Studies On DTX-loaded Pluronic P123Micelles

Docetaxel (DTX), which is of the taxane chemotherapy drug, is a semisynthetic analogue of paclitaxel derived from an extract of the needles of the European yew tree (Taxus baccata L.), belonging to the second generation of the taxoid family. Like other taxanes, DTX is an inhibitor of microtubule depolymerization, but it binds more avidly to tubulin and hence resides inside cells longer than paclitaxel, which may explain why DTX appears to be2-to4-fold more potent than paclitaxel. In the past decade, DTX has emerged as one of the most important antineoplastic agents, with proven clinical efficacy against a wide range of cancers including advanced and metastatic breast cancer, ovarian cancer, small and non-small cell lung cancer, head and neck cancer, prostate cancer, etc. DTX, which is similar to other taxanes such as paclitaxel, shows low water solubility. Its available formulation Taxotere(?)for clinical application composes of high concentration of polysorbate80. This solvent system has been associated with severe side effects such as hypersensitivity reactions which have overshadowed the clinical use of DTX.In recent years, polymeric micelles as colloidal carriers have received increasing attention of pharmaceutical researchers. Polymeric micelles are prepared from amphiphilic block copolymers with a hydrophobic inner core and a hydrophilic shell. They possess several favorable features including nanoscale size (10-100nm in diameter), drug solubilization, controlled drug release, prolonged in vivo circulation time, escaping from the recognition and uptake of reticuloendothelial system (RES) or mononuclear phagocyte system (MPS), and tumor targeting by enhanced permeability and retention (EPR) effect. Drug molecules can be loaded into the polymeric micelles as a nanosized container via physical encapsulation, chemical conjugation or electrostatic interactions.To improve the water solubility of DTX as well as to avoid the use of polysorbate80-based adjuvant, in this article DTX was loaded into polymeric micelles. Pluronic block copolymers, which compose of hydrophilic poly(ethylene oxide)(PEO) and hydrophobic poly(propylene oxide)(PPO) blocks arranged in a basic PEO-b-PPO-b-PEO, were used in this study for their non-toxicity, non-immunogenicity and biocompatibility. In addition, these block copolymers are shown to inhibit P-glycoprotein (P-gp) mediated drug efflux action and to sensitize the multidrug resistant (MDR) cancer cell lines, resulting in an increase in the cytotoxic activity of the drugs such as docetaxel by2-3orders of magnitude. Among them, Pluronic P123(PEO20-b-PPO70-b-PEO20), where40and70designate the total average number of the PEO and PPO repeat units and b stands for "block", possesses higher proportion of the hydrophobic block, promising a higher drug loading.DTX was incorporated into the hydrophobic core of Pluronic P123polymeric micelles through physical encapsulation and chemical conjugation. The physicochemical properties of the DTX-loaded polymeric micelles were evaluated. The in vitro and in vivo antitumoral activities of the DTX-loaded polymeric micelles were also assessed. This subject provides theoretical foundation and experimental basis for the development of micellar formulations of docetaxel. This study was divided into two parts to evaluate DTX-loaded polymeric micelles. The main experimental methods and results are as follows:1. Studies on Pluronic P123polymeric micelles with physical encapsulation of docetaxelDTX-loaded Pluronic P123micelles (P123micelles) were prepared by the thin-film dispersion method, followed by lyophilization. On the basis of optimization with single factors, orthogonal experiment design was employed for further optimization taking the entrapment efficiency as index. The optimal preparation was characterized in terms of morphological observation, particle size distribution, zeta potential, drug loading and encapsulation efficiency. In vitro release of DTX from the polymeric micelles was carried out in the phosphate buffer solution (PBS, pH7.4) containing0.5%Tween80(w/v) using the dialysis method. In vitro antitumor activity of P123micelles against human liver carcinama HepG2cell lines, human alveolar basal epithelial A549cell lines and mouse malignant melanoma B16cell lines was assessed by the MTT method. The in vivo antitumor activity was evaluated in Kunming mice bearing B16tumor.The analyses of DTX in vitro were determined by reversed-phase high performance liquid chromatography (RP-HPLC) method. Before freeze-drying, the encapsulation efficiency (EE) and drug loading (DL) of P123micelles were (92.07±1.77)%and (2.35±0.08)%, respectively; after lyophilisation and reconstitution, the DTX-loading and efficiency into the micelles were (2.12±0.09)%and (86.34±3.32)%, respectively. Before and after freeze-drying, transmission electron microscopy (TEM) exhibited that P123micelles were well dispersed as individual particles with spherical shape, before freeze-drying, the average diameter determined by dynamic light scattering (DLS) was38.9nm with the size distribution from9to55nm; freeze-dried P123micelles suspended in de-ionized water possessed a mean particle size of50.7nm with the size distribution from22to84nm. Before and after freeze-drying, the zeta potential was (-10.56±2.34) mV and (-12.4513.24) mV, respectively. The in vitro release profile of P123micelles followed the Weibull equation:lnln(1/(1-y/100))=0.7381nt-1.796(r=0.9935). It was obvious that the release of DTX from P123micelles was slower than that of Duopafei?. The in vitro cytotoxicity against HepG2, A549and B16cell lines showed that IC50values of P123micelles ((0.34±0.02)μM,(0.44±0.05) μM and (0.49±0.08) μM, respectively) were all significantly lower than those of Duopafei?((0.96±0.05) μM,(0.74+0.02) μM and (0.72±0.10) μM, respectively). In addition, P123micelles exhibited remarkable antitumor activity and reduced toxicity on B16tumor in vivo. The tumor inhibition ratio (TIR) of P123micelles was91.6%versus76.3%of Duopafei?(P<0.01).2. Studies on Pluronic P123-DTX conjugate micellesDTX was covalently bound to Pluronic P123through the formation of an ester bond between the hydroxyl group of Pluronic P123and the carboxylic acid group of carboxyl-terminated DTX after the2’hydroxyl group of DTX was activated by succinic anhydride. Structure of the resulting P123-DTX conjugates was confirmed by proton nuclear magnetic resonance (1H-NMR) spectroscopy in CDC13. Due to the amphiphilicity, the P123-DTX conjugates could spontaneously self-assemble in aqueous medium to become polymeric micelles. The123-DTX conjugate micelles were prepared through the dialysis method. The conjugate micelles were characterized in terms of critical micelle concentration (CMC), drug loading content, morphology, particle size and zeta potential. In vitro release was performed in phosphate buffered solution (PBS) at pH1.2,5.0,6.8,7.4and mice plasma. The in vitro antitumor activity of P123-DTX conjugate micelles against human liver carcinama HepG2cell lines, human breast cancer MCF-7cell lines and mouse malignant melanoma B16cell lines was evaluated by the MTT method. The in vivo antitumor activity was assessed in Kunming mice bearing B16tumor.1H-NMR spectra data of2’-succinyl-DTX in CDC13compared with that of DTX exhibited an additional signal at2.5-2.7ppm as multiple peaks due to the OCOCH2CH2COOH protons from the succinic anhydride group. A series of new peaks appeared in the’H-NMR spectra of P123-DTX conjugates due to the introduction of DTX moieties into the Pluronic P123. The CMC of P123-DTX conjugates determined by fluorescent spectroscopy with pyrene as the fluorescent probe was (1.34±0.07)×10-5mol/L, which was lower than that of Pluronic P123which was (1.91±0.05)×10-5mol/L. The DTX content in the P123-DTX conjugates determined by UV-Vis spectroscopy at a230nm wave-length was (13.69±0.52)%(w/w). The TEM and DLS results showed that the P123-DTX conjugates could self-assemble into uniform spherical particles with (85.3±1.59) nm.The percentage of DTX released from the conjugate micelles increased as the pH value decreased from7.4to1.2. For example, after60h incubation, the amounts of DTX released in the buffers of pH7.4,6.8,5.0and1.2were3.5,3.7,10.45and20.34%. This pH sensitivity of the release rate of DTX from the conjugate micelles could be probably attributed to the sensitivity of the cleavage of the ester bonds to pH value. In plasma, the P123-DTX conjugate micelles underwent rapid ester bond hydrolysis and degradation, which was reflected in the release of about42.50%of DTX from the conjugates in60h. The in vitro cytotoxicity against HepG2, MCF-7and B16cell lines showed that IC50values of P123-DTX conjugate micelles ((1.14±0.13) μM,(1.44±0.15) μM and (1.68±0.22) μM, respectively) were all higher than those of Duopafei?((0.66±0.02) μM,(0.54±0.06) μM and (0.83±0.11) μM, respectively). In addition, P123-DTX conjugate micelles exhibited remarkable antitumor activity and reduced toxicity on B16tumor in vivo. The tumor inhibition ratio (TIR) of P123micelles was95.94%versus79.02%of Duopafei?(P＜0.01).