In Vivo Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures.
In Vivo Evaluation of Site-Specifically PEGylated Chemically Self-Assembled Protein Nanostructures.
复制标题
体内特定于定位的化学自我组装蛋白纳米结构的体内评估。
DOI：
10.1021/acs.molpharmaceut.6b00110
复制
发表时间：
2016-07-05
影响因子：
4.9
通讯作者：
Kumarapperuma SC
中科院分区：
文献类型：
--
作者：
Shah R;Petersburg J;Gangar AC;Fegan A;Wagner CR;Kumarapperuma SC
化学自组装纳米环（CSAN）由二氢叶酸还原酶（DHFR）融合蛋白制成，并已成功地在体外用于细胞货物递送和细胞表面工程应用。然而，CSAN的体内稳定性、循环和组织分布尚未得到评价。为了在体内评估CSAN，我们设计了一种靶向DHFR分子的位点特异性聚乙二醇化表皮生长因子受体（EGFR），用二价甲氨蝶呤（bis-MTX）表征了它们在CSAN中的自组装，通过microPET/CT成像可视化了它们的体内组织定位，并通过基于组织的γ计数确定了它们的离体器官生物分布。使用非天然氨基酸诱变，将与C-末端EGFR靶向肽（LARLLT）融合的二聚体DHFR（DHFR 2）分子工程化以掺入位点特异性酮官能团。不同链长的氨氧基-PEG使用肟化学成功地缀合至蛋白质。这些蛋白质自组装成CSAN与bis-MTX DHFR二聚体，其特征在于通过尺寸排阻色谱和动态光散射。使用使用bis-MTX-FITC组装的荧光CSAN进行体外结合研究，而使用使用bis-MTX-DOTA[64 Cu]组装的放射性标记的CSAN进行体内microPET/CT成像。PEG化使小鼠巨噬细胞（RAW 264.7）对抗EGFR CSAN的摄取减少高达40%，而不改变CSAN对U-87 MG胶质母细胞瘤细胞的体外结合亲和力。在携带U-87 MG异种移植物的小鼠中，通过microPET/CT成像和生物分布研究观察到64 Cu标记的抗EGFR-CSAN的显著时间依赖性肿瘤蓄积。PEG化CSAN显示出肝脏、肾脏和脾脏的摄取减少，导致静脉内注射1小时内的高对比度肿瘤成像（9.6%ID/g），并持续增加至24小时（11.7%ID/g），而背景信号减少。CSAN在体内表现出介于快速清除小分子和缓慢清除抗体之间的特征。因此，CSAN提供了一个模块化的，可编程的和稳定的基于蛋白质的平台，可用于体内药物递送和成像应用。
Chemically Self-Assembled Nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used in vitro for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their in vivo stability, circulation and tissue distribution. In an effort to evaluate CSANs in vivo, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly in to CSANs with bivalent methotrexates (bis-MTX), visualized their in vivo tissue localization by microPET/CT imaging and determined their ex vivo organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR2) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino-acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. In vitro binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while in vivo microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA[64Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSAN’s binding affinity towards U-87 MG glioblastoma cells in vitro. A significant time dependent tumor accumulation of 64Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished. CSANs displayed an in vivo profile between that of rapidly clearing small molecules and slow clearing antibodies. Thus CSANs offer a modular, programmable and stable protein based platform that can be used for in vivo drug delivery and imaging applications.
登录
查看更多内容
影响因子：
15
作者：
Li Q;So CR;Fegan A;Cody V;Sarikaya M;Vallera DA;Wagner CR
通讯作者：
Wagner CR
DOI：
10.2217/17435889.3.5.703
发表时间：
2008-10
期刊：
Nanomedicine (London, England)
影响因子：
--
作者：
Longmire M;Choyke PL;Kobayashi H
通讯作者：
Kobayashi H
影响因子：
4.7
作者：
Basu, Amartya;Yang, Karen;Filpula, David
通讯作者：
Filpula, David
影响因子：
4.7
作者：
Lim, Reyna K. V.;Yu, Shan;Kazane, Stephanie A.
通讯作者：
Kazane, Stephanie A.
影响因子：
16.6
作者：
Gabrielse, Kari;Gangar, Amit;Wagner, Carston R.
通讯作者：
Wagner, Carston R.