Supercritical Carbon Dioxide-Peracetic Acid Treatment Inactivates Low-Level Bacterial Inocula in Collagen Analogue-Phosphorylcholine Hydrogels while Preserving Functional Properties

Terminal sterilization of hydrogels is challenging because their high-water-content, chemical structures, and cell-friendly properties are often altered by conventional sterilization methods. Hydrogel implants comprising collagen-like peptide (CLP), polyethylene glycol (PEG), and poly (2-methacryloyloxyethyl phosphorylcholine) (MPC) showed promise in promoting corneal regeneration in preclinical animal studies. However, before these CLP-PEG-MPC implants can be used clinically, they must be sterilized effectively to ensure patient safety. Here, we evaluated supercritical carbon dioxide (scCO₂) with peracetic acid (PAA) as a potential sterilization technique for hydrogels. CLP-PEG-MPC was treated with scCO₂ containing PAA and compared against 15 kGy of electron beam irradiation (E-beam) and chloroform (CF) sterilization. Several samples were spiked with 200 colony-forming units (CFU) of either Pseudomonas aeruginosa or Staphylococcus aureus prior to sterilization to compare the bactericidal efficacy of the three sterilization methods. Only scCO₂-PAA effectively inactivated all the inoculated bioburden. Non-spiked hydrogels were also sterilized and then compared for complement activation, changes to their physical and mechanical properties, and biocompatibility. Optical clarity was unchanged regardless of sterilization mode. scCO₂-PAA did not alter the mechanical properties of the hydrogels but resulted in a modest lowering of corneal epithelial cell proliferation, while E-beamed hydrogels showed reduced mechanical strength. In conclusion, newer scCO₂-PAA terminal sterilization preserves the functional properties of soft hydrogels, such as CLP-PEG-MPC, including transparency, which is needed for corneal implants.