RNA/Peptide双重适配体介导腺病毒/阿霉素肿瘤靶向递药系统构建及抑癌机制研究

Tumor-targeted therapy focused on the subjects that if the drugs were concentrated directly in the tumor site and that if the cancer cells excluding the formal cells were killed thoroughly, in which the key problems are the high targeting efficiency and the high effects of therapeutical agents. RNA and peptide aptamers are the small molecule nucleotide fragments with high affinity and highly specificity which might be designed according to the tumor membrane epitope. In the previous study, we found that the adenovirus-mediated tumor suppressor gene of PTEN and the chemotherapy drug of doxorubicin have synergistic effect on inhibition of tumor cells in vitro. Therefore, this project intends to prepare the complexes of Ad5-PTEN and doxorubicin (ADDP-Ad5) targeted to prostate cancers by aptamers of A10-3.2 and DUP-1, in which doxorubicin could bind onto the stem part of double-strand nucleotides with CG sequences in the RNA aptamer and the Ad5-PTEN was modified with the RNA aptamer of the prostate-specific membrane antigen (PSMA) positive cancer and the peptide aptamers of the PSMA-negative cancer by taking the functional PEG as the arm's linkers. Therefore, this tumor-targeted drug delivery system combined the gene therapy and the chemical therapy as a whole body. We will clarify the method of aptamers conjugating covalently with adenovirus, investigate their toxicity, targeting effect and apoptosis effect in vitro using the confocal laser and flow cytometry technology. We will further demonstrate the bio-distribution in vivo, tumor-targeting effect in vivo and tumor-inhibition effect by iodine tracer method, chemical luminescence living imaging, Q-PCR, etc. Moreover, some studies will be carried out to discover the mechanism on the tumor inhibition. It could supply an important guiding role and practical value for developing the molecular target therapy of prostate cancers.

肿瘤靶向治疗致力于将药物定向浓集至肿瘤部位，定向彻底消灭癌细胞。高效靶向性、药物有效性是关键。RNA和Peptide适配体是一种可基于肿瘤膜表位，具有高亲和力、高特异性的小分子核苷酸片段。我们已证明腺病毒介导的抑癌基因PTEN与化疗药阿霉素体外具有协同抑瘤效应。因此，本课题拟用功能性PEG为连接臂，将基于前列腺特异性膜抗原（PSMA）阳性癌的RNA适配体和PSMA阴性癌的Peptide适配体同时连接到携载抑癌基因PTEN的腺病毒表面，再利用阿霉素能与RNA双链CG序列牢固结合的特点，将基因治疗和化学治疗巧妙地整合为一体，制备由RNA和Peptide双重适配体介导的既能靶向PSMA阳性癌又能靶向PSMA阴性癌的腺病毒/阿霉素肿瘤靶向给药系统。阐明适配体与腺病毒偶联方法、体外靶向性、体外促凋亡效应、体内分布、肿瘤靶向性和抑瘤效应，探讨其抑瘤机制。对发展前列腺癌分子靶向治疗具有重要的指导作用。

适配体DUP-1能靶向于PSMA阴性的前列腺癌细胞，适配体A10-3.2能靶向于PSMA阳性的前列腺癌细胞，携载抑癌基因PTEN的重组腺病毒Ad5和阿霉素都能有效抑制前列腺癌，本研究探索了经过双重适配体修饰的并携载有盐酸阿霉素的重组腺病毒给药系统的合成与检测方法，并对给药系统的体内和体外毒性进行检测。实验进行了腺病毒的构建及生产，目标复合物A10-3.2(Dox)/DUP-1－PEG－Ad5 (ADDP－Ad5)的制备，采用SDS-PAGE电泳检测Ad5的PEG化，荧光分光光度计检测PEG化的Ad5与双适配体以及盐酸阿霉素的连接，然后用前列腺癌细胞LNCaP（PSMA+）、前列腺癌细胞PC-3（PSMA-）、人正常前列腺细胞WPMY-1和乳腺癌细胞MCF-7检测给药系统对各个细胞的生长抑制率，最后用昆明小鼠进行体内毒性实验。结果得到具有一定滴度的重组腺病毒原液，重组腺病毒进行PEG化后与双适配体以及盐酸阿霉素连接获得目标给药系统ADDP-Ad5；给药系统对LNCaP细胞和PC3细胞都有抑制作用，但是对LNCaP的作用更显著；小鼠急性毒性实验的结果表明高剂量的给药系统对肝脏和心脏有轻微的毒性作用。上述结果表明用酰化反应能将双适配体与重组腺病毒进行连接，并且可用SDS-PAGE电泳和荧光分光光度计进行检测，给药系统对前列腺癌具有明显的抑制作用，且具有适配体靶向性，对小鼠的肝脏和心脏仅造成轻微毒性。.血管靶向给药系统不仅可以阻断肿瘤营养物质的供应，还可以在一定程度上阻止肿瘤的迁移和侵袭，已经逐渐成为一种潜在的胃癌治疗方式。目前，我们已经制备了一种由双功能肽GX1介导的阴离子脂质体-腺病毒靶向载药系统(GX1-Ad5-AL)，其腺病毒中已经导入了了抑癌基因PTEN。考察该给药系统对胃癌细胞系SGC-7901和人血管内皮细胞HUVEC的增殖抑制、迁移抑制作用和选择特异性。结果显示GX1-Ad5-AL靶向给药系统对SGC-7901 和 HUVEC细胞的增殖抑制率均高于单独使用GX1或Ad5-AL；GX1-Ad5-AL对细胞迁移的抑制明显高于GX1–AL和Ad5-AL；激光共聚焦的结果中，连接有GX1的药物的摄取效果明显更好。因此，GX1能介导腺病毒阴离子脂质体携载抑癌基因PTEN，靶向胃癌血管内皮细胞，高效低毒的发挥肿瘤抑制作用。