Development and application of theranostic biomaterials: evaluation of cellular response to fluorescent nanoparticles of lanthanide and magnesium-doped calcium phosphates and its applicability as non-viral vectors for gene therapy

• Grant number: 19/16187-9
• Support Opportunities: Scholarships abroad - Research
• Effective date (Start): August 26, 2019
• Effective date (End): February 25, 2020
• Field of knowledge: Engineering - Materials and Metallurgical Engineering - Nonmetallic Materials
• Principal Investigator: Flávia Rodrigues de Oliveira Silva
• Grantee: Flávia Rodrigues de Oliveira Silva
• Host Investigator: Pall Thordarson
• Host Institution: Instituto de Pesquisas Energéticas e Nucleares (IPEN). Secretaria de Desenvolvimento Econômico (São Paulo - Estado). São Paulo , SP, Brazil
• Research place: University of New South Wales (UNSW), Australia

Abstract

Theranostic agents have emerged as a powerful multifunctional modality to integrate therapeutic and diagnostic strategies in a single all-in-one particle. The versatility of these structures based on nanomaterials finds great applicability in the field of gene therapy, a therapeutic approach that can, theoretically, be used for the treatment and prevention of any disease. It is a technique based on the transfection of therapeutic genes by replacing or silencing defective genes. The major challenge of gene therapy is the successful delivery of nucleic acids into their destination. Different carrier vehicles have been investigated: viral vectors, naked DNA and non-viral vectors. Viral vectors are by far the most studied one, although achieving higher efficiencies, their envelope protein biosafety is the major limitation. Synthetic non-viral vectors, as nanoparticles, are increasingly being considered as great alternatives. Studies have demonstrated high efficiency and biocompatibility of calcium phosphate (CaP) nanoparticles, in the form of HA and B-TCP, as vectors when compared to other non-viral gene carriers. The greatest difficulty in using the CaP as a vector is the synthesis of homogeneous and monodisperse particles of nanometric dimensions (10 to 200 nm) to allow the complexation of the therapeutic genes to the CaP vector. In this context, the development of Lanthanides and/or Magnesium-doped CaP is very interesting, because replacing Ca2+, Mg2+ and Ln3+ produces smaller (due to the atomic radius) and positively charged nanoparticles, facilitating the complexation with the gene. In addition, Ln3+ also makes the biomaterial a fluorescent vector that allows its bioimaging in vitro and in vivo, monitoring the delivery of the nucleic acid, making it a multifunctional theranostic nanomaterial. In addition, the complexation of CaPs with organic molecules, functionalizing theses nanoparticles, improves their application as nanovectors for gene therapy. The direct observation of cell-nanoparticles interaction requires fluorescence microscopic techniques, and the super-resolution multiphoton fluorescence stands out by the fact that it reveals the molecular processing of living cells, in real time, tracking an individual nanoparticle through their intrinsic fluorescence, by the Ln3+ dopant, providing the understanding of the intracellular living cells responses to the calcium phosphate non-viral vector. This abroad research fellowship application aims to continue the development of both FAPESP projects: PDIP 2017/50332-0 and BPE 2018/18928-3, in a collaboration between IPEN and UNSW-Sydney/Australia, under Prof. Pall Thordarson supervision, aiming at the continuation of the cellular interaction with the pure and Ln and/or Mg-doped CaP nanoparticles studies, before and after their funcionalization with organic or polimeric molecules, in order to understand how cells interact with nanomaterials. The understading of the internalization process of the particles, as well as their bioavailability, biodistribution, and intracellular processing, will be achieved by super resolution fluorescence microscopy techniques. Understanding the cellular response triggered by novel biomaterials is an essential part of the development and improvement of theranostic agents. These studies are being carried out at the UNSW, which has state-of-art equipment such as the super resolution multiphoton fluorescence microscope (Zeiss LSM 880 - Airyscan), which we do not have in Brazil yet. Parallelly, studies of these nanoparticles as non-viral vectors are being performed in cell tranfection tests. For successful transfection, biomaterials are required to transpose all barriers imposed by cells, and the knowledge of how cells interact with these biomaterials will elucidate the importance of the composition, structure, size, and surface charge and chemical modification of the calcium phosphate nanoparticles for its application as non-viral vectors. (AU)