Screening of Cell-Penetrating Peptides To Enhance the Therapeutic Efficacy of α-Helical Antimicrobial Peptides against Intracellular Bacterial Infections

Intracellular bacterial infections pose a major health challenge. Antimicrobial peptides (AMPs) are promising anti-infective agents, but their efficacy against intracellular pathogens is limited by poor cellular uptake. Cell-penetrating peptides (CPPs) have been employed to improve AMP delivery and intracellular bactericidal activity. However, due to the diversity in CPP types and cellular uptake mechanisms, the optimal CPPs for AMP delivery remain unknown. Herein, six representative CPPs were conjugated to the model α-helical AMP HC1, and the resulting conjugates were systematically evaluated for cellular uptake efficacy and in vitro and in vivo intracellular antibacterial activity. Bac7-HC1 emerged as the most effective conjugate against intracellular bacterial infections. The results highlight that cationic or amphipathic CPPs, particularly those with intrinsic antimicrobial activity and high positive charge, can enhance the anti-infective performance of HC1-based conjugates in this context. This work identifies Bac7-HC1 as a lead candidate for treating intracellular infections and provides preliminary insights for the design of CPP-based AMP therapeutics.