Conformational stability of human interferon-gamma on association with and dissociation from liposomes

Abstract The integrity of a therapeutic protein has to be safeguarded when formulated in delivery systems such as liposomes. In this study, we investigated the conformational stability of recombinant human interferon gamma (hIFNγ) on association with and after dissociation from liposomal bilayers using circular dichroism (CD) and steady‐state fluorescence spectroscopy as well as time‐resolved fluorescence methodology. We used hIFNγ adsorption to and desorption from empty liposomes as a model for hIFNγ‐containing liposomes prepared via the film hydration method. CD studies indicated that no changes in the secondary and tertiary protein structure occur during and after interaction of hIFNγ with the liposomes. Steady‐state fluorescence emission spectra of untreated and liposome‐desorbed hIFNγ revealed that the environment of the sole Trp residue was not affected by the adsorption/desorption process. The Trp‐36 residue remained fully quenchable by acrylamide after desorption of hIFNγ from the liposomes. Time‐resolved fluorescence studies were conducted to probe the local environment and the mobility of Trp‐36 before, during, and after interaction of hIFNγ with the liposomal membrane. Differences in rotational correlation time between free and liposomal hIFNγ were attributed to immobilization of the protein on adsorption to the liposome bilayer. Disparities were detected between the average lifetimes of liposome‐adsorbed hIFNγ and hIFNγ–liposomes, indicating that subtle changes in the Trp‐36 environment took place during preparation of the liposomes via the film hydration method compared with the adsorption of hIFNγ to the liposome surface. The results of this study indicate that association of hIFNγ with negatively charged liposomes results in minimal changes in the secondary and tertiary structure of the protein. We conclude that all techniques used point to a full retention or restoration of the protein conformation after desorption from the liposomes. © 2000 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 89: 1605–1619, 2000