基于DSPE-PEG-Alkyne的靶向*体纳米系统构建与表征

链接：https://aacrjournals.org/mct/article/12/12/2628/91625/Urokinase-Plasminogen-Activator-System-Targeted

作者：张依林; 希拉里·A·肯尼； 埃尔登·P·斯温德尔； 阿尼尔班·K·米特拉； 帕特里克·L·汉金斯； 理查德·W·安； 卡佳·格温； 安德鲁·P·马扎尔； 托马斯·V·奥哈洛伦； 恩斯特·伦吉尔通讯作者 

节选：

负载As2O3的尿激酶*体偶联纳米颗粒的合成与表征

上皮性卵巢癌细胞在原发性肿瘤和所有腹部转移瘤中均均匀表达高水平的 u-PAR（图 1A ）。对 cBio 癌症基因组学门户 ( 30 ) 上的TCGA 卵巢癌数据库 ( 29 ) 的分析表明，患有 u-PAR 基因表达改变的卵巢癌患者的总体生存期 (19.5 个月 vs. 44.3 个月) 和无病生存期 (12.0 个月 vs. 17.5 个月；补充图 S1A) 明显较差。我们之前的组织微阵列研究表明，u-PAR 在上皮性肿瘤 ( 19 ) 中高度表达，包括 90% 以上的临床卵巢癌标本 ( 20 )。这提出了一种可能性，即高 u-PAR 表达不仅可以作为上皮性卵巢癌细胞侵袭性的标志，还可以用于专门向这些细胞递送*有效载荷。对于下面描述的实验，我们鉴定了 u-PAR 表达高的（HeyA8、ES-2）和低的（SKOV3ip1、MONTY-1 和 CaOV3）卵巢癌细胞系，这些细胞系也表达 uPA（图 1B）。由于 uPA 系统在卵巢癌中高度表达（18、20、31），我们试图开发能够主动靶向表达 uPA/u-PAR 的卵巢癌细胞的新型纳米箱。选择在小鼠中产生的针对 uPA 的 kringle 结构域的单克隆*体 (ATN-291) 作为靶向配体，因为它与人 uPA 紧密结合，Kd ≈ 0.5 nmol /L，并且不会破坏 uPA/u-PAR 结合（18）。纳米箱由胆固醇、DSPC 和 DSPE-PEG 组装而成，并负载有砷/镍共沉淀物（图 1C；补充图 S1B；参考文献12、14、15）。接下来，将 DSPE-PEG-炔烃后插入双层膜中，以促进叠氮化物功能化的 uPA *体通过点击化学进行结合。该方法可确保靶向*体不会暴露在可能导致变性的高温下（图 1C；参考文献32）。为了便于表征*体-纳米箱结合物，在点击化学之前用 Alexa Fluor 647 标记*体。蛋白水解后对 Alexa Fluor 647 进行紫外/可见光分析表明，每个载砷纳米箱平均装饰有 8.5 个*体（表 1）。我们还发现，结合后，ATN-291 靶向纳米粒子仍然以纳摩尔结合亲和力与 uPA 结合。

译文：

Synthesis and characterization of urokinase antibody–conjugated nanobins loaded with As2O3

Epithelial ovarian cancer cells homogeneously express elevated levels of u-PAR in the primary tumor and all abdominal metastasis (Fig. 1A). Analysis of the TCGA ovarian cancer database (29) at the cBio cancer genomics portal (30) showed that patients with ovarian cancer with u-PAR gene expression alterations had a significantly worse overall (19.5 vs. 44.3 months) and disease-free survival (12.0 vs. 17.5 months; Supplementary Fig. S1A). And our previous tissue microarray studies demonstrated that u-PAR is highly expressed in epithelial tumors (19) including more than 90% of clinical ovarian cancer specimens (20). This raises the possibility that high u-PAR expression might not only function as a marker of aggressiveness for epithelial ovarian cancer cells but could also be used to specifically deliver a therapeutic payload to those cells. For the experiments described below, we identified ovarian cancer cell lines with high (HeyA8, ES-2) and with low (SKOV3ip1, MONTY-1, and CaOV3) u-PAR expression, all of which also express uPA (Fig. 1B). Because the uPA system is highly expressed in ovarian cancer (18, 20, 31), we sought to develop novel nanobins that would actively target uPA/u-PAR–expressing ovarian cancer cells. A monoclonal antibody raised against the kringle domain of uPA in mice (ATN-291) was chosen as a targeting ligand as it binds tightly to human uPA with a Kd ≈ 0.5 nmol/L and does not disrupt uPA/u-PAR binding (18). The nanobins were assembled from cholesterol, DSPC, and DSPE-PEG, and loaded with an arsenic/nickel coprecipitate (Fig. 1C; Supplementary Fig. S1B; refs. 12, 14, 15). Next, DSPE-PEG-alkyne was postinserted into the bilayer to facilitate the conjugation of the azide-functionalized uPA antibody using click chemistry. This method ensures that the targeting antibodies are not exposed to potentially denaturing high temperatures (Fig. 1C; ref. 32). To facilitate characterization of antibody–nanobin conjugates, the antibody was labeled with Alexa Fluor 647 before click chemistry. UV/Vis analysis of Alexa Fluor 647 after proteolysis showed that the arsenic-loaded nanobins were decorated with an average of 8.5 antibodies per nanobin (Table 1). We also found that, after conjugation, the ATN-291–targeted nanoparticle still bound uPA with nanomolar binding affinity (data not shown).

西安齐岳生物提供相关产品：

DSPE-PEG-SP94(SFSHHTPILPLC; 二硬脂酰基磷脂酰乙醇胺-聚乙二醇-肝癌靶向肽)

DSPE-PEG-THRPPMWSPVWP（二硬脂酰基磷脂酰乙醇胺-聚乙二醇-转铁蛋白靶向肽)

DSPE-PEG-HAIYPRH(二硬脂酰基磷脂酰乙醇胺-聚乙二醇-转铁蛋白靶向肽)

DSPE-PEG-ferrocene

DSPE-PEG-WGA

DSPE-PEG-Streptavidin

DSPE-PEG-BSA

DSPE-PEG-Lysozyme

DSPE-PEG-PLL

DSPE-PEG-Heparin

DSPE-PEG-Insulin

DSPE-PEG-Lectins

DSPE-PEG-lactoferrin

DSPE-PEG-Galactose

DSPE-PEG-Dextran

DSPE-PEG-Chitosan

DSPE-PEG-Mannose

DSPE-PEG-Glucose

DSPE-PEG-HA

DSPE-PEG-Alginate

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