In situ Forming Hyperbranched PEG-Thiolated Hyaluronic Acid Hydrogels With Honey-Mimetic Antibacterial Properties

The rapidly increasing resistance of bacteria to currently approved antibiotic drugs makes surgical interventions and the treatment of bacterial infections increasingly difficult. In recent years, complementary strategies to classical antibiotic therapy have, therefore, gained importance. One of these strategies is the use of medicinal honey in the treatment of bacterially colonized wounds. One of the several bactericidal effects of honey is based on the in situ generation of hydrogen peroxide through the activity of the enzyme glucose oxidase. The strategy underlying this work is to mimic this antibacterial redox effect of honey in an injectable, biocompatible, and rapidly forming hydrogel. The hydrogel was obtained by thiol-ene click reaction between hyperbranched polyethylene glycol diacrylate (HB PEGDA), synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization, and thiolated hyaluronic acid (HA-SH). After mixing 500 mu L HB PEGDA (10%, w/w) and 500 mu L HA-SH (1%, w/w) solutions, hydrogels formed in similar to 60 s (HB PEGDA/HA-SH 10.0-1.0), as assessed by the tube inverting test. The HB PEGDA/HA-SH 10.0-1.0 hydrogel (200 mu L) was resistant to in vitro dissolution in water for at least 64 days, absorbing up to 130 wt% of water. Varying glucose oxidase (GO) amounts (0-500 U/L) and constant glucose content (2.5 wt%) were loaded into HB PEGDA and HA-SH solutions, respectively, before hydrogel formation. Then, the release of H2O2 was evaluated through a colorimetric pertitanic acid assay. The GO content of 250 U/L was selected, allowing the formation of 10.8 +/- 1.4 mmol H2O2/L hydrogel in 24 h, under static conditions. The cytocompatibility of HB PEGDA/HA-SH 10.0-1.0 hydrogels loaded with different GO activities (<= 500 U/L) at a constant glucose amount (2.5 wt%) was investigated by in vitro assays at 24 h with L929 and HaCaT cell lines, according to DIN EN ISO 10993-5. The tests showed cytocompatibility for GO enzyme activity up to 250 U/L for both cell lines. The antibacterial activity of HB PEGDA/HA-SH 10.0-1.0 hydrogels loaded with increasing amounts of GO was demonstrated against various gram-positive bacteria (S. aureus and S. epidermidis), antibiotic-resistant gram-positive bacteria (MRSA and MRSE), gram-negative bacteria (P. aeruginosa, E. coli, and A. baumanii), and antibiotic-resistant gram-negative strains (P. aeruginosa and E. coli) using agar diffusion tests. For all gram-positive bacterial strains, increasing efficacy was measured with increasing GO activity. For the two P. aeruginosa strains, efficacy was shown only from an enzyme activity of 125 U/L and for E. coli and A. baumanii, efficacy was shown only from 250 U/L enzyme activity. HB PEGDA/HA-SH 10.0-1.0 hydrogels loaded with <= 250 U/L GO and 2.5 wt% glucose are promising formulations due to their fast-forming properties, cytocompatibility, and ability to produce antibacterial H2O2, warranting future investigations for bacterial infection treatment, such as wound care.

Automated summary

This study aimed to mimic the antibacterial effect of medicinal honey by preparing an injectable hydrogel using hyperbranched polyethylene glycol and thiolated hyaluronic acid, with the addition of glucose oxidase to release hydrogen peroxide for antibacterial purposes. The hydrogel exhibited promising fast-forming properties, cytocompatibility, and antibacterial efficacy, making it a potential formulation for future investigations in bacterial infection treatment, particularly in wound care.