Doxorubicin-loaded poly(ethylene glycol)–poly(β-benzyl-l-aspartate) copolymer micelles: their pharmaceutical characteristics and biological significance

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Abstract

Doxorubicin (DOX) was physically loaded into micelles prepared from poly(ethylene glycol)–poly(β-benzyl-l-aspartate) block copolymer (PEG–PBLA) by an o/w emulsion method with a substantial drug loading level (15 to 20 w/w%). DOX-loaded micelles were narrowly distributed in size with diameters of approximately 50–70 nm. Dimer derivatives of DOX as well as DOX itself were revealed to be entrapped in the micelle, the former seems to improve micelle stability due to its low water solubility and possible interaction with benzyl residues of PBLA segments through π–π stacking. Release of DOX compounds from the micelles proceeded in two stages: an initial rapid release was followed by a stage of slow and long-lasting release of DOX. Acceleration of DOX release can be obtained by lowering the surrounding pH from 7.4 to 5.0, suggesting a pH-sensitive release of DOX from the micelles. A remarkable improvement in blood circulation of DOX was achieved by use of PEG–PBLA micelle as a carrier presumably due to the reduced reticuloendothelial system uptake of the micelles through a steric stabilization mechanism. Finally, DOX loaded in the micelle showed a considerably higher antitumor activity compared to free DOX against mouse C26 tumor by i.v. injection, indicating a promising feature for PEG–PBLA micelle as a long-circulating carrier system useful in modulated drug delivery.

Introduction

Recently, particulate carrier systems have received much attention in the field of drug targeting because of their high drug loading capacity as well as the unique disposition characteristics in the body [1], [2], [3]. Distribution in the body of drug-loaded particles is determined mainly by the surface properties and size of carrier particles, and is less affected by the properties of the loaded drugs if they are embedded within matrix of particles. In this regard, the size and surface properties of carrier particles are of crucial importance in achieving modulated drug delivery with remarkable efficacy. A major obstacle in drug targeting by particulate systems is non-specific uptake by the reticuloendothelial systems (RES). Particles unrecognizable by the RES are needed to achieve longevity in blood circulation. Particulate size may also affect RES uptake, and is a determining factor for tissue penetration. Particle size distribution is crucial in the particulate disposition, because size-sieving may occur during the disposition process in the body. Thus, a narrow size distribution is desired for carrier particles in order to accomplish selective accumulation at the target site.

One such particulate system satisfying the above mentioned properties is a multimolecular assembly of block copolymers, so-called polymer micelles, with a core–shell architecture [4], [5]. A variety of flexible hydrophilic polymers can be selected as shell-forming segments, which assemble into the dense palisades of tethered chains to achieve effective steric stabilization propensities. Core segregation in an aqueous milieu is the direct driving force for micellization, and proceeds through a combination of intermolecular forces such as hydrophobic interaction [6], [7], [8], [9], [10], electrostatic interaction [11], [12], [13], metal complexation [14], and hydrogen bonding [15] of the constituent block copolymers. The segregated core embedded in the hydrophilic palisade serves as a reservoir of a variety of drugs. Compared to low-molecular-weight surfactant micelles, polymer micelles are generally more stable with a remarkable lower critical micelle concentration (CMC), and show slower dissociation, allowing retention of loaded drugs for a longer period of time, and eventually, achieving a higher accumulation of drug at the target site [4]. Further, polymer micelles are readily engineered to have sizes in the range of several tens of nanometers with a narrow size distribution, which is of great advantage in regulating biodistribution. Consequently, as described in the review articles cited in Refs. [1], [4], [5], [16], progressive interest is raised for carrier systems composed of polymer micelles.

A remarkable success in targeting of cytotoxic agents to solid tumors by polymer micelle has been obtained by our group with the system based on doxorubicin-conjugated poly(ethylene glycol)–poly(α,β-aspartic acid) block copolymer (PEG–PAsp(DOX)) [17]. PEG–PAsp(DOX) micelle with both chemically-bound and physically-entrapped DOX within the micelle core achieved prolonged circulation in a blood compartment due to the reduced uptake by the RES. Notable accumulation into solid tumors took place through the enhanced permeation retention effect (EPR effect) [18], eventually, the complete regression of solid tumor in mice was observed [19], [20]. Detailed study revealed the crucial role of physically-entrapped DOX in the cytotoxic action of the micellar drug [21], [22]. It is worth mentioning that the micelle structure was even more stabilized by increasing the amount of physically-entrapped DOX within the micellar core, reducing systemic leakage of DOX and achieving enhanced DOX accumulation into solid tumor with less toxic side effects caused by non-specific organ distribution [22], [23].

From a standpoint of drug carrier design with a wide applicability to a variety of hydrophobic drugs, it is attractive to prepare a simple block copolymer having the property to form stable polymer micelles with high efficiency in physically entrapping hydrophobic drugs in the core. Being stimulated by the early success of PEG–PAsp(DOX) micelle, we then have extended our research to develop a simpler system composed of an amphiphilic block copolymer in order to entrap DOX only in a physical fashion [9]. As reported so far, physical entrapment of DOX into the micelle of poly(ethylene glycol)–poly(β-benzyl-l-aspartate) block copolymer (PEG–PBLA) was achieved either by dialysis or by o/w emulsion method with a substantial loading level (5–18 w/w%) [24], [25]. The PBLA segment in the block copolymer is rigid and hydrophobic enough to form the solid non-polar core to entrap DOX molecules. The benzyl moiety located in the side chain may contribute in stabilizing the core through π–π interaction with entrapped DOX. Indeed, DOX in PEG–PBLA micelles was shown to be less susceptible to chemical degradation than free DOX in aqueous solution [25]. Further, as is the case with the PEG–PAsp(DOX) system (micelle with both chemically-bound and physically-entrapped DOX), PEG–PBLA micelles seem to become more stable by the incorporation of DOX, even in the presence of serum proteins [26]. Sufficient cytotoxicity against cultured P388D1 cells was also confirmed for DOX-loaded PEG–PBLA micelles [26].

Here, we report on the detailed physicochemical characteristics of DOX-loaded PEG–PBLA micelles, including structural data of loaded DOX as well as on their release properties. Further, in vivo antitumor activity and longevity in blood circulation of DOX-loaded PEG–PBLA micelles are also demonstrated in this study.

Section snippets

Materials

DOX in the form of the hydrochloride salt was purchased from Mercian (Japan) and its purity was checked by reversed-phase high-performance liquid chromatography (HPLC). Other chemicals were of reagent grade and were used as purchased.

PEG–PBLA was synthesized and characterized as described previously [7], [17]. Briefly, ring opening polymerization of β-benzyl-l-aspartate N-carboxyanhydride was initiated from the terminal primary amino group of α-methoxy-ω-aminopoly(ethylene glycol) (Mw=12 000,

Loading of DOX into the micelle with different compositions

As previously reported, DOX loading of PEG–PBLA micelles was done with considerable efficacy by an oil-in-water (o/w) emulsion method using chloroform as the organic phase [25], [26]. A similar procedure was used in this study as well. A key factor in achieving a high loading efficacy is to increase the partition of DOX from the aqueous phase to the chloroform phase, which is closely related to the protonation behavior of the 3′-NH2 group in the DOX–sugar moiety (Fig. 1). Although the pKa of

Acknowledgements

The authors would like to thank Mr. Atsushi Harada, The University of Tokyo, for his help in manuscript preparation. This work was financially supported by STA grant Biointegrated Materials for the Improvement of the Quality of Life, Science and Technology Agency, Japan.

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