A novel multi-functional chimeric peptide for enhanced safe gene delivery in immunotherapy.

Chimeric peptides hold promising potential to be introduced as an ideal gene delivery platform based on their advantages over viral carriers, including but not limited to the safety profile and specific targeting. However, their gene transfer efficiency needs improvement. Here, we designed a new multi-functional chimeric peptide for enhanced gene delivery by adding a cyclic TAT motif to a previously designed MPG2H peptide to enable the targeting of cells with independent/dependent endocytosis cell entry mechanisms. CTATMPG2H was expressed and purified using affinity chromatography; then it was characterized through a gel retardation assay, circular dichroism (CD) spectropolarimetry, transmission electron microscopy (TEM) dynamic light scattering (DLS), and zeta potential analysis. CTATMPG2H was compared with MiRGD as a chimeric peptide control in all steps. After assessing the platform stability in various conditions, its gene transfer efficiency was evaluated in the HEK293T cell line with reporter genes. Additionally, mouse bone marrow-derived dendritic cells (BMDCs) were transfected to test CTATMPG2H potential in immunotherapy. The results illustrated a safe gene transfer profile for CTATMPG2H comparable to MiRGD and Polyethyleneimine (PEI). Flow cytometry results showed up to 48% gene transfer rate for CTATMPG2H to dendritic cells with minimal toxicity (viability rate ~80%). Moreover, the in silico investigation showed that the synergistic effects of electrostatic, hydrogen, and hydrophobic interactions enhance the stability and binding affinity of peptide-pDNA complexes, ensuring robust and specific targeting of nucleic acids. This research sets a foundation for future in vivo studies and potential clinical applications, aiming for safer and more effective gene therapy strategies.