Smart silk fibroin hydrogels and its potential application as suppressor of angiogenesis and tumor progression

Common methods used to prepare silk fibroin (SF) hydrogels are based on a SF conformation transition from amorphous to β-sheet in aqueous status, which implies a longer gelation time and harsh preparation conditions. These requirements hinder the final application of SF as injectable system for cell encapsulation and drug delivery. Recently, enzyme-mediated in situ hydrogelation reactions have attracted a great deal of attention, because of their specificity and non-toxic nature. It was found that proteins with tyrosine groups can be used to prepare hydrogels in situ by using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). SF has been demonstrated to be a promising biomaterial since it contains 5 mol% tyrosine groups, which are easily chemically modified. In this study, enzymatically cross-linked SF hydrogels were prepared using high concentrate aqueous silk fibroin solution (16 wt%) mediated by a HRP/H2O2 system. The aim of the present work is to assess the conformation transition in the SF hydrogels and evaluate if this transition is allied to its potential anti-tumoral applications. The β-sheet conformation transition was analyzed by means of Transmission electron microscopy (TEM) and Thioflavin T staining. Human neuronal glioblastoma (U251) and human cervical adenocarcinoma (HeLa) cell lines were incorporated into the developed hydrogels and cultured for 1, 7 and 10 days under standard culture conditions. Cell viability and proliferation were evaluated through preliminary biological assays: Live/Dead staining and DNA quantification. 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, SF hydrogels were capable of incorporating cells and support their viability during the first 24hours of culturing. Nonetheless, the β-sheet conformation of SF induced hydrogels autofluorescence, which hindered the cell viability/death evaluation from day 7. From DNA quantification analysis it was possible conclude that no cell proliferation occurred from day 1 up to day 10. The obtained results reveal that the conformation transition is responsible for inhibiting cell growth and thus the developed SF hydrogels can be a very useful and tunable system for different biomedical applications, including suppressing angiogenesis and tumors progression in vivo.