Abstract
With the property of sol-gel transition in physiological condition and numerous advantages from peptide-based material, self assembling peptide (SAP) has been regarded as a promising 3D hydrogel scaffold or cell/gene (drug) carrier for the applications in tissue engineering and drug delivery system. The additional functional motifs with various modification ratios and multi-extended diversity can be conjugated and extended at the terminal residue sequence of specific-designed SAPs to enrich SAP possessing therapeutic functionality for different biopharmaceutical applications and serve as medical disease treatment modality. In this study, we specifically enriched RADA16, one of the widely used SAPs, with different functional motifs and evaluated the medical applications of the functionalized SAPs in hemostasis, liver tissue regeneration and brain neural tissue repair in the central nervous system. In the first part of this study, the physiochemical properties and gelation mechanism of SAPs were in detail investigated. The pKa1, pKa2, and isoelectric point (pI) was obtained by pH titration curve. The peptide showed net-charged at neutral pH value. The protein secondary structure of β-sheet arrangement was confirmed by circular dichroism (CD) spectroscopy. The morphology of nanofibers about 25nm diameter was observed by atomic force microscopy (AFM). The gelation behavior in terms of mechanical property of hydrogel was determined by oscillatory rheology test. As for in vitro experiments, the relative low cytotoxicity of peptide hydrogel was examined by Live/Dead assay and the high cell proliferation ability was shown in 3D hydrogel culture by MTS assay. The results demonstrated that the encapsulated neural stem cells well survived with 3D culture in SAP hydrogel. We also found that even with extended motifs, the gelation behavior, physical and chemical properties of functionalized SAPs were still preserved. It is believed that the functionalized SAPs can be a promising material for follow-up tissue engineering applications. In hemostasis and liver tissue regeneration study, specifically extended fragments of functional motifs derived from fibronectin and laminin, GRGDS and YIGSR, was designed to evaluate the capability of these functional SAPs in the effect of immediate hemostasis and accelerative liver tissue regeneration. The results showed that hemostasis can be achieved within 10 seconds and the histological analyses demonstrated by H/E stain and immunohistochemistry revealed that SAPs with these extended functional motifs significantly enhanced liver tissue regeneration in rat liver wound model. It is reported that SAPs enriched with the extended functional motifs not only maintained their spontaneous self-assembly property but also extensively advanced the potential therapeutic effect in hemostasis and liver tissue regeneration. In the study of brain neural tissue repair in the central nervous system, the functionalized RADA16-IKVAV peptide hydrogel was used to encapsulate neural stem cells (NSCs) for investigation of the effectiveness in brain tissue regeneration. The results showed that after 6 weeks transplantation, the regenerative extracellular matrix (ECM) of brain neural tissue was noticed. The specific neuronal protein markers, such as βIII tubulin, MAP2, NF-H were positively stained in the region of wound defect area, suggesting regenerative process of neuronal cells in the brain cerebral cortex. In summary, we have successfully developed functionalized SAPs with multiple extended motifs, including RADA16-GRGDS, RADA16-YIGSR and RADA16-IKVAV, and their physiochemical properties were also carefully evaluated for the comparison with RADA16 nanopeptides. These functionalized SAPs can not only be used alone as 3D nanoscale scaffold for immediate hemostasis and accelerative liver tissue wound healing, but also be incorporated with neural stem cells for the improvement of brain tissue repair in the central nervous system.