Intracellular Proteins Targeting with Bi‐Functionalized Magnetic Nanoparticles Following their Endosomal Escape

Abstract The specific targeting of intracellular proteins or organelles by magnetic nanoparticles (MNPs) is a major challenge in nanomedicine, as most MNPs are internalized by cells through endocytosis and remain trapped inside small intracellular vesicles, limiting their ability to reach intracellular components. Furthermore, this phenomenon limits their heating capacity in magnetic hyperthermia, and therefore their potential for cancer treatment. This study presents a strategy based on an original double functionalization of MNPs, with polyhistidine peptides (PHPs) triggering endosomal escape and antibodies targeting specific cytosolic proteins. Negatively charged γ‐Fe ₂ O ₃ @SiO ₂ MNPs with diameter smaller than 50 nm are functionalized with zwitterionic and thiol groups at their surface. These sulfhydryl groups are used to graft PHPs through a labile link, allowing the peptide to be released from the MNPs’ surface once in the cytosolic reductive environment. This severing avoids any interaction between these peptides and intracellular components, which can hinder MNPs’ intracellular mobility. The second MNPs’ surface functionalization is performed through a non‐labile link with antibodies targeting specific cytosolic proteins, namely HSP27 thermosensitive proteins, for this inaugural proof of concept. Bi‐functionalized MNPs are able to successfully target the intracellular protein of interest, opening the door to promising biomedical applications of MNPs, in cellular engineering and magnetic hyperthermia.