Poly(lactide)–vitamin E derivative/montmorillonite nanoparticle formulations for the oral delivery of Docetaxel

Abstract

Four systems of nanoparticles of biodegradable polymers were developed in this research for oral delivery of anticancer drugs with Docetaxel used as a model drug, which include the poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), the poly(lactide)–vitamin E TPGS nanoparticles (PLA–TPGS NPs), the poly(lactic-co-glycolic acid)–montmorillonite nanoparticles (PLGA/MMT NPs) and the poly(lactide)–vitamin E TPGS/montmorillonite nanoparticles (PLA–TPGS/MMT NPs). Vitamin E TPGS stands for d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), which is a water-soluble derivative of natural vitamin E formed by esterification of vitamin E succinate with polyethylene glycol (PEG) 1000. The design was made to take advantages of TPGS in nanoparticle technology such as high emulsification effects and high drug encapsulation efficiency, and those in drug formulation such as high cellular adhesion and adsorption. MMT of similar effects is also a detoxifier, which may cure some side effects caused by the formulated drug. The drug-loaded NPs were prepared by a modified solvent extraction/evaporation method and then characterized for their MMT content, size and size distribution, surface charge and morphology, physical status and encapsulation efficiency of the drug in the NPs, and in vitro drug release profile. Cellular uptake of the coumarin 6-loaded NPs was investigated. In vitro cancer cell viability experiment showed that judged by IC₅₀, the PLA–TPGS/MMT NP formulation was found 2.89, 3.98, 2.12-fold more effective and the PLA–TPGS NP formulation could be 1.774, 2.58, 1.58-fold more effective than the Taxotere^® after 24, 48, 72 h treatment, respectively. In vivo PK experiment with SD rats showed that oral administration of the PLA–TPGS/MMT NP formulation and the PLA–TPGS NP formulation could achieve 26.4 and 20.6 times longer half-life respectively than i.v. administration of Taxotere^® at the same 10 mg/kg dose. One dose oral administration of the NP formulations could realize almost 3 week sustained chemotherapy in comparison of 22 h of i.v. administration of Taxotere^®. The oral bioavailability can be enhanced from 3.59% for Taxotere^® to 78% for the PLA–TPGS/MMT NP formulation and 91% for the PLA–TPGS NP formulation respectively. Oral chemotherapy by nanoparticles of biodegradable polymers is feasible.

Introduction

Oral chemotherapy can maintain an appropriate drug concentration in the circulation to achieve prolonged exposure of the drug to the cancerous cells. This will increase the efficacy and decrease the side effects. Oral chemotherapy is an important step towards the patients' dream: “Chemotherapy at Home”, which will greatly improve their quality of life and give hope to those of late stage cancer, who have been too weak to accept any treatment at all. Oral chemotherapy provides at least a palliative treatment to give them hope and extend their life [1], [2]. Unfortunately, most anticancer drugs especially those with excellent anticancer effects such as Taxoids (Paclitaxel and Docetaxel) are not orally bioavailable, i.e. not absorbable/interactive in the gastrointestinal (GI) tract. For Paclitaxel, initial studies reported that its oral bioavailability was less than 1% [3], [4], [5], [6]. This is because the drug would be eliminated from the first-pass extraction by the cytochrome P450-dependent metabolic processes and the overexpression of plasma membrane transporter P-glycoprotein (P-gp) in the involved physiological systems especially intestine, liver, kidney, etc. Excellent work using wild-type and P-gp knock-out mice has shown the role of P-gp in multi-drug resistance and bioavailability of Paclitaxel and other anticancer drugs [7]. Measurements of the plasma Paclitaxel concentration after oral administration showed that the area under the curve of the drug concentration in the plasma versus time (AUC) was 6-fold higher in the P-gp knock-out mice than in the wild-type mice. After intravenous administration of Paclitaxel, the AUC was only 2-fold higher in the P-gp knock-out mice compared to the wild-type [7].

Possible solutions for oral delivery of Paclitaxel and other anticancer drugs are currently under intensive investigations. The general idea is to apply P-gp/P450 inhibitors such as cyclosporin A to suppress the elimination process [8], [9], [10], [11], [12]. P-gp/P450 inhibitors, however, suppress the body immune system either and thus cause medical complications. The development of specific, low-toxicity inhibitors and other drug metabolizing enzymes such as dihydropyrimidine dehydrogenase would represent a major advance in the successful formulation of oral chemotherapy [13].

Chemotherapeutic engineering may radically change the way we make drugs and the way we take drugs, and thus provide an ideal solution for oral chemotherapy [1], [14], [15], [16]. A typical example is the application of biodegradable nanoparticles (NPs) of small enough size and appropriate surface modification to improve the adhesion and absorption of the NPs to transport the drug across the gastrointestinal (GI) barrier. Although there have been a few preliminary reports, the results obtained so far are still far from comparable to the intravenous administration [17], [18], [19].

In the present study, we synthesized a novel type of biodegradable nanoparticles, i.e. those of the poly(lactide)–vitamin E TPGS copolymer incorporated with a medical clay, montmorillonite (PLA–TPGS/MMT NPs) for oral chemotherapy by using Docetaxel as a model anticancer drug due to its excellent therapeutic effects against a wide spectrum of cancers and its commercial success as the best seller among all the anticancer agents. Another three nanoparticle systems, i.e. the poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), poly(lactide)–vitamin E TPGS nanoparticles (PLA–TPGS NPs) and poly(lactic-co-glycolic acid)/montmorillonite nanoparticles (PLGA/MMT NPs), were also investigated to make comparison. In our design, the FDA-approved biodegradable polymer PLA and PLGA were employed to maintain the mechanical strength of the copolymer. The d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E, which is formed by esterification of vitamin E succinate with polyethylene glycol (PEG) 1000 and could result in a desired hydrophile–lipophile balance (HLB, about 13). TPGS has been proved to be an excellent emulsifier in nanoparticle technology, which has 67 times higher emulsification efficiency than the traditional emulsifier polyvinyl alcohol (PVA). High drug encapsulation efficiency up to 100% could also be achieved [20]. It has also been reported that TPGS could increase in vivo oral bioavailability of cyclosporine A, vancomycin hydrochloride and talinolol [21], [22], [23]. We were thus inspired to synthesize PLA–TPGS copolymers for NP formulation of small molecule drug chemotherapy [24], [25], [26].

One of the most common side effects of cancer chemotherapy is the gastrointestinal problem, which leads to mucositis and ulceration of gastrointestinal tract, and diarrhea. MMT is a potent detoxifier with excellent adsorbent properties due to its high aspect ratio. It can absorb excess water from feces and thus act as antidiarrhoeaic. MMT can also provide mucoadhesive capability for the NPs to cross the GI barrier [27]. It has also been used as drug carrier for controlled release systems [28]. MMT has been proved to be non-toxic by hematological, biochemical and histopathological analysis in rat models [29].

In the present study, Docetaxel is used as a model small molecule anticancer drug, which is a poorly water-soluble, semi-synthetic taxane analog commonly used in the treatment of breast cancer, ovarian cancer, small and non-small cell lung cancer, prostate cancer. Pre-clinical studies demonstrated that Docetaxel had several advantages over Paclitaxel. Compared with Paclitaxel, Docetaxel showed wider cell-cycle bioactivity, greater affinity to the β-tubulin binding site and greater uptake with slower efflux from the tumor cells, resulting in longer intracellular retention time and higher intracellular concentrations. Docetaxel demonstrated superior efficacy versus Paclitaxel in a randomized phase III study [30], [31].

The Docetaxel-loaded PLA–TPGS/MMT NPs along with the other three nanoparticle systems were prepared by a modified solvent extraction/evaporation method, which were then characterized by the laser light scattering for particle size and size distribution, the laser Doppler anemometry for surface charge and the field emission scanning electron microscopy (FESEM) for surface morphology. The MMT content of the NPs was measured by the thermogravimetric analyzer (TGA). The physical status of Docetaxel in the NPs was characterized by the differential scanning calorimetry (DSC). Drug encapsulation efficiency in the NPs and the in vitro drug release profile were measured by the high performance liquid chromatography (HPLC). Cellular uptake of the coumarin 6-loaded NPs by Caco-2 cells (as an in vitro GI model) and MCF-7 cells (as model cancer cells) was visualized by the confocal laser scanning microscopy (CLSM) and quantitatively assessed by the microplate reader. In vitro cytotoxicity of the Docetaxel-loaded NPs for the cancer cells was evaluated by the CCK-8 assay, which has higher sensitivity and better reproducibility than the MTT assay. In vivo pharmacokinetics was measured in animal model of male Sprague–Dawley (SD) rats, which was made in close comparison with the current clinical dosage form of Docetaxel, i.e. Taxotere^®. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC), National University of Singapore.