Elucidation and control of the mechanism of uptake and intracellular distribution of peptidic drugs bound in redox-sensitive nanogels in Caco2 cells as a model for small intestinal enterocytes

作为小肠肠上皮细胞模型，阐明和控制 Caco2 细胞中氧化还原敏感纳米凝胶中结合的肽类药物的摄取和细胞内分布机制

• 批准号: 524851962
• 负责人: Professor Dr. Jürgen Groll
• 金额: --
• 依托单位: Lehrstuhl für Funktionswerkstoffe der Medizin und der Zahnheilkunde
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/524851962?language=en
• 关键词: Elucidation; control; mechanism; uptake; intracellular

Nanohydrogels combine advantages of hydrogels (biocompatibility, high water content) and nano particles (high surface area, size in subcellular range) and represent promising drug delivery systems. My research group has had experience with redox-sensitive nanohydrogels for over 10 years. One target is the use for the transport of peptide drugs after oral administration into the intestinal wall. Recent results show that the reaction control during nanogel production influences the distribution of the nanogels and the active ingredient after uptake in the cells of the intestinal wall (enterocytes). The aim of the grant application is to understand this effect in the context of oral drug delivery, to amplify it and to explore it with regard to targeted intracellular drug transport into different organelles or the cell nucleus. Preliminary work in the form of first LSM analyzes and functional measurements regarding inhibition of glucose uptake in mouse small intestine and Caco2-cells demonstrate, that a peptide with antidiabetic properties (RS1-reg) is taken up via nanohydrogels and transported specifically to its site of action at the trans-Golgi network. The application aims to elucidate the exact mechanism of uptake of biologically active substances via polyglycidol-based nanohydrogels in Caco2 cells and the possibility of targeted control of uptake into the cytosol or the cell nucleus by optimising the material properties and reaction control during nanogel synthesis using the two examples of the antidiabetic peptide RS1-reg (site of action cytosol) and the Cas9 enzyme with 4 SV40 nuclear localisation sequences (site of action cell nucleus). For this purpose, firstly LSM and SIM microscopy on the one hand and electron microscopic investigations on the other hand will be used to systematically analyse where the example cargos accumulate as a function of time depending on a) the nanogel production method, on b) the degree of nanogel cross-linking and on c) the absence or presence of cell-penetrating peptides (TAT-peptid, penetratin, transportan). Secondly, it shall be clarified whether nanogels remain stable in contact with mucus under the basic conditions of the small intestine or whether they disintegrate. Thirdly, it is to be determined whether nanogel uptake in presence or absence of cell penetrating peptides occurs via direct membrane permeation or receptor-mediated endocytosis, and whether the protein loading is required for this. Fourthly, if endocytosis is present, receptors that are important for receptor-mediated endocytosis of the nanogels will be identified. Fifthly, the binding of the nanogels with RS1-reg peptide or Cas 9 to these receptor proteins will be characterized by surface plasmon resonance measurements. For this purpose, the membrane proteins found will be produced recombinantly and incorporated into lipid nanodiscs.

纳米水凝胶联合收割机结合了水凝胶（生物相容性、高含水量）和纳米颗粒（高表面积、亚细胞范围内的尺寸）的优点，并且代表了有前途的药物递送系统。我的研究小组在氧化还原敏感性纳米水凝胶方面有超过10年的经验。一个靶点是用于口服给药后肽类药物转运到肠壁中。最近的结果表明，纳米凝胶生产过程中的反应控制影响纳米凝胶和活性成分在肠壁细胞（肠细胞）中摄取后的分布。资助申请的目的是了解口服药物递送背景下的这种效应，放大它并探索它与靶向细胞内药物转运到不同细胞器或细胞核中的关系。关于抑制小鼠小肠和Caco 2细胞中葡萄糖摄取的第一LSM分析和功能测量形式的初步工作表明，具有抗糖尿病特性的肽（RS 1-reg）通过纳米水凝胶吸收并特异性地运输到其在trans-Golgi网络中的作用位点。该申请旨在阐明Caco 2细胞中生物活性物质经由基于聚缩水甘油的纳米水凝胶摄取的确切机制，以及通过使用抗糖尿病肽RS 1-reg的两个实例优化纳米凝胶合成期间的材料性质和反应控制来靶向控制摄取到细胞溶质或细胞核中的可能性图1示出了具有4个SV 40核定位序列的Cas9酶（细胞质作用位点）和具有4个SV 40核定位序列的Cas9酶（细胞核作用位点）。为此目的，首先一方面使用LSM和SIM显微镜，另一方面使用电子显微镜研究，系统地分析示例货物作为时间的函数积累的位置，取决于a） 所述纳米凝胶制备方法，在B） 纳米凝胶交联的程度和c） 是否存在细胞穿透肽（TAT-肽、穿透素、转运蛋白）。其次，应澄清纳米凝胶在小肠的基本条件下与粘液接触时是否保持稳定，或者它们是否崩解。第三，确定在存在或不存在细胞穿透肽的情况下纳米凝胶摄取是否通过直接膜渗透或受体介导的内吞作用发生，以及是否需要蛋白质负载。第四，如果存在内吞作用，则将鉴定对于纳米凝胶的受体介导的内吞作用重要的受体。第五，将通过表面等离子体共振测量表征具有RS 1-reg肽或Cas9的纳米凝胶与这些受体蛋白的结合。为此目的，将重组产生所发现的膜蛋白并将其并入脂质纳米盘中。