity against a wide range of solid tumors, especially drug-resistant ovarian cancer, metastatic breast cancer, and non-small cell lung cancer [1], [2], [3], [4]. Its unique mechanism of action involves the ability to promote polymerization of tubulin dimers to form microtubules, and then stabilize the microtubules by preventing depolymerization, thereby inhibiting cell replication in the late G₂ or M phase of cell cycle [5], [6], [7]. Though paclitaxel has shown high potential as an anticancer drug, its practical use is possible by overcoming its poor aqueous solubility of approximately 1 μg/ml [8]. Therefore, a 50:50 mixture of Cremophore EL (polyoxylated castor oil) and ethanol was used in the current clinical formulation [9]. However, a number of studies have reported that Cremophore EL induced serious side effects such as hypersensitivity, neurotoxicity, nephrotoxicity, and the extraction of plasticizers from intravenous infusion line [10], [11], [12]. Thus, there has been much effort in the development of Cremophore EL-free alternative carrier systems including liposomes, cyclodextrins, emulsions, mixed-micelles, microsphreres, and polymeric micelles [13], [14], [15], [16], [17], [18], [19], [20], [21], [22].

Among various carrier systems, polymeric micelles derived from amphiphilic block copolymers have been widely pursued for a wide variety of drugs [23], [24], [25], [26]. The high potential of polymeric micelles as a drug carrier lies in their unique characteristics such as nano-size and thermodynamic stability. In addition, their core–shell structure can mimic naturally occurring transport systems such as plasma lipoproteins and viruses, satisfying the structural aspect to act as a transport system in a body [23]. Especially for anticancer agents, it has been reported that highly selective delivery to specific tumor sites was achieved using polymeric micelles as carrier through a passive targeting mechanism [27]. In recent reports, Zhang et al. reported on the antitumor activity and biodistribution of paclitaxel loaded in polymeric micelles based on PEO-b-poly(dl-lactide) [22]. They demonstrated that both polymeric micellar paclitaxel and Cremophore EL-based paclitaxel exhibited comparable efficacy in inhibiting the growth of Hs578T breast tumor cells, SK MES non-small cell lung tumor cells, and HT-29 colon tumor cells. So far, most micelle-forming block copolymers investigated for drug delivery are based on hydrophilic poly(ethylene oxide) (PEO), and the structural variations have been made mainly with the hydrophobic block such as polyesters, poly(amino acids), and poly(propylene oxide) [22], [24], [25]. In recent years, we have investigated the micellar characteristics of amphiphilic block copolymers based on hydrophilic poly(2-ethyl-2-oxazoline) to systematically diversify micelle-forming amphiphilic block copolymers, thereby expecting useful properties of their micelles as a drug carrier [28], [29], [30]. We described the formation and unique properties of micelles based on diblock copolymers (PEtOz–PCLs) of poly(2-ethyl-2-oxazoline) (PEtOz) and poly(ε-caprolactone) (PCL) (Fig. 1b) [28], [29]. These block copolymers in an aqueous media self-associate to form micelles in which hydrophilic PEtOz and hydrophobic PCL constructed the outer shell and the inner core of micelles, respectively. Our choice of PEtOz as a micellar outer shell was based on its unique ability to form the complex via strong hydrogen bonding with poly(carboxylic acid)s such as poly(acrylic acid) or poly(methacrylic acid) [31]. Recently, we reported on the pH-dependent complex formation of PEtOz–PCL micelles with poly(carboxylic acid)s and the reversible micelle release from the complex [32]. This unique complexation property of PEtOz-based polymeric micelles can find a novel applicability in the delivery of poorly water-soluble drugs such as paclitaxel in that the complex of polymeric micelles and poly(carboxylic acid) can act as a matrix for a sustained release of drug-containing micelles at a physiological pH. Besides, in a previous report, the potential use of PEtOz as a water-soluble drug carrier was suggested by examining the in vivo behavior [33].

In this work, we aimed to develop polymeric micelles derived from PEtOz–PCL block copolymers as a carrier for paclitaxel. The potential of PEtOz–PCL micelles as a carrier for paclitaxel was evaluated by estimating the loading efficiency for paclitaxel and in vitro membrane toxicity of micelles, and the proliferation inhibition activity of paclitaxel-loaded micelles for KB human epidermoid carcinoma cells.