dspe-mpeg2000用于制备脂质体的实验使用方法和相关文献

其他标题：DSPE-MPEG2000用于制备脂质体的文献指导

文献名:

Effect of surface charge and density of istearylphosphatidylethanolamine-mPEG-2000 (DSPE-mPEG-2000) on the cytotoxicity of liposome-entrapped ricin: Effect of lysosomotropic agents

文献链接：

https://doi.org/10.1016/j.ijpharm.2007.08.032

实验使用方法:

脂质体制备

Liposomes composed of soya phosphatidyl choline andcholesterol in a molar ratio of 55:45 were prepared by handshaken method. Briefly, the lipids (40 pmol total lipids)weredissolved in chloroform in a 100ml round bottom fask. Thechloroform was evaporated to dryness at 37°C, under reducedpressure by using rotary evaporator (Wheaton). The thin filmso formed, was desiccated for 1 h, followed by hydration with1 ml PBS (20 mM, pH 7.4), containing ricin (3 mg/ml) and traceamounts of 125]-ricin as aqueous phase marker. The round bot-tom fask containing liposomes suspension was stored, underN2 atmosphere to avoid lipid oxidation, at 4C for overnight forcomplete hydration. The following day, liposomes were soni-cated in a bath type sonicator (Branson) at 25 °C for 30 min in10 min batches to avoid the heat generation. Negatively and positivelycharged liposomes containing ricin were preparedexactlyas described above only 10 mol% either phosphatidic acid (PA)or stearylamine (SA) were added during the preparation of lipidflm. Sterically stabilized liposomes containing ricin were pre-pared as described above by adding various (1-7.5 mol%) ofDSPE-mPEG-2000 during the preparation of lipid film.

通过握手法制备了由大豆磷脂酰胆碱和胆固醇以55:45的摩尔比组成的脂质体。简而言之，将脂质（总脂质40pmol）溶解在100ml圆底烧瓶中的氯仿中。在37°C下，使用旋转蒸发器（Wheaton）在减压下将氯仿蒸发至干。将如此形成的薄膜干燥1小时，然后用1ml PBS（20mM，pH 7.4）水合，PBS含有蓖麻毒素（3mg/ml）和微量125]-蓖麻毒素作为水相标记。将含有圆形脂质体悬浮液在氮气气氛下储存，以避免脂质氧化，在4℃下储存过夜，以实现完全水合。第二天，脂质体在25°C的浴式声波仪（Branson）中以10分钟为一批进行30分钟的声波处理，以避免产生热量。如上所述，制备了含有蓖麻毒素的负电荷和正电荷脂质体，在制备脂质膜的过程中只添加了10 mol%的磷脂酸（PA）或硬脂胺（SA）。如上所述，通过在制备脂质膜的过程中加入各种（1-7.5mol%）DSPE-mPEG-2000，制备了含有蓖麻毒素的立体稳定脂质体。

重要检测:

重要实验结果说明

examined. As shown in Fig. 5 when cells were treated with150 ng/ml free ricin a lag period of 45 min was observed withtso (time required to achieve 50% reduction in protein synthe-sis) of 200 min. It was observed that the lag period of inhibitionof protein synthesis by ricin is significantly increased followingdelivery through different charged liposomes. At 10 pg/ml ofvarious charged liposomal ricin, a lag phase of 4 h was observedfor neutral and negatively charged liposomes, on the other hand.a lag phase of 2h was observed for positively charged lipo-somes. Monensin (50 nm) reduced the lag period of free ricinfrom 45 to 15 min (i.e., three-fold reduction of the lag period),however, in the presence of monensin, the lag period of neutraland negatively chargedliposomal ricin was reduced from 4 to 1 h(four-fold reduction of lag period) and 2 h (two-fold reductionoflag period), respectively. The lag period of positively chargedliposomal ricin was reduced from 2 to 1 h (two-fold reductionof lag period). These results implied that monensin causes anenhanced and efficient release of ricin A-chain from liposomalricin located in an intracellular compartment into the cytosolleading to the rapid onset of the inhibition of protein synthesis.

如图5所示，当细胞用150 ng/ml游离蓖麻毒素处理时，观察到45分钟的滞后期，tso（实现蛋白质合成减少50%所需的时间）为200分钟。观察到蓖麻毒素抑制蛋白质合成的滞后期在通过不同带电脂质体递送后显著增加。另一方面，在10 pg/ml的各种带电蓖麻毒素脂质体中，中性和带负电荷的脂质体观察到4小时的滞后期，而带正电的脂质体则观察到2小时的滞后阶段。莫能菌素（50 nm）将游离蓖麻毒素的滞后期从45分钟缩短到15分钟（即滞后期缩短了三倍），然而，在莫能菌蛋白存在的情况下，中性粒细胞和带负电荷的脂质体蓖麻素的滞后期分别从4小时缩短到1小时（滞后期缩短四倍）和2小时（两倍缩短标志期）。带正电荷的脂质体蓖麻毒素的滞后期从2小时缩短到1小时（滞后期缩短了一倍）。这些结果表明，莫能菌素导致蓖麻毒素A链从位于细胞内隔室的脂质体释放到细胞质中，从而快速抑制蛋白质合成。

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厂家:西安齐岳生物科技有限公司

用途:科研

状态：固体/粉末/溶液

产地:西安

温馨提醒:仅供科研，不能用于人体实验!

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