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 [1]. 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 [2]. 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) [3]. 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 [4]. 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.

[1] Kim UJ et al. Biomacromolecules. 2004;5:786-792.

[2] Gan D et al. J Am Chem Soc. 2001;123:7511-7517.

[3] Sofia SJ et al. J Macromol Sci A. 2002;39:1151-1181.

[4] Yan LP et al. National Patent Nr. 106041, priority date: 06-12, 2011.