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Conformational stability of human interferon-gamma on association with and dissociation from liposomes
M L van Slooten1, A J Visser, A van Hoek
1Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Pharmacy, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands.
Insights
Recombinant human interferon gamma (hIFNgamma) maintains its protein structure when interacting with liposomes, showing full conformational stability after desorption. This ensures therapeutic protein integrity in liposomal drug delivery systems.
Area of Science:
- Biophysical Chemistry
- Drug Delivery Systems
- Protein Science
Background:
- Therapeutic protein integrity is crucial for efficacy in delivery systems like liposomes.
- Understanding protein-liposome interactions is key to developing stable formulations.
- Recombinant human interferon gamma (hIFNgamma) is a therapeutic protein requiring careful formulation.
Purpose of the Study:
- To investigate the conformational stability of hIFNgamma during and after interaction with liposomes.
- To model hIFNgamma-liposome interactions using adsorption/desorption from empty liposomes.
- To assess the impact of liposome preparation methods on protein conformation.
Main Methods:
- Circular dichroism (CD) spectroscopy to analyze protein secondary and tertiary structure.
- Steady-state and time-resolved fluorescence spectroscopy to probe the tryptophan (Trp-36) environment.
- Acrylamide quenching studies to assess Trp-36 accessibility.
Main Results:
- CD studies showed no significant changes in hIFNgamma secondary or tertiary structure upon liposome interaction.
- Fluorescence spectroscopy indicated that the Trp-36 environment and accessibility were largely unaffected by adsorption/desorption.
- Time-resolved fluorescence revealed protein immobilization on liposomes and subtle Trp-36 environment changes during liposome preparation.
Conclusions:
- Association with negatively charged liposomes causes minimal structural changes to hIFNgamma.
- All applied techniques confirm full retention or restoration of protein conformation after desorption.
- hIFNgamma's conformational integrity is maintained in liposomal formulations, supporting its use in drug delivery.
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 (hIFNgamma) 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 hIFNgamma adsorption to and desorption from empty liposomes as a model for hIFNgamma-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 hIFNgamma with the liposomes. Steady-state fluorescence emission spectra of untreated and liposome-desorbed hIFNgamma 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 hIFNgamma 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 hIFNgamma with the liposomal membrane. Differences in rotational correlation time between free and liposomal hIFNgamma were attributed to immobilization of the protein on adsorption to the liposome bilayer. Disparities were detected between the average lifetimes of liposome-adsorbed hIFNgamma and hIFNgamma-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 hIFNgamma to the liposome surface. The results of this study indicate that association of hIFNgamma 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.
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