A novel methodology for hydrogel water content determination by EPR: The basis for real-time monitoring of controlled drug release and hydrogel swelling and degradation

The Release of a Highly Cytotoxic Paullone Bearing a TEMPO Free Radical from the HSA Hydrogel: An EPR Spectroscopic Characterization

“…The 3CP/HSA hydrogel was prepared in the same ligand-to-protein molar ratio as the 1:10 HL/HSA gel and investigated using the same protocol. As determined previously [74], 3CP does not bind to HSA, and the 3CP/HSA hydrogel displays an isotropic three-line EPR signal (Figure 4, black line). Upon 3CP/HSA hydrogel dialysis in physiological saline at room temperature, it was determined that already after 1 h, 96% of 3CP had diffused out (Figure 4, red line; note that the y-axis is magnified by 100× compared to the black line).…”

“…The successful use of spin-labeled albumin hydrogels for EPR characterization of ligand binding and release, protein matrix degradation, and the rate of ligand diffusion from the hydrogel water pores was demonstrated in this work. Together with our previous findings showing their applicability for cell viability assessment [95] and hydrogel water content determination [74], the role of state-of-the-art EPR for biomedical applications is emphasized, particularly in cancer research. Finally, the fact that the HL/HSA hydrogel did not exhibit cytotoxic activity in the Colo 205 human cancer cell line during the 72 h exposure time indicates that it acts as an efficient reservoir for the active ligand, even in the presence of living cells.…”

“…The first type of hydrogel utilized for matrix degradation evaluation was spin-labeled with a fatty acid derivative 5-DS. The second, used to study the rate of water diffusion from the hydrogel pores, contained the water-soluble spin probe 3CP, which has been previously shown to be located only in the hydrogel water pores and unable to bind to HSA [74]. The results showed that HL displacement after 7-8 and 11 days is the consequence of hydrogel degradation, attributed to the weakening of the HL-HSA interaction upon physiological saline uptake.…”

“…The HL/HSA hydrogels were prepared by heating the 1:10 and 1:20 HL/HSA stock solutions for 40 min at 75 • C [74]. It should be highlighted that increased temperature does not affect the EPR spectrum of HL.…”

“…Therefore, in order to gain more information about the rate of HL diffusion from the hydrogel water pores, a third type of spin-labeled HSA hydrogel was prepared. The idea was to take advantage of the recently reported methodology for hydrogel water content determination using the spin probe 3CP [74]. This EPR-active molecule was shown to be entirely dissolved in the water phase of the hydrogel, not interacting with the protein matrix.…”