Layer-by-Layer assembly on Doxorubicin/miRNA-34a loaded nanotheranostic system for osteosarcoma treatment

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents, with over 3.4 per million cases each year worldwide. It is a very aggressive tumor that often metastasizes, especially in the lungs, and has a 5-year survival rate around 70% for localized malignancies. The current treatment consists of surgery with adjuvant chemotherapy involving a combination of different agents, but OS can develop multidrug resistance. Moreover, chemotherapy has major drawbacks such as cardiotoxicity, neurotoxicity or nephrotoxicity. Nanotechnology has been proposed as a novel and promising approach for OS treatment to overcome the limitations of chemotherapy and to guarantee a better survival rate of patients. Nanoparticles have been used as carriers for various diseases due to their small size, tunable physicochemical properties, and efficient drug encapsulation. In this project, a DOXO/miRNA-34a loaded polymeric nanotheranostic system was designed, manufactured and evaluated for imaging and treatment of OS. The NPs were composed of Chitosan (CHI) and Poly(Lactic-co-Glycolic Acid) (PLGA), encapsulating DOXO and miRNA-34a. miRNA-34a is a short non-coded RNA sequence that inhibits tumor cell growth and proliferation by inducing metabolic activity arrest and apoptosis. The manufactured cores were functionalized with alternating nanolayers of two polyelectrolytes, Chitosan (CHI) and Pectin (PEC), using the layer-by-layer assembly method. Curcumin-derived Carbon Quantum Dots (CUR-CQDs) and Curcumin/β-Cyclodextrin (CUR/β-CD) were added to the PEC layer for imaging and anti-cancer activities. Curcumin is a natural polyphenol that exhibits anti-oxidant, anti-cancer and anti-inflammatory activities and it’s been extensively studied for various diseases, including OS. However, its therapeutic effect in vivo is limited due to low water solubility, insufficient absorption, rapid degradation at physiological pH, and low bioavailability. To address these issues, CUR is often encapsulated in carriers such as such as hydrogels, liposomes, nanoparticles, and nanocomplexes that can improve its stability and solubility. β-Cyclodextrins are oligosaccharides with a cyclic bucket-shaped structure that can encapsulate lipophilic molecules, forming host-guest inclusion complexes. CUR/β-CD complexes have shown greater antioxidant effect and a higher ability to affect OS metabolism, due to their high encapsulation efficiency. The imaging capability of the nanotheranostic systems relies on the presence of Quantum Dots (QDs). QDs have been widely used for imaging, pharmacokinetics evaluation and drug delivery, due to their high stability and high absorption spectrum. However, the potential toxicity of QDs derived from non-biocompatible materials is a concern. For this instance, in this study CUR-derived carbon QDs have been utilized to ensure cytocompatibility. The physicochemical and morphological characterization of the manufactured NPs demonstrated a round shape with a mean size of 187 nm and a surface charge of +25.4 mV, suitable for cellular uptake. The encapsulation efficiency of DOXO and miRNA-34a was found to be 71% and 99%, respectively. The nanosystem was tested in vitro on 2D and 3D models using SAOS-2 and U2OS osteosarcoma cell lines. For 3D models, spheroids of the two cell lines were created to evaluate the system’s efficacy in a more biomimetic environment. SAOS-2 models showed a more pronounced synergistic effect of loaded drugs compared to U2OS models, even at lower concentrations.