"Introduction. With limited treatment options for acute brain injuries like stroke or traumatic brain injury, recent research has focused on the development of immune-modulating and pro-regenerative therapies for brain diseases. TSG-6 (tumor necrosis factor-alpha stimulated protein 6) is a multifunctional protein with attractive therapeutic potential for neurological applications. As for numerous therapeutic proteins, the in vivo efficacy of TSG-6 is limited by short half-life and reduced availability at the target site due to endogenous degradation. This limitation can be overcome by using delivery systems. Here, we describe the development and in vitro efficacy of nanosized particles for TSG-6 delivery specifically designed for use in the nervous system.
Methodology. Recombinant human (rh) TSG-6 was encapsulated into chitosan-hyaluronic acid (HA) nanoparticles and liposomes before further nanoparticle characterisation by transmission electron microscopy (TEM) and dynamic light scattering (DLS), and assessment of encapsulation efficiency. To confirm that the delivery systems improve the anti-inflammatory effect of rhTSG-6, mouse microglial BV2 cells and human CMEC/D3 brain endothelial cells were stimulated using inflammatory lipopolysaccharide (LPS) or interleukin-1 beta (IL-1β) and treated with rhTSG-6 containing nanoparticles. Cell viability was assessed by resazurin-based AlamarBlue assay and levels of inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured by ELISA.
Results. Synthesized HA-based nanoparticles and liposomes were within the nanometric range, as confirmed by TEM and DLS analysis, and have allowed efficient rhTSG-6 encapsulation. Nanoparticles did not affect cell viability and nanoparticle-delivered rhTSG-6 has successfully decreased IL-6 and TNF-α levels following inflammatory stimulation.
Conclusions. By exploiting the natural affinity of TSG-6 protein for HA, we have synthesized nanoparticles for rhTSG-6 delivery which successfully decrease inflammation in brain cells in vitro. Short-term future work will assess the biodistribution profile in vivo and enhancement of anti-inflammatory effect in a mouse model of systemic inflammation. Long-term future work will test nanoparticle-delivered rhTSG-6 as a therapeutic agent for brain tissue repair and functional recovery following ischaemic stroke."