肿瘤微环境响应性新型磷酸钙纳米载体的构建与抗肿瘤研究

Calcium phosphate(CaP) nanocarriers have been challenging in the practical application because of its poor stability, low transfection efficiency and unsatisfactory drug controlled release. Aiming at these issues, this project intends to use block polymer and calcium phosphate nanoparticles to construct a novel CaP nanocarrier for co-delivering bevacizumab and TGF-β1 siRNA. In order to improve the stability of the nanocarriers in vivo, the disulfide bond cross-linked interlayer will be used to bundle up CaP core in which entraps siRNA. Through design of multiple responses to tumor microenvironments, the nanocarriers will be obtained targeted drug controlled-release property, that is the carrier-loaded drugs will sequentially released into the tumor matrix and tumour cells respectively. The monoclonal antibody Bvz will be attached to the polymer via MMPs sensitive peptides. When the nanocarriers arrive in the tumor stroma with high expressed of matrix metalloproteinases, bevacizumab is cleaved off from the nanocarriers and binds with VEGF for anti-angiogenesis. Once the nanocarriers are uptaked by tumour cell, CaP core will be dissolved and the acid-sensitive bond that conjugates siRNA will be broken in the acidic environment of lysosomes. As the disulfide cross-linked interlayer opened in the reductive environment of intracellular, the entrapped siRNA will be released for gene therapy by silencing of TGF-β1 gene.The aim of this project is to develop long-circulating and targeted drug controlled-release calcium phosphate nanocarrier which can both response to tumor matrix and tumor cell, and hope to open a new window on anti-angiogenic therapy and gene therapy for synergistic anti-tumor therapy with calcium phosphate nanocarriers.

针对磷酸钙纳米载体存在稳定性差、转染效率低和可控释药不理想等问题。本项目拟用嵌段聚合物形成的二硫键交联捆绑磷酸钙纳米粒子构建新型磷酸钙纳米载体，实现有效控制磷酸钙晶体生长，提高载体稳定性。通过肿瘤微环境的多重响应性设计，赋予载体靶向药物控释性能，使其负载的Bvz和TGF-β1 siRNA按顺序分别释放到肿瘤基质和肿瘤细胞中。Bvz以MMPs敏感肽段连接到聚合物上，当载体进入肿瘤基质后，被MMPs酶切，使Bvz从载体上脱落并结合VEGF，产生抗血管生成作用；载体被肿瘤细胞摄取后，响应胞内还原及酸性微环境，二硫键交联层被打开、磷酸钙内核溶解和链接siRNA的酸敏化学键断裂，释放出其包载的siRNA并沉默TGF-β1基因，进行基因治疗。通过本项目研究，制备稳定高效且具有肿瘤基质和肿瘤细胞微环境多重响应性靶向药物控释磷酸钙纳米载体，为利用其开展抗血管生成疗法和基因疗法协同抗肿瘤研究提供新思路。

恶性肿瘤的发生发展是多基因及多因子参与的多步骤过程，以肿瘤多个致病靶标进行多靶点级联治疗，顺序产生多种药理活性，可以减少毒副作用和提高治疗效果。磷酸钙（CaP）纳米载体具有良好的生物相容性、生物降解性和对核酸亲和能力强等优点，可作为多种类型药物的载体。但是CaP载体存在稳定性差、转染效率低和可控释药不理想等问题。微环境响应性聚合物载体不但可以作为模板矿化CaP而形成稳定的聚合物/CaP杂化纳米粒子，而且易于实现影像可视化的药物递送及药物在靶点部位的可控释放。为此，首先，本项目制备了一种基于癌细胞内pH响应和生物可降解性聚(氨基酸) 嵌段聚合物的纳米递送系统（SPIONs/PTX-PDPL）用于肿瘤的诊疗一体化。实现在癌细胞的弱酸性溶酶体中迅速释放出化疗药物而高效诱导癌细胞凋亡，并通过MR成像灵敏地检测到少量的癌细胞。其次，设计合成了一种ROS敏感二硫缩酮键（Tk）偶联化疗药物的两亲性聚合物PTkCPT。聚合物纳米胶束的亚表层富含羧酸，作为模板参与CaP矿化，并有效负载siRNA，从而制备了一种新型且稳定的PTkCPT/CaP/siRNA杂化纳米药物。荷瘤小鼠的治疗实验结果表明该CaP杂化纳米药物不但实现了协同高效级联治疗肿瘤，而且对多数正常器官基本没有毒副作用。综上，本项目的研究工作提供了一种MRI可视化治疗肿瘤的纳米药物；制备了一种新型的稳定高效且可药物控释的磷酸钙纳米载体，为肿瘤的高效低毒治疗提供了一种级联协同治疗策略。

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