dspe-mpeg2000用于制備脂質體的實驗使用方法和相關文獻

其他標題：DSPE-MPEG2000用于制備脂質體的文獻指導

文獻名:

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

文獻鏈接：

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

實驗使用方法:

脂質體制備

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.

通過握手法制備了由大豆磷脂酰膽堿和膽固醇以55:45的摩爾比組成的脂質體。簡而言之，將脂質（總脂質40pmol）溶解在100ml圓底燒瓶中的氯仿中。在37°C下，使用旋轉蒸發器（Wheaton）在減壓下將氯仿蒸發至干。將如此形成的薄膜干燥1小時，然后用1ml PBS（20mM，pH 7.4）水合，PBS含有蓖麻毒素（3mg/ml）和微量125]-蓖麻毒素作為水相標記。將含有圓形脂質體懸浮液在氮氣氣氛下儲存，以避免脂質氧化，在4℃下儲存過夜，以實現完全水合。第二天，脂質體在25°C的浴式聲波儀（Branson）中以10分鐘為一批進行30分鐘的聲波處理，以避免產生熱量。如上所述，制備了含有蓖麻毒素的負電荷和正電荷脂質體，在制備脂質膜的過程中只添加了10 mol%的磷脂酸（PA）或硬脂胺（SA）。如上所述，通過在制備脂質膜的過程中加入各種（1-7.5mol%）DSPE-mPEG-2000，制備了含有蓖麻毒素的立體穩定脂質體。

重要檢測:

重要實驗結果說明

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.

如圖5所示，當細胞用150 ng/ml游離蓖麻毒素處理時，觀察到45分鐘的滯后期，tso（實現蛋白質合成減少50%所需的時間）為200分鐘。觀察到蓖麻毒素抑制蛋白質合成的滯后期在通過不同帶電脂質體遞送后顯著增加。另一方面，在10 pg/ml的各種帶電蓖麻毒素脂質體中，中性和帶負電荷的脂質體觀察到4小時的滯后期，而帶正電的脂質體則觀察到2小時的滯后階段。莫能菌素（50 nm）將游離蓖麻毒素的滯后期從45分鐘縮短到15分鐘（即滯后期縮短了三倍），然而，在莫能菌蛋白存在的情況下，中性粒細胞和帶負電荷的脂質體蓖麻素的滯后期分別從4小時縮短到1小時（滯后期縮短四倍）和2小時（兩倍縮短標志期）。帶正電荷的脂質體蓖麻毒素的滯后期從2小時縮短到1小時（滯后期縮短了一倍）。這些結果表明，莫能菌素導致蓖麻毒素A鏈從位于細胞內隔室的脂質體釋放到細胞質中，從而快速抑制蛋白質合成。

西安齊岳生物科技有限公司供應合成磷脂、PEG磷脂、兩親嵌段共聚物,磁性納米顆粒,納米金,熒光量子點及樹枝狀聚合物、PEG衍生物等等產品.可供常規磷脂單體、PEG磷脂,改性磷脂,熒光標記磷脂、聚合物磷脂、磷脂偶連無機納米材料及微球類材料、單分散小分子PEG磷脂偶連產品,還可根據客戶需要定制合成復雜及特殊的聚合物磷脂產品。

廠家:西安齊岳生物科技有限公司

用途:科研

狀態：固體/粉末/溶液

產地:西安

溫馨提醒:僅供科研，不能用于人體實驗!

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