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

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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.