Characterisation and toxicological evaluation of hydrogel-type polymeric biomatrials pre-and poststerilisation with a novel pulsed plasma gas-discharge system.

Abstract

This study investigated the in vitro biocompatibility and important physicochemical properties of unique N-vinyl-2-pyrrolidone–acrylic acid based copolymer hydrogels intended for use in biomedical applications. Moreover, the suitability of a novel pulsed plasma gas-discharge (PPGD) technology for the nonthermal sterilisation of such hydrogels was also evaluated. Physical hydrogels were synthesised using varying monomer concentrations in conjunction with UV340nm photopolymerisation and the photoinitiator Irgacure® 184. In addition, covalently crosslinked chemical hydrogels were prepared by supplementing the polymerisation reaction with ethylene glycol dimethacrylate based crosslinking agents. The hydrogels were analysed and characterised using a variety of appropriate analytical and mechanical testing techniques. Initial swelling and degradation studies revealed that the physical hydrogels dissolved after a short swelling period. Further analytical testing showed that considerable amounts of residual monomeric material still remained in the gels post-polymerisation. Subsequently, in vitro biocompatibility testing was performed to determine the cytotoxic and genotoxic potential of the hydrogels following direct and indirect contact with human hepatoma (HepG2) and human ileocecal adenocarcinoma (HCT-8) cells, chosen as model cell lines. More specifically, dissolved physical hydrogels were tested in appropriate cytotoxicity assays using MTT and neutral red endpoints. The presence of unreacted components in the physical networks induced severe cytotoxicity following direct cellular exposure to concentrations above 1.25 mg/ml of hydrogel solutions in cell culture medium. In addition, the cell morphology was adversely affected. Similar results were observed following cellular exposure to solid hydrogel discs in the indirect contact (agarose overlay) assay. In the alkaline comet assay the highest tested concentration of physical hydrogels (2.5 mg/ml) resulted in a slight but significant increase in DNA migration. However, this effect was believed to be exerted by the hydrogels’ cytotoxic potential rather than through an active genotoxic mechanism. Investigations carried out at the nucleotide level using the Ames assay provided no evidence of mutagenic activity associated with the physical hydrogels. Chemical hydrogels were shown to be water insoluble and their swelling capacity permitted the retention of high amounts of aqueous solution (over 20-35 times their own weight depending on the monomeric composition), yet still maintaining structural integrity. Chemical analysis suggested that the unreacted monomeric base material was efficiently removed post-polymerisation by applying an additional purification process. An investigation into the gels’ mechanical properties revealed that they may find use in wound healing applications. Promising biocompatibility data was obtained during direct and indirect contact exposure of the purified polymers to human keratinocytes (HaCaT) and HepG2 cells. No indication of significant cell death was observed using MTT, neutral red and fluorescence-based toxicity endpoint indicators. In addition, the alkaline comet assay and the Ames assay showed no genotoxic effects following cell exposure to hydrogel extracts. Findings from this study demonstrated that these chemical hydrogels are noncytoand nongenotoxic and further work should be carried out to investigate their potential as a wound healing device. Seminal investigations were carried out on the development and subsequent optimisation of the prototype PPGD system for the novel decontamination of hydrogels vi under varying operating conditions. Significant calibration and modification of this PPGD system were undertaken in order to apply this emerging technology for hydrogel sterilisation. After considerable and intensive investigations it was discovered that using O2- and N2-mediated discharges produced significant antimicrobial activities in test media at varying applied voltages. When artificially inoculated with a high microbial cell density (reaching 4.6 cfu/cm2 of either Escherichia coli or Staphylococcus aureus), PPGD effectively decontaminated the chemical hydrogels within 16 minutes of exposure at 16 kV. Preliminary material characterisation suggested that the treatment had no adverse impact on the functional material properties. However, studies into the toxicological safety showed that reactive chemical species and/or electrode degradation products can adversely influence the biocompatibility of treated hydrogels if operated for 30 min at 16 kV. Although no genotoxic effects were observed, cytotoxic changes were introduced during prolonged PPGD treatment regimes (at 30 min exposures) that were aimed at extreme microbial contamination conditions. The negative impact on the hydrogels biocompatibility was reduced by simply preincubating the hydrogels for 7 days prior to testing thus allowing reactive species to dissipate. This multidisciplinary project constitutes the first study to report on the development of novel pulsed power electrotechnologies for the nonthermal decontamination of important hydrogels. Specifically, development of PPGD as an alternative or complementary approach to hydrogel sterilisation will increase the available methods of effective decontamination. Development of this PPGD is both pressing and timely as it also coincides with a marked reduction in the amount and intensity of existing decontamination approaches that are effective, as attested by the commensurate rise in unwanted community and healthcare related infections. Findings from this project have also been presented at leading international conferences and published in leading journals