(99d) ?-Lactamase Responsive Hydrogels for Bacteria-Triggered Antibacterial Treatments

Antibiotic resistance is a major global threat increasing the difficulty of treating bacterial infections. Overuse and misuse of antibiotics has contributed to the rise in antibiotic resistance. There is a need for smart drug delivery systems that can self-regulate release of antibacterial agents to localize delivery, concentrate the drug at the site of infection, and reduce off-site toxicity to the microbiome, potentially lowering susceptibility to antibiotic resistance development. In this talk, I will describe our recent work on developing hydrogel technologies that respond to β-lactamases (βLs) (bacterial enzymes which are among the leading causes of antibiotic resistance) as a platform for bacteria-triggered release of encapsulated antibacterial agents.

We have functionalized a βL-cleavable compound with maleimides on both termini and utilized it as a crosslinker in the synthesis of responsive hydrogels through end-crosslinked polymerization with multi-arm thiol-terminated poly(ethylene glycol) (PEG) macromers via Michael-type addition. Bismaleimide PEG was used to form non-βL-degradable hydrogel controls. We observed that only hydrogels containing the βL-responsive moiety decreased in size and wet mass in the presence of βLs produced by three different bacterial species. The hydrogels degraded completely within 5 hours when incubated with 1 units(U)/mL βL from Enterobacter cloacae, 8 hours with 30 U/mL βL from Pseudomonas aeruginosa, and 9 hours with 400 U/mL with βL from Bacillus cereus (here 1 U is defined as hydrolyzing 1.0 µmol of benzylpenicillin per minute at pH 7.0 at 25°C). Control non-responsive hydrogels remained stable in βLs from all three species. Hydrogel degradation rates were further tuned by changing polymer density (complete degradation in ~13, 8, 3.5 hours for 15, 10, and 5% w/v hydrogels in 30 U/mL βL from P. aeruginosa, respectively).The release profile of fluorescent polystyrene nanoparticles (NPs) encapsulated within these hydrogels as model cargo tracked the βL-responsive hydrogel wet mass decrease rates, while no NPs were released from non-responsive hydrogels, indicating degradation-controlled cargo release. Hydrogels exhibited an on-off response when βLs were added or removed, respectively, further demonstrating their tunable βL-responsive behavior. The responsive hydrogels were found to degrade specifically when cultured with βL-producing strains of P. aeruginosa and B. cereus but remained stable in blank media or with a non-βL producing strain of Staphylococcus aureus. Again, the non-responsive hydrogels remained stable under all conditions and did not release any detectable NPs. To better mimic skin and soft tissue infections, we tested hydrogel degradation by βL-producing bacteria on infected agar or ex vivo porcine skin as opposed to shaking in broth and found similar results.

To demonstrate the versatility of this approach, we have also formulated supramolecular βL-responsive hydrogels with self-healing capabilities. These hydrogels were synthesized with the same βL-cleavable compound utilized in our PEG hydrogels, but this compound was now functionalized on both termini with adamantane moieties. The hydrogel was formed via non-covalent host-guest interactions of the adamantane moieties with polycarboxymethyl-β-cyclodextrins along with forming an interpenetrating network based on free-radical polymerization of acrylamide and N-vinylpyrrolidinone. As with the covalent PEG hydrogels, these hydrogels selectively degraded in the presence of βLs. They also have the added benefit of self-healing promoted by the host-guest interactions of the adamantane and cyclodextrin, which can increase their utility in environments subject to mechanical disruption. The βL-responsive hydrogel technologies that we developed have the potential to be used as prophylactic biomaterials, such as bandages or in situ forming injectable hydrogels, allowing for triggered therapeutic release, thereby improving management of infections, limiting exposure and reducing susceptibility to antibiotic resistance.