In the era of precision medicine, the site-specific delivery of biomacromolecular therapeutics, including nucleic acids and proteins, has emerged as a promising strategy for treating complex diseases. However, clinical translation of these therapeutics remains hindered by major physiological barriers, including inefficient translocation across cell membranes, endosomal/lysosomal entrapment and subsequent degradation, and inadequate targeting of specific subcellular organelles. Peptides have emerged as versatile functional modules for intracellular delivery owing to their programmable sequences and tunable biological functions. Although previous reviews have focused primarily on individual aspects of peptide-based delivery, particularly cell-penetrating peptides, peptide-drug conjugates, or peptide-functionalized nanocarriers, a systematic overview encompassing the entire intracellular delivery cascade remains lacking. This review summarizes recent advances in the peptide-mediated precise delivery of bioactive substances, with particular emphasis on the mechanisms underlying cell-specific targeting, membrane translocation, endosomal escape, and subsequent delivery of bioactive molecules to specific subcellular organelles, including the nucleus, mitochondria, and endoplasmic reticulum. Key design strategies are discussed, including the covalent conjugation of peptides to therapeutic payloads, supramolecular self-assembly, and the modular integration of multifunctional peptide units with stimuli-responsive nanocarriers. We further highlight recent applications of peptide-mediated precision delivery systems in cancer therapy, gene therapy, neurological diseases, infectious disease control, and diagnostic imaging. Finally, we discuss the major challenges limiting the clinical translation of these systems and identify potential directions for future research. By integrating these advances into a framework for peptide-based precision delivery, this review aims to provide a conceptual foundation for the development and clinical translation of next-generation therapeutic delivery systems based on programmable, targeted, and controllable peptide functionalities.
