| Radiation resistance of hypoxic tumor cells is one of the key points in radiation oncology and biology. It is thought that radiosensitizers are potential to overcome the problem. This area attracted the increasing attention from the medical researchers all over the world. Many kinds of radiosensitizers, such as electro-affinic compounds, biological reducers, SH inhibitors and reparation inhibitors etc., were developed. However, the side effects, such as neurotoxicity and symptoms of enteron etc., limited their clinical application. In order to increase radiosensitization without increasing toxicity, the combination of the drugs is actual, safe and effective. There were many correlative reports abroad. This study was carried out to determine the synergetic radiosensitizing effect of etanodazole and paclitaxel when administered together at clinically relevant concentrations. The study would provide a new combination of radiosensitizers to radiotherapy. Meanwhile, in order to increase further radiosensitization without increasing toxicity, the nanoparticles containing etanodazole and/or paclitaxel were prepared. The advantages of such a formulation include the controlled and targeted delivery of drugs, increased solubility and ingestion of drugs, increased therapeutic effect and reduced side effects. This study would provide a new idea for clinical application of radiosensitizers.OBJECTIVE:The object of this study was to determine the synergetic radiosensitizing effect of etanidazole and paclitaxel, to prepare active and controlled nanoparticles containing etanidazole and/or paclitaxel, to prove the radiosensitization of the drug-loaded nanoparticles and their advantage.METHODS:1. After administered together at clinically relevant concentrations of etanidazole and/or paclitaxel into the two hypoxic human tumor cell lines: a breast carcinoma (MCF-7) and a carcinoma cervicis (HeLa), cells were irradiated by 60Co gamma rays. The 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay was used to determine the number of surviving cells. Cell cycle was evaluated by Flow cytometry (FCM). Cell viability was measured by the ability of single cell to form colonies in vitro. Difference between the combination of the drugs and drug alone was studied.2. The poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles containing etanidazole and/or paclitaxel were prepared by o/w and w/o/w emulsification-solvent evaporation method. The drug loading efficiency (LE), encapsulation efficiency (EE) and release profile in vitro were measured by high-performance liquid chromatography (HPLC). The size distribution and morphology of the nanoparticles were investigated by laser diffraction analyzer and scanning electron microscope (SEM).3. After administered the free drugs and the drug-loaded nanoparticles into hypoxic MCF-7 and HeLa cells (released drug doses calculated according to the release profile in vitro), the morphology of cells was photographed using phase-contrast photomicrographe. The cellular uptake of nanoparticles by MCF-7 and HeLa cells was evaluated by transmission electronic microscopy (TEM) and fluorescence microscopy. Then the tumor cells were irradiated by 60Co gamma rays. Cell cycle was evaluated by FCM. Cell viability was determined by the ability of single cell to form colonies in vitro. Radiosensitization of the drug-loaded nanoparticles and their difference were studied.RESULTS:1. The synergistic radiosensitive effect of etanidazole and paclitaxel was less obvious during 5 d after irradiation. After hypoxic HeLa cells administered were irradiated, the cells were mainly blocked in G1 phase. The combination of the two drugs resulted in more HeLa cells blocked in G1 phase. For MCF-7 cells, there was no significant statistical difference among the groups. The synergistic radiosensitizing effect of these two drugs on MCF-7 cells was not observed, but the radiosensitizing efficiency was additive for HeLa cells irradiated at 6, 8 and 10Gy. The radiosensitizing effect of paclitaxel at 100 nM was more significant than that of etanidazole at 1 mM. HeLa cells were more sensitive to paclitaxel than MCF-7 cells.2. The prepared paclitaxel-loaded nanoparticles were spherical shape with size between 100nm and 500nm, about 300nm on average. The loading efficiency of 4.50% and the encapsulation efficiency of 85.51% were obtained. The drug release pattern was biphasic with a fast release rate followed by a slow one. The amount of cumulated paclitaxel release over 14 days was about 30%. The kinetic data showed that majority of the drug release occurred in the first day (approximately 15%). Co-culture of MCF-7 and HeLa cells with paclitaxel-loaded nanoparticles demonstrated that MCF-7 cells extremely spreaded and became fusiform, and the part of HeLa cells treated was marcid. The number of cells blocked in the G2/M phase for two tumor cell lines significantly increased. The cellular uptake of nanoparticles was observed. The paclitaxel-loaded nanoparticles and free paclitaxel more effectively sensitized hypoxic tumor cells to radiation than control. The radiosensitization of paclitaxel-loaded nanoparticles was more significant than that of free paclitaxel.3. The prepared etanidazole-loaded nanoparticles were spherical shape with size between 90nm and 190nm, about 120nm on average. The loading efficiency of 1.66% and the encapsulation efficiency of 18.02% were obtained. The drug release pattern was biphasic with a fast release rate followed by a slow one. The burst effect was very large(about 50% in 3 h). The kinetic data showed that majority of the drug release occurred in the first day (approximately 90%). The cellular uptake of nanoparticles was observed. The etanidazole-loaded nanoparticles and free etanidazole more effectively sensitized hypoxic tumor cells to radiation than control. The radiosensitization of etanidazole-loaded nanoparticles was more significant than that of free etanidazole.4. The paclitaxel, etanidazole and paclitaxel+etanidazole nanoparticles were prepared. The surface of all the nanoparticles was smooth, rounded morphology and polydispersed with a diameter between 80 nm and 150 nm, about 110 nm on average. The results indicated that a loading efficiency of 4.53% and an encapsulation efficiency of 85.52% were obtained for paclitaxel-loaded nanoparticles, 1.86% and 20.06% for etanidazole-loaded nanoparticles. For paclitaxel+etanidazole nanoparticles, the loading efficiency and the encapsulation efficiency were 4.62% and 90.51% for paclitaxel, 1.92% and 23.16% for etanidazole, respectively. The drug encapsulation efficiency of paclitaxel+etanidazole nanoparticles is slightly higher than that of single drug-loaded nanoparticles. All the drug release patterns were biphasic with a fast release rate followed by a slow one. The amount of cumulated paclitaxel release over 5 days was about 25%. The kinetic data showed that majority of the drug release occurred in the first day (approximately 15%). The kinetic data showed that majority of the drug release from etanidazole-loaded nanoparticles occurred in the first day (approximately 90%). The burst effect was very large (about 50% in 3 h). The amount of cumulated drug release from paclitaxel+etanidazole nanoparticles was slightly more than that from single drug-loaded nanoparticles. The cellular uptake of paclitaxel+etanidazole nanoparticles was observed. The paclitaxel, etanidazole and paclitaxel+etanidazole nanoparticles effectively sensitized hypoxic tumor cells to radiation, their radiosensitizing effects: etanidazole-loaded nanoparticles < paclitaxel-loaded nanoparticles |
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