EPR-effect driven tumor delivery of multifunctional boron nanomedicine for neutron capture therapy

我们合成了一种水溶性胶束药物，名为苯乙烯-马来酸(SMA)共聚物共轭氨基葡萄糖-硼酸(SGB-络合物)。基于EPR效应的肿瘤选择性蓄积和良好的体外杀瘤活性，以及多种小鼠肿瘤模型。然而，SGB-络合物的确切化学结构和在没有中子照射的情况下细胞毒性的详细作用机制尚不清楚。在2022财年期间，我重点研究了SGB-络合物的紫外可见光谱、红外光谱、核磁共振光谱和液质联用测定其分子结构。我们证实SMA-聚合物通过酰胺键结合到氨基葡萄糖上，而硼酸通过二醇键结合到氨基葡萄糖上(这项工作发表在《美国化学学会应用生物材料》杂志上)。接下来，我们试图找出SGB-络合物体外细胞毒性的可能机制。我们推测SGB复合体可能在轻度缺氧状态下诱导内质网或线粒体应激。为了支持我们的假设，我们检测了内质网应激标记磷酸化真核细胞起始因子2α(p-eIF2)、C/-EBP同源蛋白(CHOP)以及Caspase 3和7在药物治疗后的表达。有趣的是，我们发现SGB-复合体显著增加了各种ER应激标记，证实了我们的药物在低氧适应的癌细胞中诱导了ER应激。我们还测量了我们的药物治疗后线粒体膜电位，数据显示与未治疗组相比变化很小。电子显微镜图像将能够确认是否发生任何线粒体损伤。

We synthesized a water-soluble micellar drug called styrene maleic acid (SMA) copolymer conjugated glucosamine and boric acid (SGB-complex). It showed tumor selective accumulation based on EPR effect and excellent tumoricidal activity in vitro as well as various mouse tumor models. However, the precise chemical structure of SGB-complex and the detailed mode of action of cytotoxicity without neutron irradiation was not understood.During FY2022 period, I focused on the determination of molecular structure of SGB-complex by UV-vis, IR, NMR spectroscopy and liquid chromatography mass spectroscopy. We confirmed that the SMA-polymer bound to the glucosamine via amide bond and the boric acid was attached to glucosamine by di-ol bond (This works we reported at ACS Applied Biomaterials journal). Next, we try to find out the possible mechanism of in vitro cytotoxicity of SGB-complex. We hypothesized that SGB-complex might induce ER or mitochondrial stress in mild-hypoxia state. To support our hypothesis, we measure the ER stress marker phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2), C/-EBP homologous protein (CHOP), and Caspase 3 and 7 expressions after treating of our drug. Interestingly, we found the SGB-complex significantly increased various ER stress markers that confirmed our drug induces ER stress in hypoxia adapted cancer cells. We also measured the mitochondrial membrane potential after treatment of our drug, and data showed very less changes occurred compare to non-treated group. TEM image will be able to confirm whether any mitochondrial damage occur or not.