Activation of photosensitive proteins with radioluminescent nanoparticles and X-rays

Author(s)

Mariana Chaves Micheletto

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-11-28

Examining board members

Antônio José da Costa Filho; Mauricio da Silva Baptista; Alessandro Lauria

• Advisor: Antônio José da Costa Filho; Éder José Guidelli

Abstract

Photodynamic therapy (PDT) is traditionally limited due to the poor penetration of ultraviolet-visible (UV-vis) light in human tissues. However, advanced technologies allow the excitation of photosensitizers (PS) at different wavelengths, providing opportunities to discover compatible scintillators. A more efficient complex of nanoparticles and photosensitizers can be designed through a further understanding of the properties of each component of the system. This research focuses on the innovative application of X-rays as an alternative to UV-vis light for deeper tumor excitation in PDT. Scintillating nanoparticles (ScNP) are required as intermediates to convert the energy of X-rays into visible light. The role of PS is fulfilled by genetically encoded proteins. We explore the interaction of the eGFP, KillerOrange, and KillerRed proteins with ScNP LaF3:Tb3+ in terms of their physicochemical properties and energy transfer, while also investigating the structure, stability, and function of such proteins under adverse physiological conditions and X-ray irradiation. In a second system of project interest, we doped Hafnium Oxide (HfO2) nanoparticles with titanium (Ti) to enhance their luminescence properties and created a stable dispersion in PBS buffer. To stabilize the nanoparticles in PBS, we coated them with citric acid (CA). As the PS in this case, we used the miniSOG protein with its C-terminal modified by the insertion of a glutamate (E) sequence, forming miniSOG-PolyE. The proteins exhibited stability under harsh conditions experienced during cancer therapies. Biophysical characterization of the proteins, combined with tests using ionizing radiation, revealed minimal protein interaction with X-rays. We demonstrated the energy transfer from ScNP to eGFP, KO, and KR. The system consisting of eGFP, KO, and KR conjugated to LaF3:Tb3+ nanoparticles proved efficient in preventing bacterial culture growth, likely through the generation of reactive oxygen species. The genetic modification that resulted in miniSOG-Poly-E increased the binding with the HfO2 nanoparticle and stabilized the colloidal dispersion in environments similar to biological systems. We propose these systems as promising pathways for the use of genetically encoded photosensitizers in PDT applications with X-rays Further research on this topic could pave the way for more effective cancer therapies and shed light on the interaction of scintillating nanoparticles and proteins to create a conjugated nanocomposite with greater dispersion stability in biological media. (AU)

• FAPESP's process: 18/13016-6 - Activation of Photosensitive Proteins with Radioluminescent Nanoparticles and X-Rays
• Grantee: Mariana Chaves Micheletto
• Support Opportunities: Scholarships in Brazil - Doctorate