CPP–PEG‐Guided Surface Engineering of Mitochondria Enables Efficient Cellular Uptake and Respiratory Modulation

ABSTRACT Mitochondrial transplantation has emerged as a promising strategy for modulating cellular bioenergetics in mitochondrial dysfunction. However, isolated mitochondria suffer from poor stability and limited cellular uptake, restricting their therapeutic application. To address these limitations, we developed a surface engineering strategy that stabilizes isolated mitochondria while enabling interactions with target cells, providing a platform for selective organ‐ and cell‐targeting. Polyethylene glycol (PEG) with lipid/carbon chains was introduced to mitochondria‐associated membrane structures, forming a protective hydration layer on the mitochondrial surface. This PEG layer also serves as a modular platform for functionalization with biomolecules, such as peptides and antibodies, thereby broadening its biomedical applications. In this study, we examined whether mitochondrial function in target cells can be modulated using PEG‐shielded mitochondria functionalized with a cell‐penetrating peptide (CPP) via a maleimide linkage. Our results suggest that CPP‐PEG‐modified mitochondria exhibit efficient cellular internalization and are associated with increased mitochondrial respiratory activity, consistent with intracellular bioenergetic modulation. These findings suggest that spatially controlled presentation of CPP at the terminus of a PEG layer may provide an effective approach for stabilizing isolated mitochondria while modulating intracellular dynamics and functional responses. This surface engineering strategy offers a proof‐of‐concept design framework for mitochondria‐associated engineering and future bioenergetic strategies.