Therapeutic manipulation of the BCL-2 family using BH3 mimetics is an emerging paradigm in cancer treatment and immune modulation. For example, peptides mimicking the BIM BH3 helix can directly target the full complement of anti- and pro-apoptotic BCL-2 proteins to trigger apoptosis. This study has incorporated the potent BH3 α-helical death domain of BIM into peptide amphiphile (PA) nanostructures designed to facilitate cellular uptake and induce cell death. This study shows that these PA nanostructures are quickly incorporated into cells, are able to specifically bind BCL-2 proteins, are stable at physiologic temperatures and pH, and induce dose-dependent apoptosis in cells. The incorporation of a cathepsin B cleavable linker between the BIM BH3 peptide and the hydrophobic tail resulted in increased intracellular accumulation and mitochondrial co-localization of the BIM BH3 peptide while also improving BCL-2 family member binding and apoptotic reactivation. This PA platform represents a promising new strategy for intracellular therapeutic peptide delivery for the disruption of intracellular protein:protein interactions.

Despite the therapeutic promise of phospholipid-based nanocarriers, a major obstacle to their widespread clinical translation is a susceptibility to fatty acid ester hydrolysis, leading to lack of quality control and inconsistencies in self-assembly formulations. Using electrospray ionization mass spectrometry fragmentation in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have demonstrated a method to detect hydrolysis of one or both of the fatty acid esters in a PEGylated phospholipid, DSPE-PEG, in conditions commonly applied during nanocarrier production. Because such carriers are increasingly being used to deliver peptide-based therapeutics, we further investigated the hydrolysis of phospholipid esters in conditions used for solid-phase peptide synthesis and high-performance liquid chromatography of peptides. We ultimately detail a synthetic strategy to reliably produce pure phospholipid–peptide bioconjugates (peptide amphiphiles), while avoiding unintended or unnoticed hydrolyzed byproducts that could lead to polymorphic nanotherapeutics with dampened therapeutic efficacy. We believe that such an approach could help standardize phospholipid–peptide-based therapeutic development, testing, and clinical translation.

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]–[4]. Using a purified DDB2 DRC, we created a probe (“DDB2 proteo-probe”) that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.