Preventing secondary damage after brain lesion – novel macromolecular transport and membrane-anchoring systems for neuroprotective substances
Final Report Abstract
This project focused on the synthesis and formulation of polymers for polymer-drug-conjugates. The formulations are planned to be used for the transport of a novel, urea-based inhibitor targeting the membrane-bound enzyme GCPII to its target location in the brain. Excitation of GCPII over longer time can occur after brain trauma (e.g., lesions or stroke) and lead to a reaction cascade causing apoptosis of nearby brain cells. Therefore, the formulations must enable passage of the blood-brain barrier and localize the drug close to its target site of action. Different approaches were tested to achieve this: First, hydrophilic, branched polymers with a hydrophobic anchor connected on a peptoid backbone were synthesized via solid phase synthesis. Those were able to stabilize lipid nanoparticles which can mimic low-density lipoproteins and therefore be transported through the blood-brain barrier using specific LDL transporters. Further, the anchor allows to localize the compounds close to the cell surface and therefore close to their target after decomposition of the particles. Different numbers of polymeric arms with reactive end groups can be functionalized with the respective number of inhibitor molecules, which enables multivalent targeting. Three groups of compounds were synthesized during the project, each containing structures with one to three arms generated from either oligo(ethylene glycol), poly(ethylene glycol), or poly(2-ethyloxazoline), a reactive linker for the attachment of a fluorescent dye, and the hydrophobic anchor or a terminating, non-interacting control group, all connected sequencespecifically and with a defined number on a peptoid backbone. The compounds with hydrophobic anchor were able to stabilize lipid nanoparticles and bind reversibly to cell membranes. Further, they showed low cytotoxicity and low hemolytic activity. Uptake into cells was observable after their attachment to cell membranes. The attachment of the inhibitor to the structures was difficult, as full conversion of the reactive end groups of the polymeric arms was not achieved. Further, side reactions during the deprotection of the carboxylic acid groups of the inhibitor led to impurities in the final samples. Therefore, investigation of the polymer-drug conjugates remains to be done. In addition to the investigation of branched structures, linear poly(2-oxazoline)s with a hydrophobic anchor as terminating group were synthesized. The polymers were partially functionalized with glucose, connected to the polymer backbone in the 6-position of the carbohydrate molecule. These compounds were supposed to interact with GLUT1, a glucose transporter that is abundant in brain endothelial cells, to support the passage of substances through the blood-brain barrier. The polymers were able to stabilize lipid nanoparticles as well. The stability of the latter depended on their molecular weight and the degree of functionalization of the polymers. Cell tests demonstrated increased uptake of moderately glucosylated structures into GLUT1-expressing cell lines, which may be attributed to polymer-receptor interactions, but also to altered physicochemical properties of glucosylated polymers and particles. In summary, the project generated valuable results in the area of the synthesis of highly defined, multifunctional polymers as polymer-drug conjugates, despite the problems that occurred during the step of inhibitor attachment to the polymers.
Publications
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Solid-phase synthesis as strategy for the generation of multifunctional, branched polymers J. K. Elter, Martin Hrubý Contributed Lecture, EPF European Polymer Congress, Prague, Czech Republic, 2022
J. K. Elter & Martin Hrubý
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Solid-phase synthesis as a powerful tool for the generation of highly defined, branched polymers J. K. Elter, Martin Hrubý Poster presentation, Macromolecular Colloquium Freiburg, Germany, 2023
J. K. Elter & Martin Hrubý
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Solid-phase synthesis as a tool for the generation of highly defined drug carrier systems J. K. Elter, N. Sidej, V. Liščáková, P. Šácha, and M. Hrubý Contributed lecture, The 13th SPSJ International Polymer Conference, Sapporo, Japan, 2023
J. K. Elter, N. Sidej, V. Liščáková, P. Šácha & M. Hrubý
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Biodegradable solid lipid particle carriers for polymer-ligand conjugates A. Rana, V. Lobaz, J. K. Elter, and M. Hrubý Poster presentation, 85th Prague Meeting on Macromolecules (Polymers for a Sustainable Future 2024), Prague, Czech Republic, 2024
A. Rana, V. Lobaz, J. K. Elter & M. Hrubý
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Solid-Phase Synthesis as a Tool to Create Exactly Defined, Branched Polymer Vectors for Cell Membrane Targeting. Macromolecules, 57(3), 1050-1071.
Elter, Johanna K.; Liščáková, Veronika; Moravec, Oliver; Vragović, Martina; Filipová, Marcela; Štěpánek, Petr; Šácha, Pavel & Hrubý, Martin
