多西他赛载药纳米胶束的制备及其体内外评价

发布时间：2018-12-18 04:18

【摘要】：目的：多西他赛(docetaxel, DTX)是一种新型植物碱类抗肿瘤药,为了克服其在水中溶解度低的缺点,制备了一种能够包载多西他赛的聚乙二醇-聚乳酸mPEG-b-PDLLA共聚物；并且对多西他赛载药纳米胶束的进行了体内外评价。方法：以聚乙二醇单甲醚mPEG和D,L-丙交酯为原料,在辛酸亚锡的催化下,开环聚合,形成嵌段聚合物。通过傅立叶变换红外光谱(FT-IR)、核磁共振(1H-NMR)、差示扫描量热法(DSC)、 MALDI-TOF-TOF-MS、气相色谱法、电感耦合法、pH计等对聚合物mPEG-b-PDLLA进行了表征。采用溶剂挥发薄膜水化法制备了包载抗癌药物多西他赛的纳米胶束(DTX-PM)。利用Malvern Nano ZS激光粒度分析仪测定其粒径和分布；透射电镜观测纳米胶束的形态；高效液相色谱法(HPLC)测定包封率及载药量,并进行了质量标准研究；通过粒径和载药量的测定观察载多西他赛纳米胶束的稳定性。通过细胞迁移试验、MTT法和激光共聚焦实验考察载多西他赛纳米胶束对A549细胞的增殖抑制作用。裸鼠皮下接种瘤块建立肺癌动物模型后,采用尾静脉的方法分别给裸鼠注射葡萄糖水溶液、DTX-PM溶液及市售多帕菲(?)溶液,定期观察裸鼠皮下瘤体体积大小和裸鼠体重的变化。结果：通过1H-NMR得出丙交酯的摩尔百分含量为17～19%, mPEG2000的摩尔百分含量为81%～83%,共聚物平均分子量在3300左右。通过红外光谱谱图的分析证实了丙交酯和mPEG聚合形成了嵌段共聚物。MALDI-TOF-TOF-MS实验显示检测峰主要在3300 Da,纯化后杂质峰明显减少。DSC测试得到mPEG-b-PDLLA聚合物的熔点约为41.8℃,熔融焓Hm=20.3 mJ/mg。残留量检查的结果显示甲苯、二氯甲烷、乙醚等有机溶剂并无残留,丙交酯的残留含量为1.2%,锡的残留含量为1.521E+05μg/kg。 mPEG-b-PDLLA溶于水后测得pH值为5.2。多西他赛纳米胶束的粒径约16 nm左右,PDI在0.1～0.2之间；透射电镜下观察到DTM-PM的形状呈规则的球形。通过HPLC检测,得出多西他赛纳米胶束的包封率为(88.88±0.61)%,载药量为(15.00±0.69)%。质量标准研究中的含量测定方法简便、准确度高、重现性好。考察DTX-PM的复溶稳定性,结果显示胶束稳定性良好,可以满足输液要求。MTT法结果显示,高浓度时市售多帕菲(?)比DTX-PM具有更强的细胞增殖抑制作用,而在低浓度时DTX-PM具有更强的细胞增殖抑制作用；细胞迁移试验显示,市售多帕菲(?)和DTX-PM均可抑制A549细胞的增殖；激光共聚胶试验表明,胶束的入胞速度很快且能够摄取进入细胞核。给裸鼠注射葡萄糖溶液、市售多帕菲(?)溶液和DTX-PM溶液后发现DTX-PM组的裸鼠肿瘤生长速度慢于市售多帕菲(?)组,且毒性小于市售多帕菲(?)组。结果表明,DTX-PM的抗肺癌作用强于现在临床上使用的多帕菲(?)。结论：通过mPEG-b-PDLLA共聚物的结构表征,可以证实丙交酯和mPEG成功聚合成了嵌段聚合物。聚合物中有机溶剂、丙交酯和金属锡的残留量均符合要求。载多西他赛纳米胶束的形态呈球形,其粒径分布均匀,性质稳定。质量标准研究中的含量测定方法简便、准确度高、重现性好,可以用于该制剂的质量控制。多西他赛纳米胶束能够有效地抑制A549细胞的增殖,具有抗肺癌作用。
[Abstract]:Objective: To overcome the disadvantage of low solubility in water, a new type of anti-tumor medicine of plant base is prepared by using Dosizel (DTX), and a polyethylene glycol-polylactic acid (PEG-b-PDLLA) copolymer which can be used for carrying out the polysiracite is prepared. and in vivo out-of-vivo evaluation of the nano-micelle of the multi-sizinese medicament-carrying agent. Methods: The block polymer was formed by ring-opening polymerization under the catalysis of stannous octoate as the raw material with polyethylene glycol monomethyl ether (mPEG) and D (L-lactide). The polymer mPEG-b-PDLLA was characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (1H-NMR), differential scanning calorimetry (DSC), MALDI-TOF-TOF-MS, gas chromatography, inductive coupling method, pH meter and the like. The nano-micelle (DTX-PM) was prepared by the method of solvent volatilization film hydration. The particle size and distribution were determined by a Malvern Nano-ZS laser particle size analyzer, the morphology of the nano-micelle was observed by transmission electron microscope, the encapsulation rate and the drug-carrying amount were determined by high performance liquid chromatography (HPLC), and the quality standard was studied. The stability of the nano-micelle is observed by the determination of the particle size and the drug-loading amount. The proliferation of A549 cells was studied by cell migration test, MTT method and laser confocal experiment. After a lung cancer animal model was established by subcutaneous inoculation of nude mice, a method of tail vein was used to inject glucose solution, DTX-PM solution and multi-pafenone (?) in the nude mice, respectively.) The volume size of the subcutaneous tumor of the nude mice and the body weight of the nude mice were observed on a regular basis. Results: By 1H-NMR, the molar percentage content of the acrylate was 17-19%, the mole percentage of mCloth was 81-83%, and the average molecular weight of the copolymer was about 3300. Through the analysis of the infrared spectrum, the block copolymer was formed by the polymerization of the acrylate and the mPEG. MALDI-TOF-TOF-MS showed that the detection peak was mainly at 3300 Da, and the impurity peak after purification was significantly reduced. The melting point of the mPEG-b-PDLLA polymer was about 41. 8 鈩，

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