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

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 safety is the major limitation. Synthetic non-viral vectors, as nanoparticles, are increasingly being considered as alternative. Studies have demonstrated high efficiency and biocompatibility of calcium phosphate (CaP) nanoparticles, in the form of HA and ²-TCP, as vectors when compared to other non-viral gene carriers. The calcium ion of the CaP vector protects the gene from degradation and facilitates the delivery of nucleic acid into the nucleus of the target cell. 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 material.The direct observation of cell-nanoparticles interaction requires fluorescence microscopic techniques, and the super-resolution multiphoton fluorescence microscopy using stimulated emission depletion (STED) 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 study the cellular interaction with the nanoparticles of pure and Lanthanide and/or Magnesium-doped calcium phosphates in order to understand how the cells interact with nanomaterials. The internalization of the particles, as well as their bioavailability, biodistribution, and intracellular processing, through high resolution fluorescence microscopy techniques. Understanding the cellular response triggered by new biomaterials is an essential part of the development and improvement of theranostic agents. To achieve this goal, the first steps should involve molecular studies on nanoparticle-cell interactions, endocytosis, intracellular traffic, and cellular response to these materials.