When it comes to anti-aging skin care, direct peptides are some of the beauty industry’s major ballers. They’ve been around for years and have taken center stage in many a celebrity-endorsed serum. But what you may not know is that peptides can do much more than encourage collagen growth and fight signs of aging.
The molecular initiating event of skin sensitisation involves the covalent binding of electrophilic chemicals to nucleophilic centres in protein. It is thus widely recognised that the ability of a chemical to react with proteins in the skin can be used to categorize it as either a potential sensitiser or non-sensitiser, and also to determine its potency1.
A number of methods have been developed for assessing the ability of chemicals to react with proteins in the skin. These include the kDPRA (the kinetic Direct Protein Reactivity Assay) that uses a pair of peptides containing cysteine and lysine and which reacts with the test item to generate a matrix of peptide depletion values over a range of reaction times and initial concentrations. The matrix is then analysed to derive a function that correlates extent of the reaction with a combination of reactivity (k) and time, enabling chemicals to be classified as sensitisers or non-sensitisers.
However, the reactivity of impurities in the peptides can be difficult to determine, especially at high concentrations of the test chemical. Furthermore, this method relies on the assumption that a test chemical is completely free of any reactive impurity. It has been shown that reconstructed plots of peptide depletion can be used to distinguish impurities from the pure sample.
To address these issues, we have developed a simple peptide modification to enable the characterization of impurities in peptides that have undergone HPLC purification. This technique involves modifying the peptide by the introduction of an amide-containing moiety that is self-immolative, thereby eventually restoring the amine group after a series of autocatalytic reactions.
This chemistry is now being expanded to make peptides both pH- and redox-responsive for the cargo recruitment and intracellular release of a wide palette of macromolecular therapeutics. The peptide coacervates can be loaded with cargos including EGFP, AF-lysozyme, AF-BSA and mRNA and are capable of delivering these to HepG2 cells in a single phase.
The peptides were synthesised using the standard Merrifield solid-phase peptide synthesis (SPPS) technique57. Wang resin (1.0 g, 0.56 mmol) was first swollen in 15 ml of dichloromethane for 0.5 h with bubbling nitrogen flow. The swollen resin was then rinsed three times with N,N-dimethylformamide (DMF).
Peptides were isolated by lyophilization (FreeZone 4.5 Plus, Labconco) from the HPLC elutes. They were stored as a stock solution in aliquots at 80°C.