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第三周（ 3.3-3.7 ）

第三周（ 3.3-3.7 ）. 1. 用 OPSS-PEG-NHS 连接 PEG(5000)-PLA-PCL-PEI 和 REDV 。 2. 尝试 PHEMA-PCL-COOH 连接 PEI 和 MPEG 。探索 3:2,3.5:2 两种比例。. Viral Mimicking Ternary Polyplexes: A Reduction-Controlled Hierarchical Unpacking Vector for Gene Delivery.

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第三周（ 3.3-3.7 ）

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  1. 第三周（3.3-3.7） 1.用OPSS-PEG-NHS连接PEG(5000)-PLA-PCL-PEI和REDV。 2.尝试PHEMA-PCL-COOH连接PEI和MPEG。探索3:2,3.5:2两种比例。

  2. Viral Mimicking Ternary Polyplexes: A Reduction-Controlled Hierarchical Unpacking Vector for Gene Delivery Yiyan He , Yu Nie , Gang Cheng , Li Xie , Youqing Shen , and Zhongwei Gu Advanced Materials 2013, DOI: 10.1002/adma.201304592

  3. Contens • De novo synthesis of reduction sensitive polycation and hyaluronicate derivatives • Assembly and characterization of polyplexes • Stimuli responsiveness • Dual programmed stimuli responsiveness • Fluorescence resonance energy transfer (FRET) imaging • Cell culture and viability assay • In vitro transfection of pEGFP and pGL3 plasmid • In vivo gene transfection study

  4. De novo synthesis of reduction sensitive polycation and hyaluronicate derivatives

  5. Assembly and characterization of polyplexes Assembly HEPES 20mM, pH 7.4, 5 % glucose, room temperature, 20 min characterization a final DNA concentration of 3 μg mL-1 5-fold in 1 mM KCl solution Zeta potential and particle size a final DNA concentration of 3 μg mL-1 5-fold in 1 mM KCl solution Agarose gel retardation assay

  6. Figure S2. Zeta potential and particle size measurements of different polyplexes with different shielding/DNA weight ratios, the weight ratio of OEI-SeSex/DNA is 10. All the samples were prepared in HBG buffer and the measurements were performed in triplicates (n:the weight ratio of shielding/DNA). 在体系中引入透明质酸后也不会影响PEI对DNA的包裹 Figure S3. Agarose gel retardation assay of different polyplexes. (A) OEI-SeSex/DNA polyplexes with different weight ratios. (B) OEI-SeSex/DNA/HA polyplexes with OEISeSex/ DNA weight ratio of 10 and different HA/DNA weight ratios. And (C) OEISeSex/ DNA/HA-SS-COOH polyplexes with OEI-SeSex/DNA weight ratio of 10 and different HA-SS-COOH/DNA weight ratios.

  7. Stimuli responsiveness Molecular weight 做到OEI-SS x 和OEI-SeSe x初始分子量相当,探讨GSH浓度和作用时间对聚合物分子量的改变情况。 Gel retardation assay 探讨在不同浓度的GSH的存在下，DNA与两种聚合物的结合程度。 hydrodynamic diameters 在一定浓度的GSH存在下，探讨两种纳米粒粒径变化情况，从而揭示纳米粒的稳定程度 指明GSH作用！

  8. OEI-SS x OEI-SeSe x Figure 2. GPC diagrams of bioreducible polymers before and after degradation in response to redox stimuli (10, 20 μ m , and 100 μ m GSH) for 4 or 8 h. Figure S5. (A) Gel shift assay of supercoiled (S.C.) DNA released from the polyplexes. Polyplexes were incubated at indicated GSH concentrations (0, 10 μM, 20 μM, 100 μM, and 5mM) in the presence of 0.3 M sodium chloride. Weight ratio of 0.6 or 1 was selected for the polyplexes.

  9. (B) Expansion kinetics of the OEI-SeSex/DNA and OEI-SSx/DNA polyplexes (weight ratio = 10) at 37°C in the presence of indicated GSH concentrations (10 μM and 20μM).

  10. Dual programmed stimuli responsiveness 0 GSH生理条件下，pH=7 10um GSH 肿瘤内部, pH=6.5 5 mm GSH 细胞内部, pH=5.5 Zeta Potential 26.2 27 15.5 10.2 10.0 HA-SS-COOH在pH>6.5时稳定，构成双分子层，小于6.5时，就断裂，当PH到达5.5时，两个键都断，聚合物都没有压缩DNA的能力了。 C) Zeta-potential variation of different polyplexes at indicated GSH concentrations (0, 10 μ m , and 5 m m ). **: p < 0.01.

  11. morphology TEM 130nm 杂乱无章， 脱离球形结构 380nm 380nm 变化明显 DLS 没有明显变化

  12. 用荧光的方法监测在不同的还原性条件下质粒的包裹及解离情况用荧光的方法监测在不同的还原性条件下质粒的包裹及解离情况 Fluorescence resonance energy transfer (FRET) imaging 证实了当载体携载DNA到达肿瘤区域时，DNA有微弱的解离，但当到达细胞质内 (pH=5.5)，就全部解离了。 • 用CLSM的方法监测细胞内部聚合物的情况 说明两个问题1.Se-Se连接的载体在细胞内培养2h后，就会发生DNA解离，DNA可以完全释放出来，但是没有还原键的体系里，在这个时间下是不可能断裂的，也许需要长的时间。2.DPH和DOS体系中荧光强度高，说明HA能有效压缩DNA，结合程度高，与前面结果一致。 DNA/PEI(无还原键) DNA/PEI/HA(无还原键)

  13. Cell culture and viability assay Figure S10. Cell viability assays of polyplexes with different OEI-SeSex/DNA weight ratios, the weight ratio of HA-SS-COOH (HA)/DNA is 2 or without HA shielding, in (A) HepG2cells and (B) B16F10 cells（HA receptor CD44 positive cells） Cell viability assays of the OEI-SeSex, OEI, and PEI polymer in HepG2（好）cells (C). (n = 6).受体细胞的筛选

  14. In vitro transfection of pEGFP and pGL3 plasmid 不同聚合物转染后荧光素酶在HepG2细胞中的表达时聚合物与质粒比例的调整及血清含量的筛选

  15. attributed to the synergistic interplay between redox stimuli-deshielding of HA-SS-COOH and a redox-degradable OEI-SeSe x core, which leads to viruslike hierarchical unpacking and release of DNA. 不同聚合物转染后荧光素酶在HepG2细胞中的表达 与上面结果一致，DOS转染效率最高 EGFP荧光蛋白在B16F10 细胞的转染效率

  16. In vivo gene transfection study 在荷实体瘤小鼠体内注射携载基因的载体，观察肿瘤部位的变化情况 绿色荧光蛋白 牛乳糖蛋白 在各种器官内运用荧光素酶检测法监测基因表达情况， 1.DOS载体转染效率高，2.该载体可以做到区域特异性表达，对其他正常组织伤害较小。 深蓝绿色区域代表牛乳糖蛋白表达的细胞

  17. Summary 1.构建了一种在还原性条件下可以高效地分步解离和释放基因的类病毒载体。 2.该聚合物载体可以在正常生理条件下保持稳定，在肿瘤内部微弱解离，而在强还原性的细胞内部完全分解。 3.该聚合物载体具有如下特点：有效识别靶细胞，对正常组织伤害小，逐步解离和释放质粒，体内体外基因转染效率都异常高。

  18. 附录：

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