Coassembled Cytotoxic and Pegylated Peptide Amphiphiles Form Filamentous Nanostructures with Potent Antitumor Activity in Models of Breast Cancer

Self-assembled peptide amphiphiles (PAs) consisting of hydrophobic, hydvrogen-bonding, and charged hydrophilic domains form cylindrical nanofibers in physiological conditions and allow for the presentation of a high density of bioactive epitopes on the nanofiber surface. We report here on the use of PAs to form multifunctional nanostructures with tumoricidal activity. The combination of a cationic, membrane-lytic PA coassembled with a serum-protective, pegylated PA was shown to self-assemble into nanofibers. Addition of the pegylated PA to the nanostructure substantially limited degradation of the cytolytic PA by the protease trypsin, with an 8-fold increase in the amount of intact PA observed after digestion. At the same time, addition of up to 50% pegylated PA to the nanofibers did not decrease the <i>in vitro</i> cytotoxicity of the cytolytic PA. Using a fluorescent tag covalently attached to PA nanofibers we were able to track the biodistribution in plasma and tissues of tumor-bearing mice over time after intraperitoneal administration of the nanoscale filaments. Using an orthotopic mouse xenograft model of breast cancer, systemic administration of the cytotoxic pegylated nanostructures significantly reduced tumor cell proliferation and overall tumor growth, demonstrating the potential of multifunctional PA nanostructures as versatile cancer therapeutics

Gd(III)-Labeled Peptide Nanofibers for Reporting on Biomaterial Localization <i>in Vivo</i>

Bioactive supramolecular nanostructures are of great importance in regenerative medicine and the development of novel targeted therapies. In order to use supra­molecular chemistry to design such nanostructures, it is extremely important to track their fate <i>in vivo</i> through the use of molecular imaging strategies. Peptide amphiphiles (PAs) are known to generate a wide array of supramolecular nanostructures, and there is extensive literature on their use in areas such as tissue regeneration and therapies for disease. We report here on a series of PA molecules based on the well-established β-sheet amino acid sequence V₃A₃ conjugated to macrocyclic Gd(III) labels for magnetic resonance imaging (MRI). These conjugates were shown to form cylindrical supra­molecular assemblies using cryogenic transmission electron microscopy and small-angle X-ray scattering. Using nuclear magnetic relaxation dispersion analysis, we observed that thermal annealing of the nanostructures led to a decrease in water exchange lifetime (τ_m) of hundreds of nanoseconds only for molecules that self-assemble into nanofibers of high aspect ratio. We interpret this decrease to indicate more solvent exposure to the paramagnetic moiety on annealing, resulting in faster water exchange within angstroms of the macrocycle. We hypothesize that faster water exchange in the nanofiber-forming PAs arises from the dehydration and increase in packing density on annealing. Two of the self-assembling conjugates were selected for imaging PAs after intra­muscular injections of the PA C₁₆V₃A₃E₃-NH₂ in the <i>tibialis anterior</i> muscle of a murine model. Needle tracts were clearly discernible with MRI at 4 days post­injection. This work establishes Gd(III) macrocycle-conjugated peptide amphiphiles as effective tracking agents for peptide amphiphile materials <i>in vivo</i> over the timescale of days