多功能直链淀粉纳米载体用于肝癌基因治疗的影像学评估

Gene therapy is the promising therapeutic strategy,and the choice of nanocarrier for gene delivery is critical. In the attempt to overcoming the limitations of viral vector and polymeric vector for gene delivery, we aim to solve two key technical problems, the poor biodegradation and toxicity of nanocarriers and lack of sensitive indicators of evaluating the response to gene therapy vectors in vivo. Based on our previous study on natural macromolecules of amylose as gene delivery carrier, this study is to construct folate receptor targeting amylose nanoparticle loaded with superparamagnetic iron oxide nanoparticle (SPIO) for survivin small interfering RNA (siRNA) delivery. In our preliminary experiment, the targeting ability of HCC HepG2 cells and the visibility in magnetic resonance imaging (MRI) of this amylose nanoparticle have been verified.The cytotoxicity and transfection efficiency of siRNA-survivin nanoparticles are studied in cultured HepG2 cells. Survivin mRNA expression of HepG2 cells cultrured in vitro is detected by reverse trsanscription-polymerase chain reaction (RT-PCR), and survivin protein expression is detected by Western blot. The HepG2 cell growth and apoptotic rate are assessed. T2 weighted imaging (T2WI) and T1 weighted imaging (T1WI) are performed to investigate the demonstrating ability in a 3 Tesla (3T) MRI and the signal intensity alteration. The targeting ability of siRNA-survivin nanoparticles in the nude mice models bearing HCC is also explored.The MR imaging and pathology results will be obtained and analyzed, furthermore, texture parameters derived d from MR images are correlated with gene expression alteration. This study is to establish a new HCC targeting nanoparticle carrying both survivin siRNA and SPIO for inhibiting HCC growth, which can be observed with 3T MR equipment in vitro and in vivo. Texture analysis of MR images is performed to evaluate the gene therapeutic effect in vivo.

基因治疗是肝细胞癌具有前景的新型治疗模式，输送基因的纳米载体是关键。本研究突破病毒及人工合成聚合物载体的限制，解决纳米基因载体生物降解性差及疗效在体评价的指标不敏感两个关键技术问题。项目利用已有的天然高分子纳米载体研究基础，制备叶酸修饰的负载survivin小干扰RNA（siRNA）及超顺磁性氧化铁（SPIO）的多功能直链淀粉纳米载体，实现纳米载体的生物相容性和可降解性；采用纹理分析的方法，在体内实验定量分析纹理参数与基因表达的关联性，实现基因水平的疗效评估。本项目采用影像学评估该载体对肝癌细胞的靶向作用，细胞与分子生物学实验检测其沉默肝癌细胞survivin基因的效果，在活体水平观察纳米载体对肝癌动物模型的靶向性及基因输送效果，利用MRI可视化观察基因输送过程，提取纹理参数定量评价其疗效。本研究为肝癌提供一种新的基因治疗手段，阐明采用MRI纹理分析方法评价基因治疗疗效的价值。

设计并制备一种SP94与直链淀粉耦合的磁性探针，用于靶向survivin-siRNA递送和双模式成像。该探针（CSP）由直链淀粉（CA）、铁氧体磁性纳米粒子（SPIO）和与SP94肽结合的survivin-siRNA组成。SP94肽与肝细胞癌(HCC)细胞具有特异性结合。当siRNA survivin与纳米载体结合后，可表现出良好的基因释放行为。体外细胞毒性实验结果表明聚合物胶束具有良好的生物相容性。利用荧光成像和MRI成像，通过体内/外成像系统和MRI直接观察CSP和siRNA释放在体内的生物分布。由于肿瘤组织中存留了高剂量的CSP，用CSP@siRNA-SP94治疗的Huh-7肿瘤被完全根治。Western blot用于检测相关蛋白表达。我们发现caspase-3和survivin在肿瘤组织和Huh-7细胞系中下调。此外，CSP@siRNA-SP94通过靶向基因治疗肝癌，在体内介导增强肿瘤抑制作用。我们还通过提取肝癌区域的MR纹理参数，构建无创预测survivin表达水平的回归模型，筛选该纳米载体的目标人群。因此，用于双模式成像和基因治疗的靶向纳米药物在肿瘤治疗中的控制基因传递和临床诊断方面具有广阔的应用前景。