Structural studies of proteins incorporated in ordered mesoporous silica

Author(s)

Jéssica Aparecida Ferreira Pedro

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Física (IF/SBI)
• Defense date: 2022-05-27

Examining board members

José Luiz de Souza Lopes; Tereza da Silva Martins; Silvia Helena Pires Serrano

• Advisor: José Luiz de Souza Lopes

Abstract

Ordered mesoporous silicas, such as SBA-15, are excellent carriers for protein encapsulation, mainly because they have adjustable textural and structural properties, as well as the possibility of chemically modifying their surface. It is already known that SBA-15 can provide proteins with greater structural and/or functional stability when exposed to denaturing conditions, and this protective aspect is essential for their use in therapeutic applications. In addition, in recent years, the adjuvant properties of SBA-15 have also been studied through the incorporation of proteins and antigenic domains for vaccine production, including oral formulations. However, for safe and effective applications, some challenges related to the particularities of each incorporation process must still be overcome, and it is important to carry out studies aimed at the characterization of hybrids produced with different proteins. In this work, three proteins representing different hierarchical classes of protein structures (all-alpha, all-beta and low regular content) were individually incorporated into SBA-15 particles in two silica: protein ratios (10:1 and 5:1, w/w), in order to evaluate the adsorption and possible structural changes in the incorporated proteins. The secondary structures of proteins in aqueous solutions were previously analyzed by conventional circular dichroism (CD) spectroscopy and by CD with synchrotron radiation under different conditions of temperature and pH. These analyzes established the thermal stability limits of these proteins (up to 40 °C in pH 7.0 solution) that were considered during the incorporation procedure. The characterizations of SBA-15 particles were performed by Scanning Electron Microscopy (SEM), Low Angle X-Ray Scattering (SAXS) and by Nitrogen Adsorption Isotherms (NAI) measurements, which indicated particles with large surface area (781.5 m²/g) and high pore volume (1.5 cm³/g). Regardless of the immobilized protein, the incorporation process reduced the textural parameters by 20 to 30% in the composites, when considering the effect of competition with ions in the buffer solution. SAXS measurements suggested the presence of higher oligomeric states (and/or possible aggregate states) that can be related to the protein concentration used during the incorporation processes. Thermogravimetric (TG) analyzes indicated that the degradation process of incorporated proteins is initiated at higher temperatures (20 to 30°C) as a result of new intermolecular interactions established between them and the silica surface. Fourier Transform Infrared (FTIR) vibrational spectroscopy analyzes suggest that the predominant secondary structure for each protein was conserved in the composites. Furthermore, fluorescence analyzes indicated that tryptophan residues are, in general, better protected from interactions with the aqueous environment. Based on these results, the synthesized SBA-15 presented suitable characteristics as support for the three protein models. From these results, the synthesized SBA-15 proved to be adequate to support the three protein models evaluated. (AU)

• FAPESP's process: 19/19567-7 - Structural studies of proteins incorporated in ordered mesoporous silica
• Grantee: Jéssica Aparecida Ferreira Pedro
• Support Opportunities: Scholarships in Brazil - Master