Application of Cell-Penetrating Peptides (CPP) to Enhance the Efficacy of Antisense Peptide Nucleic Acids (PNA) Molecules Delivery

Peptide nucleic acids (PNAs) are synthetic molecules with a unique N-(2-aminoethyl)glycine backbone that offer increased stability and resistance to degradation compared to DNA/RNA. The ability of these compounds to bind complementarily to DNA or RNA, forming various stable hybrids, makes them hold potential for applications in gene therapy and molecular diagnostics. Nonetheless, the poor ability of PNAs to cross cell membranes required the use of delivery systems. This study aimed to establish and optimize non-covalent PNA, and cell penetrating peptide (CPP) complexes and evaluate their efficiency thereby establishing a tool for future targeted therapeutic applications. This study utilized three different CPPs to deliver PNA, demonstrating successful cell membrane crossing in vitro. We optimized PNA and CPP concentrations and evaluated the efficiency of the CPPs. The CPPs used were TP10, Tat, and TD2.2. The PNAs and CPPs were complexed via non-covalent binding. Each CPP molecule was designed to contain a nuclear localization signal (NLS) fragment at their 3’ end to enhance nuclear delivery. Research methods included cellular fluorescence measurement, high-performance liquid chromatography (HPLC) analysis, western blot, and cell viability assessment using the MTT assay. We optimized PNA and CPP concentrations and evaluated the efficiency of the CPPs, finding that TP10 was more effective in comparison with TD2.2 and Tat. This approach effectively delivered PNAs to their specific location. The obtained results demonstrated the effective delivery of PNAs combined with CPPs to their specific cellular location thus suggesting that this may pose a promising strategy for analogous targeted molecular applications.