Full Length ArticleMixed micelles based on a pH-sensitive prodrug and TPGS for enhancing drug efficacy against multidrug-resistant cancer cells
Graphical abstract
Introduction
Chemotherapy is a commonly used and beneficial method for treating cancer. The therapeutic efficacy of traditional small molecule chemotherapeutic drugs is often limited by their intrinsic properties, such as poor water solubility, rapid elimination and severe side effects [1]. More importantly, the occurrence of multidrug resistance (MDR), which confers simultaneous resistance to a variety of drugs with different targets and chemical structures, is a major obstacle to clinical success [2]. Numerous MDR mechanisms have been proposed [3]. Of these, the overexpression of P-glycoprotein (P-gp), which catalyses the efflux of a range of structurally-unrelated anticancer drugs, has received great attention. P-gp-mediated active efflux of drugs is considered to be one of the major causes of MDR and chemotherapeutic failure in cancer therapy. Considerable effort has been devoted to inhibition of drug efflux by P-gp to overcome MDR and improve drug bioavailability [4], [5].
D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E that is formed by esterification of vitamin E succinate with polyethylene glycol 1000 [6]. As an excipient, TPGS can overcome MDR by inhibition of P-gp and modulation of efflux pump activity [7], [8]. TPGS itself can form micelles in aqueous solution and can be used as a drug carrier to enhance drug efficacy against multidrug-resistant cells. However, TPGS micelles are readily dissociated in plasma because TPGS has a relatively high critical micelle concentration value (0.2 mg/mL) [9]. Therefore, TPGS has been used as a component in mixed micelles, liposomes or nanoparticles to increase micelle stability, obtain sustained and controlled drug delivery, and achieve high drug encapsulation efficiency [10], [11], [12].
Mixed micelles show improved thermodynamic stability and drug therapeutic index compared to the individual components [13]. The use of mixed micelles enables integration of multiple functionalities into a single system. To date, mixed micelles have received much attention in the area of drug delivery applications [14] and a variety of mixed micelles containing TPGS have been developed. For example, polyethylene glycol-b-distearoylphosphatidylethanolamine (PEG-DSPE)/TPGS mixed micelles were prepared and shown to improve the pharmacokinetic characteristics of gemcitabine and enhance its anticancer efficacy [15]. Monomethoxy-polyethylene glycol-co-poly-d,l-lactide (mPEG-PLA)/TPGS mixed micelles were prepared with high drug loading content and stable drug release properties [16]. Mixed micelles with pH-sensitive sequential degradation properties have been produced to release encapsulated drug within different intracellular domains of MDR cells [17]. Mixed micelle systems composed of methoxy poly(ethylene glycol)-b-polycaprolactone (mPEG-PCL)/TPGS were shown to increase drug uptake efficiency of resveratrol, resulting in high rates of apoptotic cell death against drug-resistant cancer cells (MCF-7/ADR) [18].
Preparation of a prodrug consists of grafting a molecule onto an active drug molecule to form a derivative. The most attractive property of a prodrug is the alteration of drug transport across cell membranes, which is a common mechanism of resistance to chemotherapy [19]. Currently, numerous prodrug-based nanosystems are undergoing clinical trials [20]. Prodrugs that are pH-sensitive have great potential for controlled drug delivery [21], [22], [23].
In the present study, we fabricated a mixed micelle system based on a pH-sensitive doxorubicin (DOX) conjugate prodrug and TPGS to overcome MDR. The pH-sensitive prodrug was synthesized by covalent conjugation of DOX to methoxy poly(ethylene glycol) via carbamate linkage to give high drug content. DOX, one of the most widely used anticancer agents in the clinic, was used as a model drug in this study. It can intercalate into the base pairs of the DNA helix to inhibit DNA synthesis [24]. In addition to its side effects, the clinical application of DOX has been severely hindered by MDR [25]. The pH-sensitivity of the prodrug was engineered to increase the prodrug selectivity toward cancer cells. In acidic environments, the mixed micelles are broken down to release both DOX and TPGS. Since DOX is a P-gp-substrate and TPGS is a P-gp inhibitor, the co-release of drug and P-gp inhibitor may therefore enhance the cytotoxicity of DOX.
Section snippets
Materials
Methoxy-poly(ethylene glycol) (mPEG, MW = 2000 g/mol) was purchased from TCI (Shanghai) Development Co., Ltd., China. p-Nitrophenyl chloroformate (p-NPC) was purchased from Creasyn Finechem (Tianjin) Co., Ltd., China. Doxorubicin hydrochloride (DOX·HCl) was purchased from Beijing HVSF United Chemical Materials Co., Ltd., China. TPGS was supplied by Sigma-Aldrich, USA. N,N-dimethylformamide (DMF), dichloromethane (DCM) and triethylamine (Et3N) were supplied by Tianjin Kemel Chemical Reagent Co.,
Synthesis and characterization of prodrug
The prodrug was synthesized via ammonolysis by reaction of DOX·HCl and mPEG activated with p-NPC, in which Et3N was employed to remove the hydrochloride of DOX·HCl. The synthetic route and the 1H NMR spectrum of mPEG-DOX conjugate are shown in Fig. 1. The spectrum contained an intense peak at 3.51 ppm, characteristic of the methylene protons of mPEG. The hydrogen protons of the benzene group in DOX gave signals at 7.69 and 7.91 ppm. The peak for the methoxy group on the benzene ring was at 3.99
Conclusion
In this study, mixed micelles composed of a pH-sensitive prodrug and a P-gp inhibitor were prepared. Their preparation is simple. The mixed micelles display an average hydrodynamic size of 144 nm, which is suitable for application in drug delivery systems. The prodrug has high drug loading content and pH-sensitive drug release characteristics. Unlike in MCF-7 cells, the pH-sensitive prodrug had apparent cytotoxicity only at high concentrations in MCF-7/ADR cells. The mixed micelles showed
Acknowledgements
This work was supported by the National Natural Science Foundation of China (21577071 and 51173083) and Key Program of Natural Science Foundation of Tianjin (14JCZDJC40300).
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