1、文献汇报杨岸蒲1210307403Background纳米给药系统的特点增加药物的吸收控制药物的释放改变药物的体内分布特征改变药物的膜转运机制Background细胞旁路转运（质外体途径）穿细胞转运（共质体途径）PP节M 细胞跨膜转运作用Pathways across the bloodbrain barrierBackground吞噬作用胞饮作用巨胞饮作用网格蛋白介导的内吞作用脂质微囊介导的内吞作用网格蛋白/脂质微囊非依赖性的内吞作用纳米给药系统经细胞的内吞作用吞噬作用1.外源性的纳米给药系统会被血液中的条理系统所识别。2.具有调理作用的蛋白会吸附于纳米给药系统表面，对纳米制剂进行调理化作用
2、。3.被调理化的纳米制剂经吞噬性细胞吞噬。4.纳米制剂在被细胞吞噬的过程中会诱发一系列信号转导相关变化，引起作为细胞运动与骨架基本结构的肌动蛋白发生重排，促进吞噬作用的进行。5.纳米给药系统被吞噬后即形成吞噬体结构，吞噬体会在胞浆内经历囊泡的融合过程后最终会与溶酶体融合，起到对外源性物质的消化作用。网格蛋白介导的内吞作用网格蛋白分子（clathrin），由三条重链和三条轻链构成，重链分子量为180kD，轻链分子量为35-40kD。每条重链和轻链组合在一起形成一个扩展臂，不同的扩展臂相互组合形成网格蛋白包裹的囊泡的外层空间结构。蛋白衔接素（adaptin），主要起到衔接细胞质膜与网格蛋白的作用。
3、目前研究较多的为AP180和AP2衔接蛋白动力蛋白（dynamin），使细胞膜凹陷作用脱离细胞膜，使囊泡游离进入胞浆，形成内吞囊泡。当纳米给药系统经网格蛋白介导的内吞作用摄取入胞后，形成的内吞囊泡中的网格蛋白会脱离重新回到细胞膜，囊泡会与早期内吞体融合，之后会被分选进入晚期内吞体/溶酶体，或进入循环内吞体再次转运回细胞膜，也可以到达顺式高尔基体。脂质微囊（小窝蛋白）介导的内吞作用脂质微囊（caveolae）是细胞中的一种特殊结构，广泛存在于上皮细胞、肌细胞、成纤维细胞中，在分子水平有着类似的组成和结构，在脂质微囊结构中最重要的膜蛋白为小窝蛋白（caveolin），还存在多种信号转导相关蛋白，包
4、括GPI锚定蛋白、G蛋白偶联受体、非受体型酪氨酸蛋白激酶受体、胰岛素受体等。脂质微囊介导的内吞作用相比于网格蛋白介导的内吞途径更为缓慢，且形成的内吞囊泡可以避免与溶酶体的融合。通过对纳米载体进行脂质微囊结构中受体的特异性配体修饰也可以促使纳米载体经脂质微囊介导的内吞作用入胞。v3整合素受体多存在于胞膜脂质微囊区域，采用RGD修饰PEG-PLL共聚物胶束用于DNA的胞内释放，纳米载体经修饰后的DNA转染效率明显提高且会通过脂质微囊介导的内吞途径进入细胞。脂质微囊/网格蛋白非依赖的内吞作用ARF6RhoA PLGA纳米粒一种聚乳酸聚羟基乙酸共聚物已经被美国FDA批准作为药用材料，其具有良好的生物相
5、容性和生物可降解性血管平滑肌细胞对PLGA纳米粒的摄取以网格蛋白介导的内吞作用为主鼠角膜上皮细胞对PLGA纳米粒的内吞作用是网格蛋白和脂质微囊非依赖性的本文章以PLGA纳米粒为模型制剂，研究其跨越极性上皮细胞单层的内吞及跨膜转运机理Part 1Preparation and characterization of C6-PNsPreparation and characterization of C6-PNs C6 could be utilized as the effective marker of PNs for the whole intracellular trafficking p
6、athways among organelles with different pH environments.Part 2Endocytosis of C6-PNs by MDCK epithelial cellsEndocytosis of C6-PNs by MDCK epithelial cells Figure shows the comparison of LDH released between PNs and SFM.There was no significant difference between them,indicating that PNs did not trig
7、ger the extra increase of LDH from cytoplasm.Endocytosis of C6-PNs by MDCK epithelial cellsLots of vesicle structures containing C6-PNs(green color)were found in cytoplasm,and obvious colocalization(yellow color)between C6-PNs and DiI was also observed in merged confocal micrograph.Endocytosis of C6
8、-PNs by MDCK epithelial cells The endocytosis of PNs by MDCK cells was energy-dependent,involving active process.Endocytosis of C6-PNs by MDCK epithelial cellsMCD and filipin：inhibitors of caveolae-mediated endocytosis,reduced the cell uptake of PNs significantly.Nystatin：triggers the absence of cav
9、eolaes in cells,also inhibited the internalization of nanoparticles.Hyperosmotic sucrose：inhibiting the clathrin dependent endocytosisEIPA：macropino cytosis inhibitor,had noeffect on endocytosis.The PNs could be endocytosed via multiple pathways by MDCK cells,involving both lipid raft and clathrin m
10、echanisms but not macropinocytosis.Part 3Intracellular trafficking of PNs in MDCK epithelial cellsIntracellular trafficking of PNs in MDCK epithelial cellsIntracellular trafficking of PNs in MDCK epithelial cells During endocytosis process,most of internalized PNs delivered from AEE to LE,and ultima
11、tely entered the lysosomes of MDCK cells.Part 4Exocytosis of PNs by MDCK epithelial cellsExocytosis of PNs by MDCK epithelial cellsExocytosis of PNs by MDCK epithelial cellsExocytosis of PNs by MDCK epithelial cellsMCD:significantly decreased theintracellular C6-PNs compared to control group during
12、the exocytosis test as shown,revealing that the lipid rafts in MDCK cell membrane also took effect on the exocytosis of PNs.BrefeldinA and monensin:inhibitors of transport function of Golgi complex,were found to suppress the exocytosis of PNs significantly,resulting in more C6-PNs in cytoplasm,but h
13、ad no effect on the internalization process.（ER/Golgi and Golgi/ARE/PM）Golgi complexlipid rafts These two inhibitors showed significant disruption effect on ER/Golgi and Golgi/ARE/PM pathways during the exocytosis of PNs.The exocytosis of PNs might involve these two pathways,namely,both Golgi comple
14、x and ER were important regulative organelles for the exocytic transportation of PNs.Exocytosis of PNs by MDCK epithelial cellsGenistein：the inhibitor of PTK,blocked the internalization of PNs by about 15%with significance but decreased the exocytosis without significance,indicating its difference i
15、n the inhibition of endocytosis and exocytosis of PNs.PMA and Staurosporine：the inhibitor of PKC,significantly blocked the cellular uptake of PNs,suggesting the regulation of PKC in the internalization of PNs.Exocytosis of PNs by MDCK epithelial cellsLY294002 and wortmannin：the inhibitor of PI3K,but
16、 they had no effect on the internalization of PNs,indicating that PI3K and the downstream signaling transduction did not regulate the endocytosis of PNs.In contrast,the inhibition of PI3K by LY294002 induced significant decrease of exocytosis in MDCK cells.Endocytosis:PTK PKCExocytosis:PI3KPart 5Tra
17、nscytosis of PNs across MDCK cell monolayerTranscytosis of PNs across MDCK cell monolayerTranscytosis of PNs across MDCK cell monolayer The fluorescence in porous membrane and basolateral compartment in the group of DiR-PNs was much stronger than that in free DiR group,indicating more effective tran
18、s-cellular capability of PNs.Transcytosis of PNs across MDCK cell monolayer TEER is an index of integrity for epithelial cell monolayer.So the trans-cellularpathway of PNs through MDCK cell monolayer was mainly transcytosis but not paracytosis.Transcytosis of PNs across MDCK cell monolayer endocytosis-excluded situationnormal situationTranscytosis of PNs across MDCK cell monolayer endocytosis-excluded situationnormal situationTranscytosis of PNs across MDCK cell monolayer endocytosis-excluded situationnormal situation endocytosis-excluded situationnormal situationThanks
