Mixed micelles made of poly(ethylene glycol)–phosphatidylethanolamine conjugate and d-α-tocopheryl polyethylene glycol 1000 succinate as pharmaceutical nanocarriers for camptothecin

Abstract

Micelles from the mixture of poly(ethylene glycol)–phosphatidyl ethanolamine conjugate (PEG–PE) and d-α-tocopheryl polyetheyene glycol 1000 succinate (TPGS) were prepared loaded with the poorly soluble anticancer drug camptothecin (CPT). The solubilization of CPT by the mixed micelles was more efficient than with earlier described micelles made of PEG–PE alone. CPT-loaded mixed micelles were stable upon storage and dilution and firmly retained the incorporated drug. The cytotoxicity of the CPT-loaded mixed micelles against various cancer cells in vitro was remarkably higher than that of the free drug. PEG–PE/TPGS mixed micelles may serve as pharmaceutical nanocarriers with improved solubilization capacity for poorly soluble drugs.

Introduction

Currently, polymeric micelles are popular pharmaceutical nanocarriers for the delivery of poorly water-soluble drugs, which can be solubilized within the hydrophobic inner core of a micelle (Bader et al., 1984, Jones and Leroux, 1999). As a result, micelles can substantially improve solubility and bioavailability of various hydrophobic drugs (Lukyanov and Torchilin, 2004). The small size (10–100 nm) of micelles allows for the micelle efficient accumulation in pathological tissues with the permeabilized (“leaky”) vasculature, such as tumors and infarcts, via the enhanced permeability and retention (EPR) effect (Wu et al., 1993, Maeda et al., 2000, Torchilin, 2001); in addition, this small size offers the advantage of simple sterilization of micelles by filtration through polycarbonate membranes with the cut off size of 0.2 μm.

Among other polymeric micelles, micelles prepared from conjugates of polyethylene glycol (PEG) and diacyllipids, such as phosphatidylethanolamine (PE)—PEG–PE conjugates—have demonstrated high stability and low toxicity (Torchilin, 2002, Lukyanov et al., 2002). The very low CMC value of PEG–PE compounds (in a 10⁻⁵ M range) indicates that PEG–PE micelles can preserve their integrity even upon the dilution in the blood pool during the supposed therapeutic application (Lukyanov and Torchilin, 2004). In our previous studies, we have successfully encapsulated a number of poorly soluble anticancer drugs into PEG–PE micelles with high efficiency and stability (Gao et al., 2002, Mu et al., 2005).

Camptothecin (CPT), a plant alkaloid from Camptotheca acuminate (Wall et al., 1966) demonstrated strong antitumor activity against lung, ovarian, breast, pancreas, and stomach cancers (Giovanella and Cheng, 1991, Wall and Wani, 1995) by targeting intracellular topoisomerase I, a nuclear enzyme that reduces the torsional stress of supercoiled DNA (Hsiang and Liu, 1988, Garcia-Carbonero and Supko, 2002). However, CPT is insoluble in water (Hatefi1 and Amsden, 2002) and exists in two forms depending on the pH value: the active lactone forms at pH below 5 and the inactive open ring-carboxylated CPT-Na⁺ form, which present at neutral pH values (Wall, 1969, Fassberg and Stella, 1992). Although the lactone form of CPT is crucial for its anticancer activity, at physiological pH values most CPT molecules exist in the inactive carboxylate form. Thus, sparing solubility and labile lactone ring hinder the clinical application of CPT.

Various formulation strategies have been designed to increase the solubility of CPT, stabilize the lactone ring, and reduce the systemic toxicity of this drug, which involve solid lipid nanoparticles (Yang et al., 1999), microparticles (Shenderova et al., 1997), and liposomes (Tardi et al., 2000). Polymeric micelles also can effectively solubilize and stabilize CPT (Cortesi et al., 1997, Barreiro-Iglesiasa et al., 2004, Opanasopit et al., 2004), and enhance the antitumor efficiency of the encapsulated CPT due to the spontaneous accumulation in tumor tissues via the EPR effect (Maeda et al., 2000, Jain, 1997). CPT incorporation into PEG–PE micelles recently described by us (Mu et al., 2005), allowed for the preparations with significantly higher CPT concentrations than described earlier (Cortesi et al., 1997, Barreiro-Iglesiasa et al., 2004, Opanasopit et al., 2004).

One may expect that further increase in the efficiency of micellar solubilization of CPT might be achieved by using mixed micelles as shown for some other poorly soluble drugs (Krishnadas et al., 2003). Interestingly, d-α-tocopheryl polyetheyene glycol 1000 succinate (TPGS), a derivative of the natural Vitamin E (α-tocopherol) and polyetheyene glycol 1000 is used as solubilizer, absorption enhancer and a vehicle for lipid-based drug delivery formulations (Eastman, 1998, Mu and Feng, 2003). It was also reported that succinate esters of Vitamin E, such as RRR-α-tocopheryl succinate, behave as potent, pro-apoptotic agent selective for cancer cells (Yu et al., 2001, Neuzil et al., 2001). TPGS itself can hardly be used as micelle-forming material to carry CPT because of the relatively low CMC value (approximately 1.3 × 10⁻⁴ M for industrial grade TPGS (Eastman, 1998), which may result in micelle dissociation in the blood, however its lipophilic portion is relatively bulky, which might allow for better drug solubilization if TPGS is used as a component of the mixed micellar drug delivery system. One can hypothesize that mixed micelles made of TPGS and PEG–PE if used as CPT carriers may allow for the better drug encapsulation and high anticancer efficiency.

Here, we describe the preparation of CPT-loaded mixed PEG–PE/TPGS micelles, some of their properties and increased in vitro cytotoxicity against several tumor cell lines.