两性离子修饰的树状大分子纳米平台的构筑及其乳腺癌诊疗应用

Development of multifunctional nanoplatforms that have antifouling property and can simultaneously inhibit tumor metastasis is crucial for precision tumor theranostics. In this project, we aim to use zwitterion-modified generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers as a platform, modify RGD peptide onto their surfaces via a polyethylene glycol (PEG) spacer, and use the zwitterion and RGD-modified G5 dendrimers as templates to entrap gold (Au) or CuS nanoparticles (NPs). Dendrimer-entrapped Au NPs (Au DENPs) will be further labelled with radioisotope 99mTc for targeted dual mode precision single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging of human triple-negative breast cancer (TNBC) cells overexpressing alphavbeta3 integrin. Dendrimer-entrapped CuS NPs (CuS DENPs) will be further labeled with radioisotope 131I for tri-mode SPECT/CT/photoacoustic (PA) imaging and combinational photothermal therapy and radiotherapy of human TNBC cells. Simultaneously, nanoplatforms with dendrimer terminal remaining amines non-acetylated will also be used as vectors for compaction of hypermethylated in cancer 1 (HIC1) gene, thereby allowing for effective inhibition of lung metastasis of TNBC. Compared with the related technologies reported in literature, we modify dendrimers simultaneously with zwitterion and RGD peptide to endow the particles with excellent antifouling property, decreased uptake by reticuloendothelial system, and improved targeted tumor penetration ability. On the other hand, we also employ the combinational gene therapy approach, which is beneficial not only for the cancer cell killing but also for the simultaneous inhibition of cancer metastasis. Overall, our project has prominent novelty and advantages.

发展具有抗污性能和抗肿瘤转移的多功能纳米平台对肿瘤的精准诊疗至关重要。本项目拟采用两性离子修饰的第五代聚酰胺胺树状大分子为平台，在其外围通过聚乙二醇间隔基修饰RGD靶向肽，以其为模板包裹合成金或硫化铜纳米颗粒。包裹纳米金的树状大分子外围进一步标记核素99mTc，用于表面高表达alphavbeta3整合素的三阴性人乳腺癌细胞的靶向SPECT/CT双模态精准成像；包裹纳米硫化铜的树状大分子外围进一步标记131I，用于乳腺癌细胞的SPECT/CT/PA三模态成像和光热/放疗联合治疗。树状大分子末端氨基未乙酰化的纳米平台，还将作为载体压缩肿瘤高甲基化基因1，抑制乳腺癌的肺转移。和文献方法相比，本项目采用两性离子和RGD肽同时修饰树状大分子，赋予纳米平台抗污性能（有助于减少网状内皮系统吞噬）和提高靶向肿瘤穿透能力；采用联合的基因治疗方法有助于在杀死癌细胞的同时并抑制其转移，因而具有突出的创新和优势。

发展具有抗污性能和抗肿瘤转移的多功能纳米器件对肿瘤的精准诊疗至关重要。本项目依据实验设计，完成了具有抗污性能树状大分子的基因载体设计、双模态造影剂构建及诊疗一体化纳米平台的制备，并成功用于肿瘤模型（包括三阴性乳腺癌）的精准诊断、基因治疗及不同组合的联合治疗及诊疗一体化：.1）.筛选出了具有较好抗污性能的1,3-丙砜(1,3-PS) 两性离子化表面修饰，进一步修饰RGD靶向肽，获得的树状大分子包裹的纳米金颗粒(Au DENPs)具有长循环时间，减少的网状内皮系统吞噬，增强的肺癌转移模型CT/MR双模态诊断；合成了两性离子巯基-N,N-二甲基-半胱胺-羧酸甜菜碱修饰的树状大分子包裹CuS纳米颗粒，并负载化疗药物阿霉素(DOX)用于肿瘤的响应性化疗/光热联合治疗；合成了1,3-PS和RGD肽修饰的树状大分子包裹的CuS纳米颗粒并负载HIC1 pDNA，实现了PA成像导引的三阴性乳腺癌模型的光热/基因联合治疗，并有效地抑制了肿瘤肺转移。.2）.合成了两性离子羧酸甜菜碱和溶酶体靶向试剂吗啡啉-聚乙二醇修饰的Au DENPs，可作为血清增强型纳米载体，负载肿瘤HIC1 pDNA用于抑制癌细胞的迁移；构建的Au DENPs载体可负载CpG、PD-L1 siRNA或PD-1 siRNA用于肿瘤细胞(或T细胞)的递送，实现肿瘤的免疫治疗；设计了功能性树状大分子或核壳树状大分子，在其表面共价连接或内部物理包裹DOX，通过肿瘤微环境的pH响应性，实现癌细胞的增强化疗，也可同时包裹DOX和负载基因抑制剂，实现癌细胞的化疗/基因联合治疗。 .3）.除了完成上述标书指定任务外，我们构建了双载药的树状大分子/碳点纳米体系，实现了超声增强的多药耐药肿瘤模型的荧光成像和化疗；研制了第五代树状大分子、核壳结构树状大分子及第三代含磷树状大分子-铜络合物用于放疗增敏的肿瘤化疗与MR成像、原位脑胶质瘤的化学动力学治疗和MR成像以及超声增强的肿瘤MR成像和化疗。.总之，项目已超额成功完成规定指标。本研究为探索新型的、复合型、多功能纳米药物体系应用于肿瘤的精准诊断和诊疗一体化提供了借鉴，具有重要的理论和现实意义。.