Unveiling Sticholysin II and plasmid DNA interaction: Implications for developing non-viral vectors.

Non-viral gene delivery systems offer significant potential for gene therapy due to their versatility, safety, and cost advantages over viral vectors. However, their effectiveness can be hindered by the challenge of efficiently releasing the genetic cargo from endosomes to prevent degradation in lysosomes. To overcome this obstacle, functional components can be incorporated into these systems. Sticholysin II (StII) is one of the pore-forming proteins derived from the sea anemone Stichodactyla helianthus, known for its high ability to permeabilize cellular and model membranes. In this study, we aimed to investigate the interaction between StII, and a model plasmid (pDNA) as an initial step towards designing an improved vector with enhanced endosomal escape capability. The electrophoretic mobility shift assay (EMSA) confirmed the formation of complexes between StII and pDNA. Computational predictions identified specific residues involved in the StII-DNA interaction interface, highlighting the importance of electrostatic interactions and hydrogen bonds in mediating the binding. Atomic force microscopy (AFM) of StII-pDNA complexes revealed the presence of nodular fiber and toroid shapes. These complexes were found to have a predominantly micrometer size, as confirmed by dynamic light scattering (DLS) measurements. Despite increase in the overall charge, the complexes formed at the evaluated nitrogen-to-phosphorus (N/P) ratios still maintained a negative charge. Moreover, StII retained its pore-forming capacity regardless of its binding to the complexes. These findings suggest that the potential ability of StII to permeabilize endosomal membranes could be largely maintained when combined with nucleic acid delivery systems. Additionally, the still remaining negative charge of the complexes would enable the association of another positively charged component to compact pDNA. However, to minimize non-specific cytotoxic effects, it is advisable to explore methods to regulate the protein's activity in response to the microenvironment.

Optimisation of the superplastic forming of a dental implant for bone augmentation using finite element simulations.

OBJECTIVES: The phenomenon of superplasticity has made it possible to form complex shapes that require extremely high degrees of ductility in titanium alloy with minimal internal stresses. Combined with the use of an investment casting material as the die material, which makes possible the forming of re-entrant angles, it is possible to produce membranes for ridge augmentation. The aim is to characterise the metal alloy sheet and simulate the superplastic forming process in three dimensions to produce process parameters, namely gas pressure as a function of time, to accurately adapt the titanium sheet to the bone surface. METHOD: The surface of the die was digitised using a 3D laser scanning system (UBM-Keyence LC2450). Ti-6Al-4V sheet of 140 mm diameter was modelled using a grid of triangular membrane elements. This mesh was automatically refined during the simulations. Finite element simulation was carried out using the Superflag software program (University of Wales Swansea) Three different options for gas pressure control were adopted, namely, target flow stress, target strain rate and target energy dissipation. The pressure cycles produced from the simulation were used to form titanium alloy sheet at 900 degrees C using argon gas. The deformed regions of the formed sheet were then examined to determine the regions of contact with the die and to characterise surface damage. RESULTS: Comparison of the simulations with experiment showed that there was good agreement between simulated and experimental thickness distributions in most parts of the sheet that were examined. Interrupted tests showed that in the intermediate positions of the forming sheet the simulations were slightly ahead of the experiment. The target stress option was found to produce the best degree of adaptation and the sheet formed using this cycle showed good surface quality, whereas in highly deformed regions using the other target options, the sheet was found to have formed microcracks. The use of a solid lubricant on the surface of the forming sheet was not found to have a significant influence on the adaptation of the titanium alloy sheet except in areas of high deformation where the sheet perforated. SIGNIFICANCE: The finite element membrane formulation is well adapted to the superplastic forming of a ridge augmentation membrane prosthesis. The simulation accurately describes the evolution of the shape of the prosthesis and its thickness distribution with time, which allows the manufacturer to select an appropriate initial thickness of titanium alloy sheet prior to attempting to form the component. The investment dies are found to have sufficient strength to withstand the forming operation if a suitable orientation of the titanium sheet with respect to the die is adopted. A metal surface of good quality can be produced in the formed prosthesis using the appropriate gas control option.