Sonodynamic Therapy with HMME@AMP Conjugate: Efficient Bactericidal Efficacy through Bacterial Structural Damage and Critical Gene Downregulation in Pathogenic Bacteria.

To combat the growing challenge of bacterial infections, sonodynamic antibacterial technology has gained increasing attention. However, current approaches still face limitations, including suboptimal efficacy and a narrow antibacterial spectrum (only targeting a single bacterial species). To overcome these drawbacks, this study designed a composite sonosensitizer, HMME@AMP, by conjugating hematoporphyrin monomethyl ether (HMME) with the antimicrobial peptide LL-37. This composite achieves efficient inhibitory effects against representative Gram-negative and Gram-positive bacteria through sonodynamic therapy (SDT). Using optimized ultrasound parameters (0.5 W/cm, 1 MHz, 60% duty cycle), we evaluated the antibacterial activity and mechanisms of HMME@AMP through agar culture, flow cytometry, bacterial weight measurement, scanning electron microscopy (SEM), whole-genome sequencing, and quantitative real-time PCR (qPCR). Results demonstrated that HMME@AMP exhibited strong, concentration-dependent antibacterial effects against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) under ultrasound irradiation. At a concentration of 120 μg/mL, bacterial weight measurement results showed that the bacterial weight inhibition rates reach 56.4% (for P. aeruginosa) and 87.3% (for), while flow cytometry indicated survival rates below 3 and 7%, respectively, confirming its excellent inhibitory ability against P. aeruginosa and S. aureus. SEM imaging revealed ultrasound-induced nanoscale pores and membrane collapse, indicating combined physical damage and ROS-mediated oxidative stress. Genome sequencing and qPCR further showed that HMME@AMP downregulated critical genes in P. aeruginosa (e.g., PA0876, PA4896, involved in phenazine synthesis and iron homeostasis) and S. aureus (e.g., SAOUHSC-02494, linked to ribosome function), disrupting bacterial metabolism and proliferation. This study demonstrates that HMME@AMP-mediated SDT achieves potent antibacterial effects through dual mechanismsphysical disruption and genetic regulationoffering a promising, antibiotic-free strategy for treating deep-tissue infections.