本文選題：pH敏感脂質體 + siRNA�。� 參考：《河北醫(yī)科大學》2014年碩士論文

【摘要】：國際抗癌聯(lián)盟發(fā)布的數據表明，全球癌癥發(fā)病形式嚴峻，發(fā)病率與死亡率呈持續(xù)上升趨勢，中國的癌癥發(fā)病率幾乎占了全球的一半，目前我國癌癥發(fā)病率為285/10萬，平均每天每分鐘有6人被診斷為惡性腫瘤。在我國，乳腺癌已成為女性最常見的惡性腫瘤之一，目前已步入每年2％～3％的快速增長期。目前的抗腫瘤藥物靶向性差、毒副作用大、治療效果不佳等缺點。 脂質體是一種類似于生物膜結構的雙分子層微囊，由于其具有細胞類似結構，進入人體后主要被網狀內皮系統(tǒng)吞噬，使藥物靶向聚集在肝、脾、肺等組織中。但脂質體作為藥物載體仍存在對疾病的靶向性差，穩(wěn)定性欠佳等缺點。pH敏感脂質體在低pH值時脂質體由有穩(wěn)定結構變?yōu)椴环�(wěn)定結構。使得pH敏感脂質體內吞進細胞后質膜破裂或與其他質膜的融合，這使藥物在未達到溶酶體前釋放，避免的了溶酶體的降解，更有效的將藥物傳遞到細胞中。由于腫瘤間質液的pH值顯著低于周圍正常組織，所以研究pH敏感脂質體對腫瘤的治療作用有很大臨床價值。 目的： 由于腫瘤細胞外呈微酸環(huán)境，設計了在低pH內可插入細胞膜的多肽pHLIP，通過合成DSPE-PEG-pHLIP。實現對入微酸環(huán)境下的腫瘤細胞膜的靶向定位。組氨酸八聚體帶正電，可協(xié)助小分子進入細胞，本研究中，將硬脂酸和組氨酸八聚體偶合，形成帶陽離子的聚合物，由靜電吸附作用與siRNA形成SA-H8/siRNA復合物，提高siRNA的載藥效率。 方法： 在文獻和預實驗的基礎上，初步確定了pH敏感型聚合物SA-H8的合成方法,在室溫條件下，充氮氣避光攪拌32h，通過MS對其進行表征。 確定合成導向性脂材（DSPE-PEG-pHLIP）的合成方法，在室溫條件下，充氮氣避光攪拌48h，通過測定多肽中剩余巰基的含量確定反應終點，并對其進行MS表征。 在文獻基礎上，采用薄膜分散法制備脂質體，分別以香豆素和siRNA為模型藥物，以粒徑和包封率為考察指標，確定最佳處方工藝，以制劑的外觀、粒徑電位和形態(tài)作為指標，考察制得脂質體的穩(wěn)定性。 以人乳腺癌細胞（MCF-7）為細胞模型，分別在pH6.5和pH7.4培養(yǎng)基的環(huán)境中，給予壓縮載藥脂質體和普通載藥脂質體，孵育后，將細胞用PBS處理后，用流式細胞儀對載體體外細胞攝取性質進行分析。 結果： 經MS分析，合成得到SA-H8和DSPE-PEG-pHLIP，冷凍干燥后成白色絮狀固體,產率分別為48.9%和52.2%；制備香豆素脂質體，所得脂質體粒徑較均一，得到微黃色發(fā)有乳光的均勻混懸液，并且較穩(wěn)定，測得該脂質體的包封率為91.0%；制備siRNA的脂質體制劑在透射電子顯微鏡下均勻分布，粒子中間呈亮光狀。 流式細胞分析結果顯示，相比普通脂質體，pHLIP修飾載體的細胞攝取具備顯著的pH響應特性，，pH6.8條件下攝取明顯高于pH7.4環(huán)境。 結論： 以香豆素和siRNA為模型藥物，以SA-H8陽離子聚合物為siRNA載體，用DSPE-PEG-pHLIP修飾脂質體表面，采用薄膜分散法制備脂質體，穩(wěn)定性良好。以人乳腺癌MCF-7為細胞模型，pHLIP多肽作為導向性膜材修飾脂質體，在低pH值時，人乳腺癌細胞的攝取率明顯高于未經修飾的脂質體，DSPE-PEG-pHLIP靶頭對siRNA細胞攝取有促進作用。
[Abstract]:UICC released data show that the serious global cancer incidence rate has continued to rise, and the incidence rate of cancer mortality, Chinese accounted for almost half of the world, China's current cancer incidence rate of 285/10 million, an average of every minute of every day 6 people are diagnosed with malignant tumor in our country, breast cancer it has become one of the most common malignant tumors of women, has entered the year 2% ~ 3% period of rapid growth. The antitumor drug targeting, toxicity, poor treatment and other shortcomings.
Liposome is a kind of similar to the biofilm structure of bilayer microcapsules, because it has the similar structure of the cell, enters the human body mainly by the reticuloendothelial system phagocytic, drug targeting aggregation in the liver, spleen, lung and other tissues. But liposomes as drug carriers still exist for diseases of poor targeting stability the disadvantages of poor.PH sensitive liposome lipid at low pH value by stable structure becomes unstable. The structure of pH sensitive liposome fusion plasma membrane into the cell after swallowing or with other membrane, the drug release has not reached the lysosomes before to avoid lysosomal degradation, will be more effective drug delivery into cells. The tumor interstitial fluid pH was significantly lower than that of the surrounding normal tissue, so the study of therapeutic effect of pH sensitive liposomes on tumor has great clinical value.
Objective:
Because the tumor cells was slightly acidic environment, the design of peptide pHLIP in low pH can be inserted in the cell membrane, through the synthesis of DSPE-PEG-pHLIP. of tumor cell membrane into the acidic environment of the target location. Histidine eight dimers are positively charged, can help small molecules into cells, in this study, the stearic acid and histidine eight polymer coupling, formed with cationic polymer, by electrostatic adsorption and the formation of siRNA SA-H8/siRNA complex, improve the drug loading efficiency of siRNA.
Method錛

本文編號：1735281