基于PAMAM联合包载siRNA与PTX靶向给药系统的构建

发布时间：2019-01-21 19:23

【摘要】：目的：本文以PAMAM为载体，以具有主动靶向功能的HA为被膜材料，构建PAMAM/HA用于递送靶向VEGF mRNA的siRNA，诱导RNAi，通过抗血管生成控制肿瘤生长。由于单一的抗血管生成疗法无法快速、完全消除肿瘤及PAMAM/HA无法完全避免体内RES吞噬作用的缺陷，在上述复合物的基础上，将HA进行PEG化后包覆PAMAM用于联合递送靶向VEGF的siRNA和抗肿瘤化疗药物PTX，构建兼具靶向及长循环双重功能的联合载药系统siRNA/PTX/PAMAM/HA-PEG，以期增强体内抗肿瘤作用，实现低毒高效的抗肿瘤效果。方法：本文构建了不同HA包覆修饰量的siRNA/PAMAM/HA（SPH）复合物，琼脂糖凝胶电泳进行siRNA阻滞实验，，激光粒度分析仪测定粒径及电位，MTT法测定细胞毒性，采用荧光显微镜和流式细胞仪对复合物的摄取进行考察，从而筛选出最佳的HA包覆量。通过酰胺化反应合成HA-PEG，经纯化后通过1H-NMR对产物结构进行表征。通过静电吸附及物理包埋法构建siRNA和PTX共递送的复合物载药系统siRNA/PTX/PAMAM/HA-PEG（SPPG），通过siRNA阻滞实验、稳定性实验、粒径、电位、形貌和包封率等对复合物进行表征。MTT法测定细胞毒性，流式细胞仪、HPLC定量考察复合物的摄取情况，RT-PCR测定复合物的RNAi效果，通过流式细胞仪和共聚焦显微镜分别考察复合物的内吞机制和细胞内定位。采用小动物活体荧光成像技术观察复合物在体内的分布情况。结果：激光粒度分析仪测定结果表明，随着HA包覆比例的增加，SPH的粒径增加，zeta电位降低。MTT和细胞摄取实验表明，HA的包覆使复合物的细胞毒性下降；HA的包覆能增加细胞对该复合物的摄取，增强复合物的主动靶向性，并且当HA的包覆量为25%（电荷比）时，细胞摄取量最高。1H-NMR检测结果显示成功合成HA-PEG。siRNA/PTX/PAMAM（SPP）、siRNA/PTX/PAMAM/HA（SPPH）、siRNA/PTX/PAMAM/HA-PEG（SPPG）的粒径依次增大，电位降低。细胞实验表明，HA的包覆使细胞对复合物的摄取最高，RNAi效果最强；而HA-PEG的包覆摄取虽然比未有包覆的复合物低，RNAi效果二者却未有明显差异。摄取机理研究结果表明，三种复合物的细胞摄取均为能量依赖的转运过程，SP经网格蛋白途径入胞，SPH及SPG经网格蛋白及小窝蛋白多种途径入胞。动物实验表明，SPPH及SPPG均可不断向肿瘤部位蓄积，具有主动靶向功能；且SPPG能够在肿瘤部位获得较长时间的累积，具有一定的长循环作用。结论：本文以PAMAM为载体，通过合成HA-PEG，构建了毒性较低的用于siRNA及PTX共递送的载药系统SPPG，在保留HA主动靶向的基础上，兼备了长循环功能，将基因疗法与传统的化疗相结合，有望实现较高的体内抗肿瘤作用。
[Abstract]:Aim: in this paper, PAMAM was used as carrier and HA with active targeting function as membrane material to construct siRNA, which was used to deliver targeted VEGF mRNA to induce RNAi, to control tumor growth through anti-angiogenesis. Because the single antiangiogenic therapy can not be rapid, the tumor and PAMAM/HA can not completely avoid the defects of RES phagocytosis in vivo, on the basis of the above complex, In order to enhance the anti-tumor effect in vivo, PEG coated HA was used to deliver the siRNA targeting VEGF and the anti-tumor chemotherapeutic drug PTX, to construct a combined drug delivery system siRNA/PTX/PAMAM/HA-PEG, with both targeting and long circulation functions, so as to enhance the anti-tumor effect in vivo. To achieve low toxicity and high efficiency of anti-tumor effect. Methods: siRNA/PAMAM/HA (SPH) complexes with different amount of HA coating were constructed. Agarose gel electrophoresis was used for siRNA block assay. Particle size and potential were measured by laser particle size analyzer. Cytotoxicity was determined by MTT method. The uptake of the complex was investigated by fluorescence microscope and flow cytometry, and the best HA coating was obtained. HA-PEG, was synthesized by amidation reaction and the structure of the product was characterized by 1H-NMR. By means of electrostatic adsorption and physical entrapping method, a compound drug carrier system of siRNA and PTX was constructed by means of siRNA block experiment, stability test, particle size, potential. The morphology and encapsulation efficiency were used to characterize the complex. MTT assay was used to determine the cytotoxicity, flow cytometry and HPLC were used to quantitatively investigate the uptake of the complex, and RT-PCR was used to determine the RNAi effect of the complex. The endocytosis mechanism and intracellular localization of the complex were investigated by flow cytometry and confocal microscopy, respectively. The distribution of the complex in vivo was observed by in vivo fluorescence imaging of small animals. Results: the results of laser particle size analyzer showed that with the increase of HA coating ratio, the diameter of SPH increased and the zeta potential decreased. The results of MTT and cell uptake tests showed that the coating of HA decreased the cytotoxicity of the complex. The coating of HA can increase the uptake of the complex, enhance the active targeting of the complex, and when the encapsulation amount of HA is 25% (charge ratio), The results of 1H-NMR showed that the particle size of successfully synthesized HA-PEG.siRNA/PTX/PAMAM (SPP), siRNA/PTX/PAMAM/HA (SPPH), siRNA/PTX/PAMAM/HA-PEG (SPPG) increased in turn and the potential decreased. Cell experiments showed that the uptake of complex by HA was the highest and that of RNAi was the strongest, but that of HA-PEG was lower than that of uncoated complex, but there was no significant difference between the two groups. The results of uptake mechanism showed that the uptake of the three complexes was an energy-dependent transport process. SP was transfered through grid protein pathway, SPH and SPG via grid protein and fossa protein. Animal experiments showed that both SPPH and SPPG could accumulate to the tumor site continuously and had active targeting function, and SPPG could accumulate in the tumor site for a long time and had a certain long circulation effect. Conclusion: in this paper, a novel drug delivery system, SPPG, with low toxicity for siRNA and PTX delivery, was constructed using PAMAM as carrier and HA-PEG, as a carrier. The drug delivery system SPPG, could retain the active targeting of HA and possess long cycle function. The combination of gene therapy and traditional chemotherapy is expected to achieve higher anti-tumor effect in vivo.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R94

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