Cholesterol Conjugated\nElastin-like Recombinamers:\nMolecular Dynamics Simulations, Conformational Changes, and Bioactivity

Current models for elastin-like recombinamer (ELR) design\nstruggle\nto predict the effects of nonprotein fused materials on polypeptide\nconformation and temperature-responsive properties. To address this\nshortage, we investigated the novel functionalization of ELRs with\ncholesterol (CTA). We employed GROMACS computational molecular dynamic\nsimulations complemented with experimental evidence to validate the <i>in silico</i> predictions. The ELR<sup>CTA</sup> was biosynthesized\nand characterized by using fluorescence assays, circular dichroism,\ndynamic light scattering, and differential scanning calorimetry. The <i>in silico</i> and <i>in vitro</i> data showed that\nCTA promotes the formation of intramolecular hydrogen bonds that favor\nβ-sheet secondary structures. Compared with an unmodified ELR<sup>VKV</sup>, CTA enhanced the hydrophobicity and stability of the system,\nallowing the formation of monodisperse nanoaggregates at physiologically\nrelevant temperatures. Importantly, calorimetry assays revealed that\nELR<sup>CTA</sup> interacted and intercalated with the lipid bilayers\nof the DPPC liposomes. To demonstrate the implications of these changes\nfor biomedical applications, ELR<sup>CTA</sup> and DPPC-ELR<sup>CTA</sup> hybrid nanoparticles were tested with cancer and immune cell lines.\nInteractions with the cell membranes demonstrated a synergistic effect\nof the composition and size of the modified recombinamer aggregates\non the internalization. The results indicated the potential use of\nELR-based nanoparticles for localized and systemic drug delivery.\nThis work sets a new precedent to design elastin-inspired biomaterials\nwith predictable self-assembly properties and develop novel drug delivery\nstrategies.