ABSTRACT
This study aims to present an ultrasound-mediated nanobubble (NB)-based gene delivery system that could potentially be applied in the future to treat bone disorders such as osteoporosis. NBs are sensitive to ultrasound (US) and serve as a controlled-released carrier to deliver a mixture of Cathepsin K (CTSK) siRNA and cerium oxide nanoparticles (CeNPs). This platform aimed to reduce bone resorption via downregulating CTSK expression in osteoclasts and enhance bone formation via the antioxidant and osteogenic properties of CeNPs. CeNPs were synthesized and characterized using transmission electron microscopy and X-ray photoelectron spectroscopy. The mixture of CTSK siRNA and CeNPs was adsorbed to the surface of NBs using a sonication method. The release profiles of CTSK siRNA and CeNPs labeled with a fluorescent tag molecule were measured after low-intensity pulsed ultrasound (LIPUS) stimulation using fluorescent spectroscopy. The maximum release of CTSK siRNA and the CeNPs for 1 mg/mL of NB-(CTSK siRNA + CeNPs) was obtained at 2.5 nM and 1 µg/mL, respectively, 3 days after LIPUS stimulation. Then, Alizarin Red Staining (ARS) was applied to human bone marrow-derived mesenchymal stem cells (hMSC) and tartrate-resistant acid phosphatase (TRAP) staining was applied to human osteoclast precursors (OCP) to evaluate osteogenic promotion and osteoclastogenic inhibition effects. A higher mineralization and a lower number of osteoclasts were quantified for NB-(CTSK siRNA + CeNPs) versus control +RANKL with ARS (p < 0.001) and TRAP-positive staining (p < 0.01). This study provides a method for the delivery of gene silencing siRNA and CeNPs using a US-sensitive NB system that could potentially be used in vivo and in the treatment of bone fractures and disorders such as osteoporosis.
ABSTRACT
Recently, Fe-Mn-based alloys have been increasingly catching the attention of the scientific community, because of their tunable and outstanding mechanical properties, and suitable degradation behavior for biomedical applications. In spite of these assets, their corrosion rate (CR) is, in general, too low to satisfy the requirements that need to be met for cardiovascular device applications, such as stents. In fact, the CR is not always the same for all of the degradation stages of the material, and in addition, a finely tuned release rate, especially during the first steps of the corrosion pattern, is often demanded. In this work, a resorbable bimodal multi-phase alloy Fe-3Mn-1Ag was designed by mechanical alloying and spark plasma sintering (SPS) to accelerate the corrosion rate. The presence of several phases, for example α-Fe, α-Mn, γ-FeMn and Ag, provided the material with excellent mechanical properties (tensile strength UTS = 722 MPa, tensile strain A = 38%) and a higher corrosion rate (CR = 3.2 ± 0.2 mm/year). However, higher corrosion rates, associated with an increased release of degradation elements, could also raise toxicity concerns, especially at the beginning of the corrosion pattern. In this study, The focus of the present work was the control of the CR by surface modification, with nitrogen plasma immersion ion implantation (N-PIII) treatment that was applied to mechanically polished (MP) samples. This plasma treatment (PT) improved the corrosion resistance of the material, assessed by static degradation immersion tests (SDITs), especially during the first degradation stages. Twenty-eight days later, the degradation rate reached the same value of the MP condition. Nitrogen compounds on the surface of the substrate played an important role in the corrosion mechanism and corrosion product formation. The degradation analysis was carried out also by potentiodynamic tests in modified Hanks' balanced salt solution (MHBSS), and Dulbecco's phosphate buffered saline solution (DPBSS). The corrosion rate was higher in MHBSS for both conditions. However, there was no significant difference between the corrosion rate of the PT in DPBSS (CR = 1.9 ± 0.6 mm/year) and in MHBSS (CR = 2 ± 1.4 mm/year). The cell viability was assessed with human vein endothelial cells (HUVECs) via an indirect metabolic activity test (MTT assay). Due to the lower ion release of the PT condition, the cell viability increased significantly. Thus, nitrogen implantation can control the in vitro corrosion rate starting from the very first stage of the implantation, improving cell viability.
