基于DSPE-PEG-Alkyne的靶向*體納米系統(tǒng)構(gòu)建與表征

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

作者：張依林; 希拉里·A·肯尼； 埃爾登·P·斯溫德爾； 阿尼爾班·K·米特拉； 帕特里克·L·漢金斯； 理查德·W·安； 卡佳·格溫； 安德魯·P·馬扎爾； 托馬斯·V·奧哈洛倫； 恩斯特·倫吉爾通訊作者 

節(jié)選：

負(fù)載As2O3的尿激酶*體偶聯(lián)納米顆粒的合成與表征

上皮性卵巢癌細(xì)胞在原發(fā)性腫瘤和所有腹部轉(zhuǎn)移瘤中均均勻表達(dá)高水平的 u-PAR（圖 1A ）。對 cBio 癌癥基因組學(xué)門戶 ( 30 ) 上的TCGA 卵巢癌數(shù)據(jù)庫 ( 29 ) 的分析表明，患有 u-PAR 基因表達(dá)改變的卵巢癌患者的總體生存期 (19.5 個(gè)月 vs. 44.3 個(gè)月) 和無病生存期 (12.0 個(gè)月 vs. 17.5 個(gè)月；補(bǔ)充圖 S1A) 明顯較差。我們之前的組織微陣列研究表明，u-PAR 在上皮性腫瘤 ( 19 ) 中高度表達(dá)，包括 90% 以上的臨床卵巢癌標(biāo)本 ( 20 )。這提出了一種可能性，即高 u-PAR 表達(dá)不僅可以作為上皮性卵巢癌細(xì)胞侵襲性的標(biāo)志，還可以用于專門向這些細(xì)胞遞送*有效載荷。對于下面描述的實(shí)驗(yàn)，我們鑒定了 u-PAR 表達(dá)高的（HeyA8、ES-2）和低的（SKOV3ip1、MONTY-1 和 CaOV3）卵巢癌細(xì)胞系，這些細(xì)胞系也表達(dá) uPA（圖 1B）。由于 uPA 系統(tǒng)在卵巢癌中高度表達(dá)（18、20、31），我們試圖開發(fā)能夠主動(dòng)靶向表達(dá) uPA/u-PAR 的卵巢癌細(xì)胞的新型納米箱。選擇在小鼠中產(chǎn)生的針對 uPA 的 kringle 結(jié)構(gòu)域的單克隆*體 (ATN-291) 作為靶向配體，因?yàn)樗�與人 uPA 緊密結(jié)合，Kd ≈ 0.5 nmol /L，并且不會(huì)破壞 uPA/u-PAR 結(jié)合（18）。納米箱由膽固醇、DSPC 和 DSPE-PEG 組裝而成，并負(fù)載有砷/鎳共沉淀物（圖 1C；補(bǔ)充圖 S1B；參考文獻(xiàn)12、14、15）。接下來，將 DSPE-PEG-炔烴后插入雙層膜中，以促進(jìn)疊氮化物功能化的 uPA *體通過點(diǎn)擊化學(xué)進(jìn)行結(jié)合。該方法可確保靶向*體不會(huì)暴露在可能導(dǎo)致變性的高溫下（圖 1C；參考文獻(xiàn)32）。為了便于表征*體-納米箱結(jié)合物，在點(diǎn)擊化學(xué)之前用 Alexa Fluor 647 標(biāo)記*體。蛋白水解后對 Alexa Fluor 647 進(jìn)行紫外/可見光分析表明，每個(gè)載砷納米箱平均裝飾有 8.5 個(gè)*體（表 1）。我們還發(fā)現(xiàn)，結(jié)合后，ATN-291 靶向納米粒子仍然以納摩爾結(jié)合親和力與 uPA 結(jié)合。

譯文：

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pg電子官方生物提供相關(guān)產(chǎn)品：

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

DSPE-PEG-THRPPMWSPVWP（二硬脂�；�磷脂酰乙醇胺-聚乙二醇-轉(zhuǎn)鐵蛋白靶向肽)

DSPE-PEG-HAIYPRH(二硬脂�；�磷脂酰乙醇胺-聚乙二醇-轉(zhuǎn)鐵蛋白靶向肽)

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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