Micellar carriers based on block copolymers of poly(ε-caprolactone) and poly(ethylene glycol) for doxorubicin delivery

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Abstract

Diblock copolymers of poly(ε-caprolactone) (PCL) and monomethoxy poly(ethylene glycol) (MPEG) with various compositions were synthesized. The amphiphilic block copolymers self-assembled into nanoscopic micelles and their hydrophobic cores encapsulated doxorubicin (DOX) in aqueous solutions. The micelle diameter increased from 22.9 to 104.9 nm with the increasing PCL block length (2.5–24.7 kDa) in the copolymer composition. Hemolytic studies showed that free DOX caused 11% hemolysis at 200 μg ml−1, while no hemolysis was detected with DOX-loaded micelles at the same drug concentration. An in vitro study at 37 °C demonstrated that DOX-release from micelles at pH 5.0 was much faster than that at pH 7.4. Confocal laser scanning microscopy (CLSM) demonstrated that DOX-loaded micelles accumulated mostly in cytoplasm instead of cell nuclei, in contrast to free DOX. Consistent with the in vitro release and CLSM results, a cytotoxicity study demonstrated that DOX-loaded micelles exhibited time-delayed cytotoxicity in human MCF-7 breast cancer cells.

Introduction

Polymeric micelles from amphiphilic block copolymers [1], [2] are supramolecular core-shell-type assemblies of tens of nanometers in diameter, which can mimic naturally occurring biological transport systems such as lipoproteins and viruses [3]. Recently, polymeric micelles as carriers of hydrophobic drugs have drawn increasing research interests, due to their various advantages in drug delivery applications. First, polymeric micelles are highly stable in aqueous solution because of their intrinsic low critical micelle concentration (cmc), which prevents the drug-entrapped micelles from dissociation upon dilution in the blood stream after intravenous injection. Furthermore, the nano size of polymeric micelles can facilitate their extravasations at tumor sites while avoiding renal clearance and non-specific reticuloendothelial uptake. In these micellar delivery systems, the hydrophobic core of the micelles is a carrier compartment that accommodates anti-tumor drugs, and the shell consists of a brush-like protective corona that stabilizes the nanoparticles in aqueous solution [3], [4], [5]. The micelle cores are usually constructed with biodegradable polymers such as aliphatic polyesters and polypeptides, and water soluble poly(ethylene glycol) is most frequently used to build the micelle corona because it can effectively stabilize the nanoparticles in blood compartments and reduce the uptake at the reticuloendothelial sites (e.g. liver and spleen) [4], [5], [6], [7], [8], [9]. By encapsulating drugs within the micelles, solubility limits for hydrophobic drugs can be exceeded.

Anti-tumor drugs, such as doxorubicin (DOX) and paclitaxel, are widely used in cancer chemotherapy. Besides their low water solubility, major drawbacks of these drugs are the acute toxicity to normal tissue and inherent multi-drug resistance effect. To reduce the acute toxicity of the free drugs and improve their therapeutic efficacy, various liposome [10], [11] and polymeric micelle systems were designed as delivery vehicles. The use of polymeric micelles as carriers of hydrophobic anticancer drugs has advanced greatly by the work of Kataoka group and others [12], [13], [14], [15], [16], [17], [18], [19], [20]. Hydrophobic drugs can be incorporated into the micelle inner core by both chemical conjugation and physical entrapment [4], depending on the chemical structure of drugs. For instances, paclitaxel was encapsulated into micelle cores usually by physical entrapment driven by hydrophobic interactions between the drug and the hydrophobic components of polymers. In contrast, DOX can also be chemically bound to the core of polymeric micelles through amidation of DOX amino groups, yielding high loading content. By this way, Kataoka and coworkers have achieved an efficient DOX delivery system based on doxorubicin-conjugated poly(ethylene glycol)–poly(aspartic acid) block copolymer (PEG–PAsp-(DOX)) [14]. The conjugation with DOX converted the hydrophilic poly(aspartic acid) into hydrophobic blocks that formed the hydrophobic micelle core and physically entrapped free DOX as well. Notable micellar accumulation [21] in solid tumors which eventually led to the complete tumor regression [22] in mice was achieved by the prolonged circulation in blood as well as the enhanced permeability and retention (EPR) effect [23]. However, the authors reported that DOX conjugated to poly(aspartic acid) played no direct role in anti-tumor activity in the murine tumor model, and instead the unconjugated DOX entrapped in micelle cores exerted the anti-tumor effects [14]. Very recently, DOX conjugation to the micelle cores through acid-cleavable linkage, such as a hydrazone bond, was reported to be an effective way to enhance the bioavailability of the chemically bound DOX [24], [25]. The hydrazone linkage was cleaved in the endosomes/lysosomes (pH around 5) to yield free DOX molecules, which then functioned as the physically entrapped DOX. Compared to the chemical conjugation strategy, physical entrapment of drugs in the micelle cores may be advantageous in terms of easy polymer preparation, simple micelle fabrication, and enhanced drug bioavailability. Although several micellar systems based on non-ionic amphiphilic block polymers such as PEO–PPO–PEO [15] and PEG-b-PBLA [26] have been reported, physically entrapped DOX delivery with polymeric micelles based on the well-known block copolymers of poly(ethylene glycol) and biodegradable polyesters is still very limited.

In the present work, diblock copolymers of MPEG and PCL were synthesized with various compositions for DOX delivery. Micelles formed with this class of copolymers were efficient carriers of paclitaxel, as reported by Kissel and coworkers [27]. Here, we investigated the relationships between the copolymer composition and the DOX-loading content (DLC) as well as the physicochemical properties of these DOX-loaded micelles, including the micelle size and DOX-release profiles. Furthermore, we evaluated the potential of these micelles as efficient DOX carriers by examining their blood compatibility, cellular internalization efficiency, and cytotoxicity against the cultured MCF-7 tumor cells.

Section snippets

Materials

ε-Caprolactone was purchased from Aldrich (Saint Louis, MO) and purified by vacuum distillation over calcium hydride (CaH2). Toluene (from Aldrich) was dried by refluxing over sodium and distilled under dry argon. Monomethoxy poly(ethylene glycol) (MPEG, from Aldrich) was first purified by precipitation from tetrahydrofuran (THF, from Aldrich) into hexane (from Aldrich), and then the vacuum-dried precipitates were further dehydrated by azeotropic distillation with dry toluene. Stannous(II)

Characterization of diblock copolymers

In the 1H NMR spectra of block copolymers dissolved in CDCl3, the characteristic chemical shifts corresponding to both PCL (1.38, 1.65, 2.31, and 4.06 ppm) and MPEG (3.39 and 3.64 ppm) were observed. The lengths of PCL blocks were calculated from the integral values of characteristic peaks of PEG (e.g. CH3O at ∼3.39 ppm) and PCL (e.g. C(O)CH2 at ∼2.31 ppm), using the known molecular weights of MPEGs. For all block copolymers, a unimodal distribution was observed in the GPC chromatograms

Conclusions

Biodegradable diblock copolymers of MPEG-b-PCL of different molecular weights and compositions were synthesized for the delivery of an anticancer drug, DOX. These amphiphilic polymers self-assembled into core-shell-structural micelles that are less than 100 nm in diameter and have hydrophobic PCL cores capable of encapsulating DOX. The PCL length rather than the DOX-loading had significant influence on the micelle size. However, the effect of PCL length on DLC was significantly less than

Acknowledgements

This research is supported by the National Institutes of Health (R01-CA-90696 to J.G.). X.T.S thanks a postdoctoral support from the Ohio Biomedical Research and Technology Trust fund.

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