Molecularly Imprinted Polymers via RAFT Polymerisation for Biological Applications
by Luise Fanslau
Date of Examination:2024-10-24
Date of issue:2024-12-03
Advisor:Prof. Dr. Philipp Vana
Referee:Prof. Dr. Philipp Vana
Referee:Prof. Dr. Alec M. Wodtke
Persistent Address:
http://dx.doi.org/10.53846/goediss-10908
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Abstract
English
Antibodies are of great importance in therapeutic, diagnostic and research fields. Producing antibodies synthetically has been a longstanding goal of researchers and may be achieved using molecularly imprinted polymers (MIPs). Conventionally, MIPs are produced by means of free radical polymerisation (FRP), which is straightforward but lacks control over polymer size, homogeneity, and architecture. The use of reversible-deactivation radical polymerisation, specifically reversible addition-fragmentation chain-transfer (RAFT) polymerisation, offers a potential solution to these issues. However, the synthesis of RAFT-MIPs has been thus far limited to imprinting of small organic molecules, preventing their application as antibody alternatives in biological contexts. Therefore, new fabrication strategies are required. In this work, the potential of RAFT polymerisation was explored for the fabrication of protein-targeting MIPs for biological applications. Initially, a RAFT-MIP synthetic strategy was developed suitable for imprinting biomolecules. The versatility of the developed RAFT polymerisation was demonstrated through successful combination with three different MIP preparation strategies. First, this approach was used to fabricate RAFT-MIPs with the purpose of binding c-Myc-tagged proteins. Second, the RAFT polymerisation strategy was combined with the solid-phase method for MIP synthesis to fabricate tumor necrosis factor α (TNFα)-targeting RAFT-MIPs. Third, the developed RAFT-MIP fabrication strategy was adapted to graft crosslinked RAFT polymer onto Au nanorod (AuNR) surfaces for the combination of MIP properties with the diagnostic and therapeutic potential of AuNRs based on near-infrared light. TNFα-RAFT-MIPs exhibited improved TNFα template binding properties compared to a biotin-RAFT-MIP control as well as compared to TNFα-FRP-MIPs, showcasing the potential of RAFT polymerisation to create template-specific MIPs and enhance MIP binding properties compared to FRP approaches. In addition, this work provides further improvements regarding MIP fabrication. An in silico strategy based on molecular dynamics (MD) simulation was developed to analyse and quantitatively compare interactions between monomer candidates for the imprinting process and the imprinting template. The results of this in silico approach guided the selection of monomers for the imprinting syntheses carried out throughout this work. In addition, the process for selecting and designing peptide template molecules was enhanced through the application of MD simulation. With that, a novel hybrid linear-cyclic peptide template was designed, which was used to fabricate MIPs targeting the protein mesothelin. Lastly, this work presents a novel strategy to fabricate a solid-phase material for MIP synthesis based on core-shell SiO2-magnetic nanoparticles. Compared to routinely employed solid-phase materials, this material increased template functionalisation per unit mass of material by two orders of magnitude, which is expected to reduce the required synthetic scale of solid-phase MIP production. To the author’s knowledge, this thesis presents the first account of protein-targeting RAFT-MIPs synthesised under biotolerant conditions. Together with the progress made in terms of monomer selection, template design and fabrication of solid-phase material, this work is a valuable contribution to the MIP field that can be applied for various biological targets, simplifying the MIP fabrication process and advancing (RAFT-)MIPs as synthetic antibody alternatives.
Keywords: RAFT polymerisation; synthetic antibodies; molecularly imprinted polymers
