Self-Assembling Diblock Copolymers of Poly[N-(2-hydroxypropyl)methacrylamide] and a β-Sheet Peptide

The self-assembly of hybrid diblock copolymers composed of poly(HPMA) and β-sheet peptide P11 (CH3CO-QQRFQWQFEQQ-NH2) blocks was investigated. Copolymers were synthesized via thiol-maleimide coupling reaction, by conjugation of semitelechelic poly(HPMA)-SH with maleimide-modified β-sheet peptide. As expected, CD and CR binding studies showed that the peptide block imposed its β-sheet structural arrangement on the structure of diblock copolymers. TEM and AFM proved that peptide and these copolymers had the ability to self-assemble into fibrils. Keywords: diblock copolymers, conjugated polymers, reversible addition fragmentation chain transfer (RAFT), self-assembly, HPMA, β-sheet peptide Introduction Molecular self-assembly of synthetic block copolymers has been lately the focus of research activity of numerous investigators.[1–3] While synthetic copolymers are still of interest, the possibility to prepare hybrid block copolymers based on combination of proteins or peptides and synthetic polymers has become an attractive way to expand the diversity of structures and functions in synthetic materials. Peptide/protein-polymer hybrid copolymers are able to overcome some of the disadvantages of the individual components. For example, a structural protein or peptide block (such as coiled-coil or β-sheet) may control the nanostructure of the synthetic component,[4–6] whereas a synthetic polymer block may increase the stability[7] and the biocompatibility of the protein/peptide segment.[8,9] A series of interesting reports has described the synthesis, as well as the self-assembly[10–15] of different β-sheet peptide/poly(ethylene glycol) (PEG) hybrid diblock copolymers. Two main approaches to obtain hybrid copolymers can be identified: polymerization strategies, using a peptide macroinitiator to synthesize the polymer block, and coupling strategies, using a preformed synthetic polymer to react with selective functionalities on the peptide.[16,17] However, when analyzing the self-assembly mechanism, a common trend arises: the hierarchical organization of the β-sheet motifs in the peptide blocks mediates the self-organization of the conjugates. As a result, fibrils can be formed by stacking of the β-sheets on top of each other.[18] Studies showed not only that the self-organization of different β-sheet peptides into fibrils was preserved after conjugation with PEG, but moreover, the lateral aggregation of these fibrils was prevented.[10,14] It seems that the PEG coating provides solubility and facilitates self-assembly into uniform fibrils with no lateral aggregation. This is particularly important in case of PEG-coated amyloid fibrils that exhibit a strong reduction in the formation of amyloid plaques,[14] associated with e. g. Alzheimer’s disease. While various β-sheet peptides have been successfully conjugated with PEG, there is no report on their conjugation with poly[N-(2-hydroxypropyl)methacrylamide] [poly-(HPMA)], a non-immunogenic, neutral, hydrophilic polymer currently employed in the delivery of anticancer drugs. Compared to PEG conjugates, imposing a certain secondary structure on a poly(HPMA)-peptide conjugate might be a challenging task, since in water, poly(HPMA) has a random coil conformation, while PEG adopts an extended conformation.[19] However, previous work on HPMA copolymers containing coiled-coil peptide grafts demonstrated that self-assembly of the hybrid poly(HPMA)-peptide copolymer systems is possible, and moreover, it can even trigger the formation of a hydrogel.[20,21] In this article, the synthesis and the self-assembly of hybrid diblock copolymers, based on poly(HPMA) and β-sheet peptide blocks, are described. We hypothesized that the β-sheet-forming peptide block will impose its structural arrangement on the overall structure of the diblock copolymers. The β-sheet peptide was synthesized using solid-phase methodology and manual 9-fluorenyl-methoxycarbonyl (Fmoc)/tert. butyl ester (tBu) strategy, and purified by reverse phase HPLC. The sequence P11-2 (CH3CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH2, CH3CO-QQRFQWQFEQQ-NH2, herein termed P11), previously designed by Aggeli et al.,[22] was used. We chose this well-characterized, short peptide sequence for the fact that in aqueous solutions, it can form antiparallel β-sheets self-organized into fibrils,[23] as well as for the easiness of its synthesis. A novel approach to prepare the hybrid diblock copolymers was developed. Well-defined semitelechelic poly(HPMA) polymers terminated with thiol functional groups were prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization in methanol followed by aminolysis. Then, the N-terminus of the P11 peptide was modified with a maleimide group, and finally, in the last step, the conjugation was accomplished via the maleimide-thiol coupling reaction. Circular dichroism spectroscopy (CD) and Congo Red (CR) binding studies were performed to characterize the secondary structure of the peptide and of the peptide domain in diblock copolymers. The morphology of the materials was investigated by transmission electron microscopy (TEM), and atomic force microscopy (AFM).