Development of nanotheranostic systems via Layer-by-Layer assembly for the treatment of osteosarcoma

Osteosarcoma is a rare primary bone cancer with a worldwide incidence of 3.4 million people per year and it is most often diagnosed in children. Due to the limits of current cancer treatments, the development of nanotheranostic platforms, which combine the therapeutic potential of drug compounds with the diagnostic potential of imaging probes in the same vehicle, has attracted the attention of researchers in recent decades, enabling improvements in drug delivery to specific sites and real-time vehicle tracking. Regarding the field of bioimaging, Quantum Dots (QD) are promising nanoparticles (NPs), but with poor biocompatibility. In this context, the use of carbon-based QD (CQDs) can improve cytocompatibility; thus, my project has been focused on the development of CQDs derived from different natural sources, such as chitin (CHCQDs) and ground coffee (GCCQDs). Morphology, optical and physico-chemical properties of each CQDs were analysed by Fourier Transformed Infrared spectroscopy (FTIR-ATR), X-ray photoelectron spectroscopy (XPS), ζ-Potential, High-resolution transmission electron microscopy (HRTEM), and UV-Visible (UV-Vis), showing a round-shaped structure, negative surface charge, and excitation-dependent emission. Finally, their cytocompatibility was evaluated on neo-dermal fibroblasts by Live/Dead and PrestoBlue assays, showing high cell viability up to a concentration of 500 µg/mL of CQDs in culture medium. Next, two different nanotheranostic systems by exploiting the immersive self-assembly layer-by-layer technique (LbL) have been developed. LbL represents an easy and versatile technology used in various fields to create multi-layered nanocoating. In this work, chitosan (CH) and Chondroitin Sulfate (CS) were used as polyelectrolytes for the deposition of seven layers for both systems produced. The first system involved the fabrication of a calcium phosphate (CaP) core containing Doxorubicin (DOXO) stabilized by the deposition of Poly(allylamine hydrochloride) (PAH) through a pumping system. Then, DOXO and GCCQDs were incorporated into the layers after dissolution in CH and CS, respectively. The second system was a drug codelivery (NPs_DD) consisting of a negative core of CHBOCQDs coated with CH and CS as polyelectrolytes containing DOXO and Docetaxel (DTX). As the outer layer, pure CH was deposited in both systems. Using the saturation method, the final particles loaded with drugs and CQDs showed a size of about 100 nm with a spherical and mostly uniform morphology. The encapsulation efficiency (EE) of DOXO in the CaP system with seven layers (CaP_7D) was 47.98%, while NPs_DDs showed an EE of less than 30% for both drugs. The 7 layers in the systems ensure time-modulated drug release. In fact, both types of NPs are affected by the burst release effect in the first 24 hours (greater than 50%), but the remaining amounts of drugs are released in the next 28 days. Cell growth inhibition of U2OS and Saos-2 cells caused by NPs was evaluated using by Live/Dead, PrestoBlue and immunostaining assays. In the case of Saos-2, the synergistic effect of the two drugs in the NPs_DD could be appreciated even for low particle concentrations. However, this did not occur for U2O2 cells. CaP_7D showed an absence of Saos-2 cell viability for a NPs concentration of 500 µg/mL. A slightly different trend was observed for U2O2 cells. Finally, TEM analysis of the Saos-2 confirmed the therapeutic efficacy of these NPs by exploiting the enhanced permeability and retention (EPR) effect of cancer cells.