The Functional Upstream Domain (FUD) peptide is a potent inhibitor of fibronectin assembly and a therapeutic candidate for disorders linked with hyperdeposition of fibronectin-modulated ECM proteins. Most recently, experiments involving subcutaneous (s.c.) administration of a PEGylated FUD (PEG-FUD) of 27.5 kDa molecular weight yielded a significant reduction of fibronectin and collagen deposition in a murine model of renal fibrosis. The benefits of FUD PEGylation need to be studied to unlock the full potential of the PEG-FUD platform. This work studies the impact of PEGylating the FUD peptide with differently sized PEG on its absorption from the site of injection following s.c. delivery using non-invasive in vivo fluorescence imaging. The FUD and mFUD (control) peptides and their 10 kDa, 20 kDa, and 40 kDa PEG conjugates were labeled with the sulfo-Cy5 fluorophore. Isothermal titration calorimetry (ITC) and confocal fluorescence microscopy experiments verified FUD and PEG-FUD fibronectin binding activity preservation following sulfo-Cy5 labeling. Fluorescence in vivo imaging experiments revealed a linear relationship between the absorption apparent half-life (t1/2) and the MW of FUD, mFUD, and their PEG conjugates. Detected drug signal in the kidney and bladder regions of mice suggests that smaller peptides of both the FUD and mFUD series enter the kidney earlier and in higher amounts than their larger PEG conjugates. This work highlights an important delayed dose absorption enhancement that MW modification via PEGylation can contribute to a drug when combined with the subcutaneous route of delivery.

中文翻译：

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通过体内荧光成像探测PEG化FUD肽纳米药物的皮下吸收。

功能上游结构域（FUD）肽是纤连蛋白组装的有效抑制剂，是与纤连蛋白调节的ECM蛋白过度沉积有关的疾病的治疗候选物。最近，涉及皮下（sc）施用分子量27.5 kDa的PEG化FUD（PEG-FUD）的实验在肾纤维化的小鼠模型中显着减少了纤连蛋白和胶原蛋白的沉积。需要研究FUD PEG化的好处，以释放PEG-FUD平台的全部潜力。这项工作使用无创体内荧光成像技术研究了sc递送后，用大小不一的PEG聚乙二醇化FUD肽对其从注射部位吸收的影响。FUD和mFUD（对照）肽及其10 kDa，20 kDa，用磺基-Cy5荧光团标记40kDa PEG缀合物。等温滴定热法（ITC）和共聚焦荧光显微镜实验证实了磺基Cy5标记后FUD和PEG-FUD纤连蛋白的结合活性得以保持。体内荧光成像实验揭示了表观吸收半衰期（t1 / 2）与FUD，mFUD及其PEG缀合物的分子量之间存在线性关系。在小鼠的肾脏和膀胱区域检测到的药物信号表明，FUD和mFUD系列的较小肽比较大的PEG缀合物更早进入肾脏，且进入肾脏的数量更高。这项工作突显了重要的延迟剂量吸收增强，即与皮下递送途径结合使用时，通过PEG化的MW修饰可能有助于药物。等温滴定热法（ITC）和共聚焦荧光显微镜实验证实了磺基Cy5标记后FUD和PEG-FUD纤连蛋白的结合活性得以保持。体内荧光成像实验揭示了表观吸收半衰期（t1 / 2）与FUD，mFUD及其PEG缀合物的分子量之间存在线性关系。在小鼠的肾脏和膀胱区域检测到的药物信号表明，FUD和mFUD系列的较小肽比较大的PEG缀合物更早进入肾脏，且进入肾脏的数量更高。这项工作突显了重要的延迟剂量吸收增强，即与皮下递送途径结合使用时，通过PEG化的MW修饰可能有助于药物。等温滴定热法（ITC）和共聚焦荧光显微镜实验证实了磺基Cy5标记后FUD和PEG-FUD纤连蛋白的结合活性得以保持。体内荧光成像实验揭示了表观吸收半衰期（t1 / 2）与FUD，mFUD及其PEG缀合物的分子量之间存在线性关系。在小鼠的肾脏和膀胱区域检测到的药物信号表明，FUD和mFUD系列的较小肽比较大的PEG缀合物更早进入肾脏，且进入肾脏的数量更高。这项工作突显了重要的延迟剂量吸收增强，即与皮下递送途径结合使用时，通过PEG化的MW修饰可能有助于药物。