INTRODUCTION The link between the inflammatory response and the promotion of cancers is well established; notably in endothelial cancers such as oral, pancreatic and colon. Epidemiological studies have shown that chronic inflammation is a significant causative factor for these cancers. The use of designed, nanostructured materials formed from self assembling peptides as scaffolds is a rapidly growing research area[1]. A particularly promising application involves materials that can mediate the local tumour environment of oral cancers through attenuation of the inflammatory response, whilst simultaneously providing a stable healthy extracellular matrix (ECM) mimic to promote regeneration. Several studies showed promising anti-tumorigenic effects using non-steroidal anti-inflammatory drugs. Hence, a therapeutic opportunity lies in developing a biocompatible material that can achieve a spatially confined, sustained, non-steroidal and selective suppression of the immune system. Hydrogels formed by bioinspired synthetic organic molecules known as self-assembling peptides (SAP) are highly suitable materials for cancer therapy, they have been shown to form nanofibrillar matrices of similar morphology which are functional both in vitro and in vivo through the inclusion of bioactive and biocompatible peptide sequences in the SAP during synthesis[2]. The formation of SAP hydrogels is a thermodynamically driven process[3]; the noncovalent forces that govern their assembly can be used to physically incorporate larger bioactive molecules. Here, we demonstrate a method to include a powerful non-steroidal anti-inflammatory polysaccharide within the matrix itself.
MATERIALS AND METHODS All N-fluorenylmethyloxycarbonyl (Fmoc) self-assembling peptides (SAPs) were synthesised stepwise using solid [phase peptide synthesis. Hydrogels were prepared at 20 mg/mL. 100 µL of deionised water with 50 µL 0.5 M sodium hydroxide (NaOH) was used to dissolve 10 mg of peptide. 0.1 M hydrochloric acid (HCl) was then added dropwise, always while vortexing, until the pH of the solution reached 7.4.. A polyanionic polysacharride, fucoidan (marinova, TAS) was incorporated into the scaffold via coassembly, confirmed by a using a suite of characterisation techniques, including: 1H, 13C Nuclear magnetic resonance spectroscopy; Small angle neutron scattering; Atomic Force Microscopy; Transmission Electron Microscopy; Dynamic Scanning and Isothermal Titration Calorimetry; Fourier Transform-Infra Red, fluorescence and Circular dichroism spectroscopy; and parallel plate rheometry. The anti inflammatory properties of this material were tested for their effectiveness using SCC25 epithelial cancer cells, challenged with Lipopolysaccharide. Cells were cultured in 24 well tissue culture plates, with cell specific culture conditions.
RESULTS AND DISCUSSION We use spectroscopic and material analyses to show that coassembly facilitates the binding of fucoidan molecules to the SAP nanofibrils.We demonstrate the scaffold supports the culture of healthy cells whilst concomitantly inducing apoptosis in cancerous cells. We determine this process is underpinned by the significant (orders of magnitude) and sustained downregulation of proinflammatory gene and protein expression related to cell division and cyctokine production, even when challenged with proinflammatory lipopolysaccharide.
CONCLUSION This work represents the potential for SAP hydrogels to provide sustained delivery of soluble macromolecules while maintaining their effectiveness as a tissue engineering scaffold material. Our findings highlight an innovative material approach for the distribution and sustained presentation of functional molecules on a nanoscale structure via self-assembly, showing their significant benefit for local cancer immunotherapy and drug delivery vehicles.
References:
[1] Nisbet, D. R.; Williams, R. J., Self-assembled peptides: characterisation and in vivo response. Biointerphases 2012, 7, (2), 1-14.
[2] Modepalli, V. N.; Rodriguez, A. L.; Li, R.; Pavuluri, S.; Nicholas, K. R.; Barrow, C. J.; Nisbet, D. R.; Williams, R. J., In vitro response to functionalized self‐assembled peptide scaffolds for three‐dimensional cell culture. Peptide Science 2014, 102, (2), 197-205.
[3] Li, R.; Horgan, C. C.; Long, B.; Rodriguez, A. L.; Mather, L.; Barrow, C. J.; Nisbet, D. R.; Williams, R. J., Tuning the mechanical and morphological properties of self-assembled peptide hydrogels via control over the gelation mechanism through regulation of ionic strength and the rate of pH change. RSC Advances 2015, 5, (1), 301-307.