Chronic wounds are those whose healing process is stalled or progresses slowly. These wounds pose significant physical, psychological, and financial burdens on patients and healthcare systems. Monitoring and controlling protease activity is considered a strategy for more efficient management of such wounds. This research aimed to develop a casein-based structure that could provide information about protease activity by disappearing or degrading, and simultaneously control their activity. Casein, which constitutes about 80% of milk proteins, was used as a relatively inexpensive protein that has been employed in very few studies as a biomaterial for medical applications and can be degraded by a wide range of proteases. In this research, initially structures based on casein coagulation at its isoelectric point were fabricated, which were stable in deionized water but not in phosphate-buffered saline (PBS) solution. Thermal treatment was employed as an eco-friendly method to stabilize them in PBS solution. This process resulted in the loss of the structures’ porosity, making them resemble non-porous films. Considering this, and the relatively challenging and time-consuming process of sample fabrication using the coagulation method, films were later fabricated using the solvent casting method, and their potential for the intended purposes was eva‎luated. A casein film was produced by casting a 12% (w/w) casein solution containing glycerol (50% of casein weight). It was shown that the duration of thermal treatment significantly affected the mechanical properties, water absorption, and degradation rate, with an increase in treatment time from 30 minutes to 3 hours leading to a 4.2-fold increase in stress tolerance, 2-fold reduction in water absorption, and a significant increase in resistance to hydrolysis in proteolytic environments, as quantified by the ninhydrin assay. The sample heat-treated for 30 minutes, selected as the optimal sample, showed sensitivity at a level of 0.2 µg/mL to human neutrophil elastase (HNE) by disintegrating. Given that concentrations higher than this amount have been reported for HNE in chronic wounds, the developed structure is expected to show sufficient sensitivity to excessive HNE activity in wounds. The effect of the optimal film on the activity of Bacillus protease (BSP) and porcine pancreas elastase (PPE) was also eva‎luated. The activity of these two proteases in the fluids resulting from the incubation of casein film with them was 30-36% lower than the control groups. The antioxidant properties of the fluids from the incubation of films with BSP, PPE, and HNE proteases were measured using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, and inhibitory effects ranging from 45% to 96% were observed. The cytotoxicity of the degradation products of the casein film on L929 fibroblast cells was assessed, and no toxic effects were observed; rather, cell viability seemed to increase in the group exposed to degradation products. The morphology of L929 fibroblast cells cultured on the film was also eva‎luated, and cells adhered well to the sample. Overall, the findings of this study indicate the potential of the developed casein film for detecting and controlling excessive protease activity, the beneficial effects of its degradation products for wound healing, and its role as a substrate for cell adhesion.

لاله قاسمي مباركه , فرزانه علي حسيني

صديقه برهاني , مهشيد خرازيهاي اصفهاني , محمد رفيعي نيا