INTRODUCTION

Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion and metastasis formation. Thus, the therapeutic suppression of angiogenesis could be the key to prevent tumor progression¹. We describe the use of injectable, in situ cross-linkable SF hydrogels to suppress angiogenic activity and tumor progression, using in vitro and in vivo models. Recently, it was found that proteins (e.g. silk fibroin) with tyrosine groups can be used to prepare hydrogels in situ via an enzyme-mediated cross-linking reaction, using horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂)². Silk fibroin (SF) contains 5 mol% tyrosine groups, which are easily chemically modified, demonstrating the promising application of this protein for the production of injectable systems and non-toxic hydrogels.

EXPERIMENTAL METHODS

The present work provides a novel route for obtaining enzymatically cross-linked SF hydrogels within a few minutes in physiological conditions, using high concentrate aqueous SF solution (16 wt%), mediated by a HRP/H₂O₂ complex³. The dominant conformation presented by the SF hydrogels was assessed over time, allied to its potential anti-tumoral applications. The β-sheet conformation transition was analyzed by means of Transmission Electron Microscopy (TEM), Thioflavin T and Congo red staining. Human neuronal glioblastoma (U251) and human cervical adenocarcinoma (HeLa) cell lines were encapsulated within the SF hydrogels and cultured for 14 days under standard culture conditions. Cell viability and proliferation were assessed through ATP and DNA quantification assays, respectively. In order to evaluate the in vivo biocompatibility of the SF hydrogels, a subcutaneous implantation was performed in mice model for a period of 2 weeks, followed by H&E staining.   

RESULTS AND DISCUSSION

The SF hydrogels presented ionic strength and pH stimuli responsiveness. The β-sheet conformation transition analysis revealed that the fast-formed hydrogels presented mainly an amorphous conformation and transparent appearance during the first week, but a conversion to a dominant β-sheet conformation and opaque appearance was verified from day 7.

Additionally, U251 and HeLa cells were successfully encapsulated within the hydrogels. ATP quantification showed a cell-dependent decrease of cell viability over culture period and from DNA quantification analysis it was possible to conclude a decrease of cell proliferation after 14 days of culture. The in vivo results showed that the SF hydrogels did not induce any acute inflammatory reaction after 2 weeks of subcutaneous implantation (Figure 1). The SF hydrogels underwent a β-sheet conformation transition after 7-10 days of in vitro cell encapsulation and 2 weeks of in vivo implantation, revealing that the observed conformation transition may be responsible for inhibiting cell viability and proliferation.

CONCLUSION

This study provides a straightforward approach to produce injectable SF hydrogels with superior biocompatibility and fast-gelation property to be used as a whole or combined with bioactive agents. Thus, the enzymatically cross-linked SF hydrogels can be a very useful and tunable system for different biomedical applications, including suppressing angiogenesis and tumors progression in vivo.