基于legumain的双靶向去氢骆驼蓬碱前药系统构建及其抗肝肿瘤活性研究

Harmine is a uygur anti-cancer drug, it has effect on liver tumor, but it has no preparations now because of its toxic to nerve system. In this study, a new harmine dual-targeted prodrug system was prepared. Legumain is a highly conserved lysosomal/vascuolar cysteine protease, which is primarily expressed in tumor cell and tumor associated macrophages. Also, Legumain is reported to cleave C-terminally aspartate(Asn) residues at pH below 5. Furthermore, Magnetic nanoparticles has many advantages in drug delivery，it can target to tumor when some magnetic field is put in tumor in vitro. So according to those backgrounds, a kind of dual-targeted harmaine prodrug system was prepared, in which harmine was conjugated with AAN peptide because the carboxyl group of AAN would react with amine of harmine , and then AAN peptide was conjugated with magnetic nanoparticles. When this kind of harmine-AAN-magnetic nanoparticles complex was given, harmine had no side effects because its structure changed and harmine would accumulate in tumor with the help of magentic targeting and legumain targeting. After harmine arrived at tumor, harmine released and took its anti-tumor effect because legumain hydrolyzed AAN peptide in tumor. This system had dual-targeted effect, whcih would be targeted into tumor because of legumain and magnetic nanoparticles and then the side-effect of harmine would be decreased too.

去氢骆驼蓬碱为新疆特有维吾尔习用药，其能有效地治疗肝肿瘤和消化道肿瘤等多种肿瘤，因存在神经系统毒副作用，其应用受到限制。本课题在前期基础上，基于肿瘤细胞表面和肿瘤相关巨噬细胞表面高表达legumain酶，且legumain酶对AAN肽序列有专一水解作用的特点，结合磁纳米粒的靶向性，设计一种去氢骆驼蓬碱双靶向前药系统，该系统利用去氢骆驼蓬碱9位仲胺基可修饰且为活性位点的特点，将去氢骆驼蓬碱与AAN肽偶联，再在AAN另一端偶联磁纳米粒，生成去氢骆驼蓬碱-AAN-磁纳米粒复合物，该复合物在体内，因去氢骆驼蓬碱结构发生变化，导致其不会产生神经系统毒副作用，类似于前药，并借磁纳米粒和legumain的双重靶向到达肿瘤细胞，在肿瘤细胞，肿瘤表面的legumain酶将去氢骆驼蓬碱与磁纳米粒断裂，药物进入肿瘤细胞发挥作用。该系统利用分子靶向和磁靶向双重靶向作用，结合前药原理，有效达到高效减毒效果。

本研究围绕项目计划书内容，对项目计划书内容作了部分调整。首先对去氢骆驼蓬碱的毒性进行研究，考察了其毒性和减轻神经系统毒性的潜在药物，首次发现，去氢骆驼蓬碱除神经系统毒副作用外，还存在心血管系统毒副作用，中枢抑制药物可抑制或减轻其神经系统毒副作用。在此基础上，课题组对去氢骆驼蓬碱进行结构修饰，得到去氢骆驼蓬碱衍生物，进一步地，将该衍生物与CGGAAN肽进行偶联，并将CGGAAN肽与磁纳米粒进行偶联，得到去氢骆驼蓬碱CGGAAN前药系统，并分别考察了去氢骆驼蓬碱衍生物和前药系统对肝肿瘤细胞的抑制作用，结果发现，初步合成的前药系统的抗肿瘤活性并不理想，因此课题组一方面继续合成了活性更强的去氢骆驼蓬碱衍生物7，以及衍生物7与CGGAAN偶联的前药系统，并继续进行活性评价。另一方面，课题组在去氢骆驼蓬碱衍生物7的结构基础上，进一步将去氢骆驼蓬碱衍生物7偶联NO供体化合物，得到去氢骆驼蓬碱NO供体偶联物，发现与去氢骆驼蓬碱原形药物相比，NO供体偶联物抗肿瘤作用更强，毒性更小，在体内能通过释放NO以及去氢骆驼蓬碱的抗肿瘤作用达到协同增效的作用。此外在原计划的基础上，课题组还做了四方面工作，一是延伸性地制备了CGGAAN肽和线粒体靶向肽双修饰的去氢骆驼蓬碱脂质体并进行体内外性质考察，结果表明双修饰脂质体抗肿瘤作用更强，毒性减小。第二，采用阿霉素为模型药，构建阿霉素－AAN－磁纳米给药系统，结果表明该给药系统具有缓释、靶向、毒性减小的特点。第三，构建了载去氢骆驼蓬碱的牛血清白蛋白纳米颗粒，进而研究了去氢骆驼蓬碱对脑胶质瘤的体内外作用。第四，对骆驼蓬种子中12个有效成分包括去氢骆驼蓬碱的抗肿瘤作用机制采用网络药理学和分子对接技术进行了探索，发现PI3K－AKT信号通路是其抗肿瘤作用的重要信号通路。本研究对Legumain酶靶向的给药系统进行了多方面探索，进而合成出活性更好的去氢骆驼蓬碱衍生物NO供体偶联物，并对去氢骆骆驼蓬碱的多种纳米载药系统进行了研究，探索了减轻去氢骆驼蓬碱毒性的多种方法，为去氢骆驼蓬碱抗肿瘤作用的开发奠定了基础。