Intracellular carboxymethylchitosan/poly(amidoamine) nanocarriers loaded with dexamethasone enhances osteogenic differentiation of RBMSCs in vitro

Biocompatible and water-soluble carboxymethylchitosan/poly (amidoamine) dendrimer, CMC/PAMAM nanoparticles (6–250 nm in diameter) were synthesized to find applications as a Drug Delivery System (DDS) of dexamethasone (Dex) aimed at modulating the stem cells behaviour namely, proliferation and differentiation both in vitro and in vivo. In this work, we have demonstrated that these nanoparticles are internalized with high efficiency by different cell types, i.e. cell lines (human osteoblast-like cells, SaOs2) and primary cultures (Rat Bone Marrow Stromal Cells, RBMSCs) by means of using the fluorescent probe fluorescein isothiocyanate (FITC) and under fluorescence-activated cell sorting (FACS) and fluorescence microscopy (FM) analysis. This work has demonstrated that the FITC-labelled CMC/PAMAM nanoparticles are efficiently internalized by RBMSCs even when loaded with Dex, after a few hours of incubation. Moreover, FACS and FM analysis have shown that the nanoparticles were internalized by RBMSCs in the presence of an endosomal inhibitor (colchicine), which is an indication that internalization mechanism is not exclusively endocytotic. Complementarily, we also assess the osteogenic potential of the Dex-loaded CMC/PAMAM nanoparticles (0.01– 1 mg.ml1 ) when present in the culture medium and by means of using RBMSCs, in vitro. Mineralization studies, ALP quantification, DNA content, Alizarin red and ALP staining were performed to investigate the osteogenic differentiation of RBMSCs (1, 7 and 14 days). Results have shown that the dexamethasone-loaded CMC/PAMAM nanoparticles may find applications in TE, namely as an intracellular DDS since enhanced the osteogenic differentiation of RBMSCs, in vitro.