Development of tannic acid and catalase-based hollow polymer capsules to prevent oxidative stress and modulate extracellular matrix activity in interleukin-1beta induced inflammation model of nucleus pulposus

摘要

Introduction: There is growing evidence that excessive reactive oxygen species (ROS) are associated with intervertebral disc degeneration (IVD). High levels of ROS inhibit proliferation, induce premature senescence and promote catabolic phenotype of nucleus pulposus (NP) cells1 ,2. Thus, development of a therapeutic system that efficiently regulates ROS levels is an attractive strategy to promote disc regeneration. Herein, antioxidant hollow polymer capsules based on catalase and tannic acid were fabricated and their potential to prevent oxidative stress and modulate extracellular matrix activity in interleukin-1β (IL-1β) induced inflammation model of NP was evaluated. Materials and Methods: Hollow polymer capsules were fabricated via the layer-by-layer approach employing polyfallylamine hydrochloride) (PAH), dextran (DEX) and tannic acid (TA) as polyelectrolytes and catalase-loaded CaCO3 micropartides as sacrificial templates. These capsules were fully characterized in terms of morphology, physico-chemical properties and H_2O_2 scavenging capacity. NP cell viability and metabolic activity in the presence of these capsules was analyzed by Live/Dead® and AlamarBlue® assays, respectively. Oxidative stress in NP cells stimulated with IL-1β3 was evaluated with CellRox® fluorogenic probes under confocal microscopy. The mRNA expression of catabolic enzymes of MMP-3 and ADAMTS-5, and matrix component of aggrecan and collagen Ⅱ were also determined by RT-qPCR. Results and Discussion: As depicted in Figure 1, hollow polymer capsules loaded with catalase and functionalised with a layer of tannic acid were successfully fabricated via a layer-by-layer approach. Concentration of H_2O_2 was reduced from 10 to -4 uM in the presence of catalase-loaded capsules, but also when catalase was not encapsulated, indicating the scavenging effect of the external layer of tannic acid. In presence of 50 uM H_2O_2 solution, the reduction was greater when catalase was encapsulated, justifying the combined effect of catalase and tannic acid at higher H_2O_2 concentrations. Figure 2 demonstrates that the developed hollow capsules were able to prevent oxidative stress and to attenuate the catabolic activity in NP cells. According to confocal micrographs (Figure 2a), the fraction of NP cells that were positively stained to CellRox® increased from 0.27 to 0.89 when cells were stimulated with IL-1β. However, in the presence of 100 or 400 μg/ml of hollow polymer capsules the fraction decreased to 0.32 and 0.08, respectively. In inflamed NP cells the expression of MMP-3 and ADAMTS-5, major proteolytic enzymes responsible for ECM degradation in intervertebral disc, was attenuated in the presence of hollow polymer capsules. Conclusion: The hollow polymer capsules fabricated showed antioxidant properties due to the combined effect of encapsulated catalase and the external layer of tannic acid. These capsules reduced oxidative stress and were able to attenuate the expression of catabolic enzymes in a IL-1β induced inflammation model of NP cells.