Biofilm Microenvironment‐Sensitive Anti‐Virulent and Immunomodulatory Nano‐on‐Nanodroplets to Combat Refractory Biofilm Infection Through Toxin Neutralization and Phagocytosis

Abstract Biofilm‐associated wound infection is principally perceived as the bacterial defense mechanism that hinders antibiotic penetration, causes toxin impairment, and suppresses the immunological responses of the host immune system. Several antibiofilm agents have been developed, but the least of these agents can simultaneously cornerstone on the biofilm‐associated immunosuppression and bacterial toxin‐induced cellular dysfunction. Inspired by the fusogenic property of nanodroplets and immunomodulatory functions of metal nanoparticles, biofilm targeted anti‐virulent immunomodulatory cationic nanoparticle shelled nanodroplets (C‐AgND) is fabricated to completely disintegrate and eradicate the Staphylococcus aureus (S. aureus) biofilm. The specific binding of C‐AgND neutralizes the negatively charged EPS layer, causing their destabilization followed by penetration of the nanoformulation into the biofilm matrix, killing the persister cells. Consequently, C‐AgND eliminates the virulence property of the S. aureus biofilm through α‐hemolysin neutralization. C‐AgND promotes a strong immunomodulatory effect by polarizing macrophages into their M1 phenotype to induce phagocytosis of the disintegrated biofilm‐released residual cells, rejuvenating the host's innate immune responses for the complete eradication of the biofilm. Moreover, the ex vivo skin wound infection model illustrates an excellent biofilm eradication efficacy of C‐AgND in comparison to the commercial ones, rendering them to be a promising replacement of existing antibiofilm agents in clinical application.