磷脂/磷酸钙壳核组装序贯给药克服脑胶质瘤细胞耐药研究

The down-regulation sequential administration of phospholipid and calcium phosphate assembly hybrid nanoparticles to overcome glioma cells drug resistance. Based on the growth of glioma cells heterogeneity and structural heterogeneity, existing local sustained-release chemotherapy has disadvantage in the ease of use of the drug clinically, drug permeability, controlled-release profile. Calcium phosphate (CaP)/polymer nanospheres have been studied for use in drug delivery systems. In this study, two different mechanisms of temozolomide (TMZ) and paclitaxel (PTX) were used as model drugs. We developed a new strategy to prepare multifunctional nanoparticles of biodegradable polymers. TMZ was adsorbed on the surface of porous CaP. Polymeric micelles loading PTX used to modulate the size of CaP particles were chosen as a base material because this system has already shown long-circulating properties and has been successfully utilized for systemic administration of small molec-ular drugs including anticancer drugs. Phospholipid (PL)-PEG-VEGF was chosen as a base material to prepare calcium phosphate/phospholipid(PL)-PEG-VEGF hybrid porous nanospheres (CaP/PL-PEG-VEGF NSs) for anticancer drug delivery. PEG has been given US Food and Drug Administration (FDA) approval for use in a variety of biomaterials applications and phospholipids are one of the main ingredients of cell membranes. Calcium phosphate has excellent biocompatibility, biodegradability and very low toxicity. Application Percol discontinuous density gradient centrifugation glioma cell lines U87 subsets, clarify the different cell subset to sequential administration nanoparticles. Constructing two-phase composite temperature-sensitive gel sequential releaseing nano-drug delivery systems,relalized the effect of hemostasis immediately and automatically fitness of the tumor cavity forming. The glioma interstitial administration nano-gel sustained release reservoir, following released of temozolomide and paclitaxel killing glioma cells in different cycle cell populations, thereby reducing the occurrence of drug resistance. The project will design different size and morphology of nanoparticle, jointing low-frequency ultrasound supporting role to improve the drug tissue permeability. Mimicking glioma growth microenvironment, developmenting tumor cells in vitro 3D culture model, evaluating methods of drug permeability, and discussing the parameters of nanoparticles influence drug permaability. Exploreing the inhibition of tumor cell by ion channel mediated nanoparticles, reveals the apoptotic mechanism mediated by nanoparticles targeted tumor cells mitochondrial ion channels. The results provide some theoretical basis for a reasonable choice for design and development of ion channels or mitochondria-targeted new drug delivery system.

针对现有局部缓释化疗系统在药物渗透性、控释及临床易用性方面的不足，拟选用不同作用机理的替莫唑胺（TMZ）和紫杉醇（PTX）作模型药，发展以磷脂-PEG和PTX为核、磷酸钙和TMZ为壳的可降解纳米载体。分离脑胶质瘤细胞系U87亚群，验证各细胞对纳米粒的敏感性。构建复合温敏凝胶序贯释药的给药系统，实现脑胶质瘤间质给药凝胶纳米缓释储库，使植入过程能一步达到止血、自动适合瘤腔成形的效果。纳米粒从凝胶储库中扩散渗透，先后释放TMZ和PTX而杀死脑胶质瘤细胞不同周期的细胞群，从而降低耐药性。本项目将从纳米粒粒径大小，联合低频超声辅助作用改善药物组织渗透性。设计肿瘤细胞三维培养模型，建立渗透性评价方法，优化纳米粒特性对渗透性的影响。探讨离子通道介导的纳米粒对肿瘤细胞的抑制作用；揭示离子通道介导的线粒体为靶点的纳米粒对肿瘤细胞的凋亡机制。研究结果可为新型给药系统的合理选择和开发设计提供理论依据。

神经胶质瘤是高度致死性的脑肿瘤，目前对神经胶质瘤的化疗倾向于联合用药，根据细胞动力学和药物对细胞周期的特异性，联合应用两种或以上的药物以提高疗效。本项目成功构建并研究了PTX和TMZ共载的纳米复合温敏凝胶共递送系统对脑胶质瘤的协同治疗效果。.本研究采用溶剂-乳化蒸发法制备载紫杉醇磷酸钙/mPEG-DPPE纳米粒（PTX-NPs），自组装法制备载替莫唑胺磷酸钙/mPEG-DPPE纳米粒（TMZ-NPs），制得的PTX-NPs和TMZ-NPs的粒径分别为24.9 ±1.31 nm和28.9 ± 1.09 nm，且大小均一；透射电镜结果证实PTX-NPs和TMZ-NPs呈类球形，粒径在20-30 nm，分布均匀。有一定缓释能力，离心法测得它们的包封率为92.5%和84.5%。采用药物相互作用指数（CDI）分析PTX和TMZ联合应用的最佳协同作用比例，通过MTT法证实PTX和TMZ对胶质瘤细胞的增殖抑制具有协同作用，且PTX与TMZ 质量比为1:5时，对U87细胞的协同抑制作用最强；质量比为1:100时，对C6细胞的协同抑制作用最强。且与其他实验组相比，按以上比例制备的PTX/TMZ-NPs的对U87和C6细胞凋亡诱导作用最强；同时，建立U87皮下胶质瘤模型进行体内药效学研究，实验结果与体外结果一致，即PTX/TMZ-NPs组的肿瘤生长速度最慢，且对小鼠的全身毒副作用最小。.利用普朗尼克F127、海藻酸钠（SA）和羟丙基甲基纤维素（HPMC-100M）制备纳米复合温敏凝胶给药系统，以胶凝37℃条件下的胶凝时间和溶蚀速度为检测指标，通过均匀化设计处方制备的凝胶表现出弹性凝胶的流变学性质。细胞实验表明，相比PTX/TMZ-NPs组，纳米复合凝胶组对U87和C6细胞的协同增殖抑制和凋亡诱导作用更强。同时，建立大鼠C6原位胶质瘤模型和胶质瘤术后模型，并于术后模型的瘤腔内给药，结果显示：相比于PTX/TMZ和PTX/TMZ-NPs组，纳米复合温敏凝胶组的抗胶质瘤生长效果最好，且全身毒副作用最小，纳米复合温敏凝胶是通过诱导胶质瘤细胞自噬性死亡发挥抗胶质瘤作用。.综上，PTX和TMZ共载纳米复合温敏凝胶给药系统是良好的PTX和TMZ共递送系统，有望成为脑胶质瘤治疗全新的治疗策略。该课题成果应用趋向为肿瘤治疗及相关药物制剂开发；应用单位为相关医院及药物制剂公司。