Introduction: ROS (Reactive Oxygen Species) are key signature pathological signals expressed in inflammatory diseases, such as ischemic conditions and arteriosclerosis[1]. Collagen is a body constituent protein which has shown to be responsive to protease cleavage. We hypothesize that a composite system, made up of collagen type I and a synthesized polythioether will ensure both protease and ROS responsiveness, respectively. The objectives of this study are to synthetize a polythioether and to design the optimal conditions to fabricate a mixed natural/synthetic hollow sphere construct that can be used to deliver a therapeutic. Through the balance of a collagen/poly-thioether ratio, a natural-mimic platform, with specific ROS-responsiveness can be fabricated.
Materials and Methods: Polythioether synthesis: Polypropylene sulfide (PPS) was synthesised by the living emulsion polymerization method and further functionalized with N-Hydroxysuccinimide (NHS). The product was characterized by NMR, FT-IR, GPC and DSC.
Hollow spheres fabrication: Collagen type I-PPS composite hollow microspheres were fabricated using a polystyrene template method[2]. Several collagen/PPS concentration ratio were employed. Collagen-4armPEG composite hollow spheres and collagen-dithiolPEG were used as controls. Hollow spheres were characterized by SEM and FT-IR.
Cell viability studies: PPS-toxicity studies were performed on H9C2 rat cardiomyoblasts. Non-treated cells, cells treated with PEG 2000 and dithiolPEG were used as controls. Metabolic activity studies were performed on the collagen-PPS composite hollow spheres. Collagen-armPEG, collagen-dithiolPEG composite hollow spheres, polystyrene templates and untreated cells were used as controls. Metabolic activity was evaluated by alamarBlue® assay.Statistical analysis was conducted using SPSS Software.
ROS responsiveness: Both the pristine polymer and the final hollow sphere constructs were exposed to hydrogen peroxide (H2O2) water solutions at different concentrations, at 37°C, to mimic the ROS exposure. SEM and FT-IR were employed to assess ROS-induced nanoporation and sulfur oxidation, respectively.
Results and Discussion: PPS was synthesized and functionalized. The oxidation of thioether bonds to sulfone was assessed via NMR and FT-IR. The synthesized compound demonstrated a highly responsive behavior to ROS, as the swelling switch took place at H2O2 concentration corresponding to the physiological range. Assays on cell metabolic activity showed that, while PEG 2000 had no effect on cells, PPS reduced cell metabolic activity. Thus, appropriate protocols for effective hollow spheres fabrication were optimized. Hollow spheres exhibited a ROS-responsive nanoporation, with different pore size depending on the H2O2 concentration applied (Fig.1). Cell viability studies demonstrated that the final construct does not affect cell metabolic acitivity (Fig. 2).
Conclusions: Polypropylene sulfide was synthesized and exhibited a high ROS-responsiveness. Collagen-PPS hollow spheres were fabricated, revealing uniform size, ROS-responsive nanoporation and compatibility with metabolic activity on a cardiomyoblast cell line. This is a promising technology for targeted responsive drug delivery in inflammatory diseases.
Seventh Framework Programme Grant Agreement no.: 317304; Centre for Microscopy & Imaging at the National University of Ireland Galway funded by Irish Government’s Programme for Research in Third Level Institutions, Cycles 4 and 5, National Development Plan 2007-2013
References:
[1] Lee S.H., et al., Adv. Healthcare Mater., 2013, 2: 908-915
[2] Browne S., et al., Biomaterials, 2015, 69: 133-147