In situ assembly of peptides in living cells

The self-assembly of peptides into well-defined nanostructures represents a versatile strategy for developing biomedical agents. However, cellular internalization of pre-assembled peptide nanostructures is often hampered by insufficient uptake and low selectivity toward target cells, leading to off-target concerns and limited therapeutic efficacy. The past few years have witnessed the development of in situ assembly in living cells as a new endeavor for optimizing the performance of peptide assemblies. In situ assembly of peptides refers to intracellular assembling processes regulated by biocompatible stimuli, thereby enhancing peptide internalization and locally forming assemblies at target sites. Recently, we have developed strategies for establishing in situ assembly of peptides in living cells dictated by stimuli-responsive unnatural amino acids. Here we detail the procedure for developing in situ peptide assembly systems in living cells through incorporating stimuli-responsive unnatural amino acids. We present a thorough workflow encompassing peptide design and synthesis as well as the characterization of peptide responsiveness to glutathione or sulfatase. Self-assembly conditions are described for experiments in solution, in murine macrophage cells, human cervical carcinoma cells and murine breast cancer cells. The applications of in situ-formed peptide assemblies for subcellular targeting and cancer therapy in murine breast tumor-bearing BALB/c mice are also presented. The described methodology provides a robust platform for developing in situ-formed peptide assemblies and advancing the clinical translation of peptide-based supramolecular therapeutics. Performance of this Protocol requires expertise in peptide chemistry, materials engineering and biomedical engineering, taking an expected 92-115 d to complete.