Multifunctional Framework Nucleic Acid Vehicle for Antibiotic Sensitization and Treatment of Methicillin‐Resistant Staphylococcus aureus

ABSTRACT The increasing prevalence of methicillin‐resistant Staphylococcus aureus (MRSA) due to antibiotic misuse necessitates novel therapeutic strategies to counter multidrug‐resistant infections. Here, we introduce a self‐assembling, aggregation‐enhanced tetrahedral DNA nanostructure (tFNA) platform that achieves targeted drug delivery through controlled aggregation and sustained release, effectively restoring MRSA susceptibility to β‐lactam antibiotics. These tetrahedral frameworks, termed tFNAs‐ASOs‐ceftriaxone sodium (TACs), serve as a dual‐functional system that co‐encapsulates antisense oligonucleotides (ASOs) targeting the mecA gene and the β‐lactam antibiotic ceftriaxone sodium (Cef). Aggregation of TACs plays a pivotal role in maximizing drug retention and stability, prolonging the localized release of both ASOs and antibiotics while maintaining high bioavailability at the infection site. Characterization studies, including size distribution, zeta potential, and fluorescence quenching assays, confirm their robust aggregation stability and encapsulation efficiency, ensuring controlled drug kinetics and prolonged therapeutic effects. Upon interaction with bacterial cells, the locally concentrated TACs facilitate efficient ASO‐mediated mecA silencing, thereby disrupting PBP2a expression and re‐sensitizing MRSA to β‐lactams. Simultaneously, the aggregated ceftriaxone sodium reservoir ensures sustained inhibition of bacterial cell wall synthesis, leading to effective bacterial clearance. In addition, TACs display potent antibiofilm activity by penetrating the biofilm matrix and delivering therapeutics directly to the embedded bacterial population, thereby overcoming the diffusion barriers. In vivo, TACs exhibit superior therapeutic efficacy in an MRSA‐induced pneumonia mouse model, significantly improving survival rates, reducing bacterial burden, and mitigating lung tissue damage. These findings highlight the transformative potential of tFNAs as an intelligent drug aggregation and release system, offering a novel paradigm for optimizing antibiotic therapy against multidrug‐resistant pathogens.