Abstract
Introduction: Nisin, a lantibiotic holds promise as a local therapeutic agent for periodontal disease due to its broad spectrum of antimicrobial activity and strong safety profile. It has also been suggested that nisin may have clinical applications via modulation of the host immune response, however, a direct effect on periodontal disease requires further investigation. The constant washing of the oral cavity by saliva complicates the sustained delivery of topical pharmaceutical agents to periodontal tissues. The encapsulation of nisin within a chitosan nanosphere is proposed to overcome some of the shortfalls associated with intraoral drug delivery, by providing a mechanism for sustained release at effective concentrations.
Methods: We developed nisin Z-loaded chitosan nanospheres using the ionic gelation method. Nisin’s encapsulation efficiency, release profile, as well as the size, and dispersity of nanospheres were characterised. The minimum inhibitory concentrations and minimum bactericidal concentrations were determined against two oral bacteria (Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis)). In vitro investigations evaluated the immunomodulatory effects of nanosphere treatment on human gingival fibroblasts (HGF). A Human XL Cytokine Array (ARY022B, R&D Systems, Minneapolis, USA) was used to investigate the effect of nanosphere treatment on 105 signalling proteins. A further experiment investigated the effect of nanosphere treatment on the expression of T-helper cell differentiation cytokines by HGFs stimulated with P. gingivalis- Lipopolysaccharide (LPS). The cytotoxicity of nanosphere concentrations on HGFs was also evaluated using a Presto blue assay.
Results: An optimal formulation (20,000 IU/ml nisin, 1:40 TPP/chitosan ratio) produced nanospheres measuring 397 nm, with high encapsulation efficiency (91%), and sustained nisin release (for 48 hours). A MIC of 5 mg/ml against S. mutans and 40 mg/ml against P. gingivalis indicated effectiveness against these oral bacteria. Importantly, these concentrations exhibited no lasting cytotoxic effects. HGFs treated with nisin Z loaded and unloaded chitosan nanospheres reduced resting expression of interleukin 8 (IL8), plasminogen activator inhibitor 1 (PAI1), angiogenin, and fibroblast growth factor 19 (FGF19), while increasing monocyte chemoattractant protein 1 (MCP1), demonstrating effects which may be either protective or harmful in the context of periodontal disease. It was also observed that chitosan as part of the formulation reduced Dickkopf WNT signalling pathway inhibitor 1 (DKK1), a secreted inhibitor of beta-catenin-dependent Wnt signalling expression. Interestingly, LPS stimulation did not significantly alter cytokine expression in HGFs.
Conclusions: This study demonstrated the successful development of nisin Z loaded chitosan nanospheres with sustained release potential. Whilst this formulation did not show a beneficial immunomodulatory effect. It may have a role in the treatment of periodontal disease due to the observed bactericidal concentrations against oral bacteria without cytotoxicity to HGFs. A novel finding from this study was the reduction in DKK1 expression by HGFs cultured with chitosan nanospheres. This reduction of DKK1 has not been previously described and could suggest a pathway in which chitosan promotes bone health