环境响应载体材料：跨膜型细胞因子基因的输送和调控

A structure invertible polymer, PEG-modified dendrimers which to retain and exploit the properties of both PEG and low generation dendrimer vectors was synthesized. The M-PEG was modified with cysteamine via CDI-mediated coupling reaction to introduce disulfide bond into the M-PEG terminal. The M-PEG-S-S was then conjugated with an amine-reactive, heterofunctional linear PEG to give M-PEG-SS-PEG (PSP). Finally, the hydroxyl groups at the terminal M-PEG-SS-PEG were reacted with primary amines of G2 through CDI-mediated coupling reaction to yield M-PEG-SS-lPEG-G2 (PSPG). It possessed an interior hydrophilic, a non-fouling M-PEG arm and low-generation dendrimer terminals that are linked by sensitive disulfide linkers. Our design consideration was that while dendrimer interior provide cavities, and the surface primary amines are able to interact with DNA by electrostatic force. The neutral M-PEG part not only provides a shielding layer to prevent non-specific serum interactions after structure inversion. M-PEG also offers a branched network to assemble low generation derndrimer to mimic high generation dendrimers through responsive disulfide bonds in order to enhance endosomal escape capability. Transmembrane-cytokine gene was carried by PSPG to the tumor cells. Here, the transmembrane (TM) fragment and cytokine gene was genetically engineered fusion gene, which incorporating the cytokine gene on the surface of tumor cells. It would permit the TM-cytokine gene to passively anchor onto the cell membranes and constructed the expression vector of TM-cytokine gene. It was mediated by the stimulation of cellular immunity, regulation of secretion of cellular factors and inhibition tumor growth.

本研究用多臂聚乙二醇(M-PEG)为内核，通过双硫键桥臂链接外侧低代数阳离子材料合成具有环境响应性的基因载体材料。当材料外层带正电荷的阳离子材料与带负电荷的细胞因子基因结合后，则发生结构翻转，使其嵌入M-PEG内臂的空隙中，形成一个纳米微球。载体所携带基因是跨模型细胞因子重组基因，其由跨膜区和功能区组成，跨膜的TM片段用于肿瘤细胞膜表面的“锚定”作用，而功能域部分（如：IFN-α）则用于激活和活化T-细胞等，通过对免疫细胞的激活引起IFN-α、IL-2、GM-CSF和G-CSF等细胞因子的分泌。当载体材料携带跨模型-细胞因子基因接近肿瘤组织时，由于微环境pH的变化，连接内核和阳离子的双硫键桥臂发生断裂，使内侧的阳离子部分及携带的基因被输送入肿瘤组织中进行释放。未进入组织的微球再次进行体循环，进行二次输送，以实现对肿瘤组织的数次释药，多重刺激，产生自体免疫，并进行肿瘤的免疫治疗。

1）构建了具有环境响应性材料，研究采用多臂聚乙二醇(M-PEG)为内核，通过易生物降解的键桥臂连接外侧低代数阳离子材料组成载体材料。当材料外层带正电荷的阳离子材料与带负电荷的细胞因子基因结合后，发生结构翻转，使其嵌入M-PEG内臂的空隙中，形成纳米微球。此时M-PEG由内核转为外层，形成一个保护外壳，易于微球在体内循环。当微球接近肿瘤组织时，由于微环境pH的变化，链接内核和阳离子的桥臂发生断裂，使内侧的阳离子部分及携带的基因被输送入肿瘤组织中进行释放。.2）通过生物学技术，制备编码跨膜区片段特异性引物（Transmembrane, TM）,经PCR技术扩增，用剪接重叠延伸法制备多种跨模型-细胞因子重组融合基因。构建质粒为：pDoubleEx-SEA-TM， pDoubleEx-SEA-TM-IFNγ， pDoubleEx-SEA-TM-IL12A， pDoubleEx-SEA-TM-GM-CSF， pCDNA3.1-IFNγ， pCDNA3.1-IL12A， pCDNA3.1-GM-CSF.3）在体外细胞学研究中，以多种细胞为研究模型，对材料携带核酸对肿瘤细胞的生长周期和凋亡的影响，特别是核酸分子进入细胞后免疫因子，特异蛋白和siRNA的变化进行研究。.将载体材料携带重组基因后进行转染，提取肿瘤细胞膜，与C57小鼠的腹腔原代巨噬细胞细胞。分别进行M1和M2型标示分子的分型，同时进行相关效应细胞因子的表达的研究。.对相关跨膜因子及免疫因子作了测定，进行炎症相关因子：TNF-α、IL-1β、IFN-γ、IL-6等的检测。研究结果表明：所设计的载体材料可以与 TSA 质粒通过静电作用形成纳米微粒，可有效地进入细胞。通过 Western blot 实验证实 TSA 质粒可在肿瘤细胞内表达出 TSA 蛋白。.4）动物实验结果表明，载体材料介导的的 TSA 质粒可在体内肿瘤细胞表达TSA 蛋白，使 CD4/CD8 处于正常范围，同时上调 TNF-α、IFN-γ，激活机体的免疫功能。流式实验表明：polymer/TSA+HPC/GFT 对 CD8 T 淋巴细胞的增值产生较大的影响。且载体材料携带的 TSA 治疗组的小鼠 CD4/CD8 均处于正常小鼠的免疫指标。 Elisa实验也表明：小鼠淋巴细胞分泌 α-TNF、γ-IFN 含量可表明材料携带的 TSA 对免疫系统的调节作用。