Cell-penetrating peptides (CPPs) have gained attention as an efficient strategy for improving the intracellular delivery of nanocarriers, particularly in drug and gene delivery applications. Among nanocarriers, magnetite nanoparticles (MNPs) are widely explored due to their biocompatibility, ease of functionalization, and potential in targeted delivery. However, the functionalization method and specific motif placement in CPP conjugates can significantly influence cellular uptake and endosomal escape, thereby affecting the efficiency of bioactive molecule delivery. This study investigates the impact of the positional arrangement of a cell-penetrating motif (LFVCR) on the internalization efficiency, endosomal escape, and biocompatibility of MNP-based nanobioconjugates.
Methods
Three distinct nanobioconjugates were synthesized by attaching the LFVCR motif at different positions (N-terminal, middle, and C-terminal) within designed CPP sequences. The peptides were immobilized onto polyethylene glycol (PEG)-functionalized MNPs via carbodiimide-based coupling. Physicochemical characterization was performed using Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Biocompatibility was assessed via hemolysis, platelet aggregation, and cytotoxicity assays on Vero cells. Cellular uptake and endosomal escape were evaluated using fluorescence-based confocal microscopy, with and without inhibitors of clathrin-mediated endocytosis.
Results
The study demonstrated that motif positioning significantly influenced cellular uptake and intracellular trafficking. The nanobioconjugate with the motif positioned at the N-terminal exhibited the highest uptake efficiency but showed moderate endosomal escape. Conversely, the C-terminal motif placement resulted in improved endosomal escape, likely due to optimized exposure of critical residues for interaction with cellular uptake pathways. All nanobioconjugates displayed high biocompatibility, with negligible hemolysis and no significant platelet aggregation. Comparative analysis with reference CPP-based nanocarriers further highlighted the improved performance of the designed nanobioconjugates, making them suitable candidates for drug and gene delivery applications.
Conclusions
These findings underscore the importance of motif positioning in CPP-based nanocarriers for optimizing intracellular delivery. The enhanced internalization and endosomal escape properties of the designed nanobioconjugates highlight their potential for efficient delivery of bioactive molecules. Future work will focus on in vivo validation and expansion to other bioactive cargoes, facilitating their translation into therapeutic applications.