Tuning Non-Covalent Interactions to Optimize Supramolecular Biomaterials

Chen, Charlotte Hui

doi:https://doi.org/10.21985/n2-ny67-5p74

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Tuning Non-Covalent Interactions to Optimize Supramolecular Biomaterials

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Built from non-covalent interactions, supramolecular biomaterials are highly dynamic and tunable, and recent work has shown that they are uniquely capable of mimicking functional biological structures. In this work, supramolecular biomaterials built from self-assembling peptide amphiphiles (PA) were investigated with the goal of precisely tuning their cohesive interactions to optimize biological function. First, this work examined self-sorting versus co-assembly in a multicomponent PA system, where fluorescent PAs conjugated to either fluorescein isothiocyanate (FITC) or carboxytetramethylrhodamine (TAMRA) fluorophores were mixed with non-fluorescent diluent PA. The diluent PA self-assembled into a matrix of negatively charged nanofibers, which FITC-conjugated PA did not co-assemble with but TAMRA- conjugated PA did since the FITC fluorophore is negatively charged while the TAMRA fluorohpore is zwitterionic. This caused the FITC- and TAMRA-conjugated PA to undergo “matrix-mediated” self-sorting, which was suppressed when Na+ counterions were added to screen the negative charge of FITC residues. In another part of this work the cytotoxicity of cationic PAs was systematically altered, by changing the way the molecule’s alkyl segment was conjugated to its peptide chain. When the alkyl tail was conjugated to the peptide’s N-terminus, the PA assemblies exerted a rapid cytotoxic effect by physically disrupting the cell membrane. Conversely, when the alkyl tail was conjugated to the peptide’s C-terminus, the PA supramolecular assemblies exerted a slower cytotoxic effect by sequestering cholesterol from the cell membrane, which initiated programmed cell death. In the PA causing slower cytotoxicity, switching the orientation of the lysine linker between the alkyl tail and peptide chain hindered cholesterol sequestration and rescued cell viability. Next, this work examined the damage and self-repair of infinitely long PA nanofibers in response to freezing or freeze drying stress, which are important processes for the clinical translation of the biomaterials investigated. Freezing or freeze drying was found to physically break long nanofibers into shorter ones, which are not optimal for supporting cell adhesion, but thermal energy was found to re-elongate them and rescue their biological function. The long nanofibers are a thermodynamically preferred state that the system can return to but kinetic traps may impede this “repair” process. Furthermore, freeze drying can increase the charge density on the PA molecules, introducing charge repulsion that shifts the thermodynamic minimum to shorter nanofibers, which interferes with self-repair. Finally, this work presented a possible off-the-shelf PA formulation for clinical use for specific application in spinal fusion surgeries. In this formulation the PA is stored as a dried powder to improve its stability, and rehydrated immediately prior to use. The rehydrated PA solution contained short metastable nanofibers that successfully reduced the therapeutic bone morphogenetic protein-2 (BMP-2) protein dose by a factor of 100 in a rat spinal fusion model without requiring thermal elongation of the nanofibers. This suggests that nanofiber length is not a critical property for promoting successful bone growth, and that nanoscale supramolecular interactions may be more crucial to achieving this desired biological function. In conclusion, this work has shown that that while non-covalent interactions can lead to complex self-assembly behaviors in supramolecular biomaterials, with sufficient mechanistic knowledge, they can be precisely tuned and controlled to produce clinically viable therapies.

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