Thermoresponsive pNIPAAM Hydrogels: eco-friendly synthesis and antibacterial properties

Introduction Stimuli-responsive hydrogels have found applications in various fields due to their easily tunable properties. Among them, poly(N-isopropylacrylamide) (pNIPAAM) hydrogels are well-known for their biocompatibility and temperature-sensitive behavior. Above their lower critical solution temperature, which is close to human physiological temperature, pNIPAAM hydrogels undergo polymer network contraction, which lead to the release of encapsulated active substances. This property makes them highly suitable for controlled drug release applications. Previous studies have demonstrated that a system based on vitamin C and hydrogen peroxide can initiate polymerization of various monomers. In this study, a novel initiator system, utilizing peroxidase isolated from food waste material, hydrogen peroxide, and vitamin C, was employed for the polymerization of N-isopropylacrylamide. The use of peroxidase extracted from food waste aligns with circular economy principles by valorizing waste materials. Antibacterial properties of obtained pNIPAAM hydrogels with encapsulated gentamicin were further analysed. Experimental pNIPAAM hydrogels were synthesized using an initiation system composed of peroxidase isolated from food waste material, hydrogen peroxide, and vitamin C. The polymerization was conducted under ambient conditions, leading to successful hydrogel formation with reduced reaction time. The composition of synthesized pNIPAAM hydrogels were confirmed using Fourier-transform infrared spectroscopy (FTIR). The effects of peroxidase activity on hydrogel swelling in phosphate buffer pH 6.8 at different temperatures were investigated. Additionally, the antibacterial properties of these hydrogels were evaluated. Gentamicin was incorporated post-synthesis by immersing xerogels in an aqueous gentamicin solution. Two model bacteria were chosen for this analysis: Escherichia coli K12 (E. coli; Gramm negative) and Staphylococcus capitis (S. capitis; Gramm positive). Fresh cultures of both bacteria were diluted to the same optical density (OD600=0.1) prior starting the experiment. Two mL of bacteria culture was added in falcons with samples. After 1 h, OD of all samples were measured and 5 μL of each suspension was seeded onto agar in 24 well plates. Measurement of OD and bacteria seeding was performed also after 8 h and 24 h of incubation with hydrogel samples. Control was bacterial culture starting at OD600=0.1 without any contact with tested samples during 24 h. After seeding, petri dishes were placed at 37 °C and they were imaged 24 h later using light microscope. The pNIPAAM hydrogel samples were UV sterilized prior the analysis. Results The composition of the synthesized pNIPAAM samples was confirmed by the presence of characteristic peaks of poly(N-isopropylacrylamide) in the FTIR spectra of all samples: the peak at 1524 cm⁻¹ corresponding to bending vibrations of the -NH group, the peak at 1639 cm⁻¹ corresponding to C=O stretching vibrations, and the peak at 3281 cm⁻¹ corresponding to N-H vibrations. The equilibrium swelling ratio of the hydrogels decreased with increase in temperature, confirming the thermoresponsive nature of pNIPAAM. Higher peroxidase activity led to increased swelling ratios. The pNIPAAM hydrogels were effective against E. coli after incubation of 24h.