(351d) Bacteria-Triggered Antibiotic Releasing Hydrogels for Treatment of Wound Infections

Wound infections have become a pressing global health concern due to the increasing prevalence of antibiotic resistance and the stagnation of new drug discovery. The misuse and overuse of antimicrobial drugs have worsened this crisis, necessitating the development of new biomaterials with infection-triggered drug delivery capabilities that have the potential to limit exposure to these antimicrobials while still providing effective infection eradication. Hydrogels, three-dimensional networks of hydrophilic polymers that can effectively entrap antimicrobial agents, have gained significant attention in the field of controlled drug delivery due to their potential for tunable release. Hydrogels can be designed for bacteria-specific triggered drug release by incorporating degradable elements recognizable by bacteria-produced enzymes, such as β-lactamases. β-lactamases are among the leading causes of antibiotic resistance, and their β-lactam ring hydrolysis activity can be utilized to develop enzyme-responsive hydrogels that release their cargo upon sensing β-lactamases. Here we developed β-lactamase-responsive hydrogels and investigated their ability to eradicate wound infections on demand.

To formulate β-lactamase-responsive hydrogels (R-hydrogel), a maleimide functionalized β-lactamase-cleavable cephalosporin (β-lactam core) was used to crosslink multi-arm-poly(ethylene glycol) (PEG)-thiol macromers. Non-responsive hydrogels (NR-hydrogel) were also prepared with maleimide-PEG-maleimide as a crosslinker. The backbone polymer of the hydrogels was tagged with Cy5 dye in order to track hydrogel degradation. For effective entrapment inside the hydrogel, ciprofloxacin, a broad-spectrum fluoroquinolone antibiotic, was actively loaded into saturated hydrogenated soy phosphatidylcholine (HSPC) liposomes using a transmembrane gradient of ammonium ions. These ciprofloxacin liposomes (CIP-LIPO) exhibited a hydrodynamic diameter of ~104 nm and drug loading of ~7.5% (w/w); for drug-loaded hydrogels, CIP-LIPO were incorporated during hydrogel gelation. The antibacterial properties and degradation of R-hydrogels were tested against Pseudomonas aeruginosa, a common gram-negative pathogen that leads to difficult to treat wound infections. The luminescent strain, P. aeruginosa Xen-41, was confirmed to produce β-lactamases and used in our studies. The degradation of R-hydrogels triggered by β-lactamases was observed within three days of incubation with Xen-41 on agar, while no degradation was observed for NR-hydrogels. R-hydrogels did not degrade when exposed to a non-β-lactamase-producing bacteria, Staphylococcus aureus Xen-29.

The behavior of R-hydrogels, degradation, and antibacterial activity were investigated in vivo in a Xen-41-infected murine superficial skin wound model induced using a tape-striping approach. The progression of infection was monitored daily for both treated and non-treated groups using an in vivo imaging system (IVIS). IVIS showed an increase in bioluminescence from Xen-41-infected wounds over four days of infection. When non-drug loaded R-hydrogels were applied to infected wounds, a dissipation of the R-hydrogel fluorescence signal (i.e., Cy-5) was observed over four days due to hydrogel degradation. The NR-hydrogels did not degrade during the four-day study on infected wounds. When R-hydrogels loaded with CIP-LIPO (5 μg ciprofloxacin/hydrogel) were applied, the infection was eradicated within one day of treatment (i.e., no bacteria luminescence observed); the infection did not reoccur over four days of monitoring the animals. In contrast, the clinically available treatment, Silvasorb (500 μg/mouse), reduced the infection but failed to prevent the reoccurrence of infection after one day of treatment. Our results demonstrate the potential of enzyme-responsive hydrogels for use in on-demand elimination of infections, which can reduce unnecessary exposure to antimicrobials and lower susceptibility to antibiotic resistance.