Introduction: Biological materials enjoy extraordinary properties that have been achieved through hundreds of years of evolution, in which natural selection has allowed only the best systems to survive. As such, further understanding of the rules that governs the assembly and the interactions among the different motives present in the Nature is required in order to make the most of their potential for designing advance functional materials.
Following this idea, here we present the development of bioinspired protein-based polymers whose composition is governed by a multi-block designed, characterized by the presence of elastin-like, silk-like and zipper domains. As such, the developed biomaterials will be referred as ELR (elastin-like recombinamer)[1], SELR (silk-elastin-like recombinamer)[2] or ZELR (Zipper-elastin-like recombinamer)[3]. The purpose of this work is to elucidate the interplay between these domains when included in the same molecule as well as to provide a rational basis for the development of extracellular matrix (ECM) analogues.
Materials and Methods: The synthesis of the recombinamers was achieved by recombinant DNA techniques[3], assessed by agarose gel electrophoresis, and expressed in E. coli. Physicochemical characterization of the obtained material included polyacrylamide gel electrophoresis (SDS-PAGE), MALDI-TOF mass spectrometry, and differential scanning calorimetry (DSC) to determine the transition temperature (Tt). Mechanical features were studied by rheological measurements of the hydrogels, and conformational properties were evaluated by infrared spectroscopy (FTIR) and circular dichroism (CD). The behaviour at the nanoscale level was assessed by transmission electron microscopy (TEM) and atomic force microscopy (AFM).
Results and Discussion: The developed bioinspired materials were able to form injectable hydrogels under physiological conditions. Moreover, regarding the mechanical properties of the formed hydrogels, a broad range of storage modulus, from 100 Pa to 10000 Pa, was covered. Such differences were found to be a direct consequence of the different bioinspired molecular designs performed, which had also an impact at the conformational and nanometric level. Thus, while ZELRs self-assembled into entangled and locked micelles, SELR folding resulted in the progressive formation of a nano-fibrillar network, emulating the organization of the native ECM. The conformational study revealed the presence of β-sheet structures in the SELR, whereas α-helices were detected in ZELRs. It was found that the β-sheet formation and the emerged nano-fibrillar structure experienced by SELR were responsible of the dynamic behaviour observed at the macroscale level, characterized by an increase of the stiffness over time. Regarding ZELR, the careful selection of the zipper, with cysteine residues likely to form disulphide bonds, allowed the reversibility of the gel system depending on the redox environment. Noticeable, both SELR and ZELR formed hydrogels with mechanical properties suitable to be used as cell niches. They were also stable in an excess of aqueous medium at physiological temperature for long periods of time of up to several months.
Conclusion: To conclude, results point to the huge potential of these systems as a basis for the development of injectable biomaterial platforms towards a fully functional and biomimetic artificial extracellular matrix.
EU FP7 THE GRAIL Project (Grant no. 278557); PRI-PIBAR-2011-1403; MAT2012-38043; MAT2013-42473-R; MAT2013-41723-R; VA152A12; VA155A12; VA313U14; BIO/VA43/14; CIBER-BBN; Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León; European Regional Development Fund (ERDF); European social Fund (ESF)
References:
[1] Girotti A, Fernandez-Colino A, Lopez IM, Rodriguez-Cabello JC, Arias FJ. Elastin-like recombinamers: biosynthetic strategies and biotechnological applications. Biotechnology journal 2011; 6:1174-86.
[2] Fernandez-Colino A, Arias FJ, Alonso M, Rodriguez-Cabello JC. Self-organized ECM-mimetic model based on an amphiphilic multiblock silk-elastin-like corecombinamer with a concomitant dual physical gelation process. Biomacromolecules 2014; 15:3781-93.
[3] Fernández-Colino A, Arias FJ, Alonso M, Rodriguez-Cabello JC. Amphiphilic Elastin-Like Block co-Recombinamers Containing Leucine-Zippers: Cooperative Interplay between Both Domains Results in Injectable and Stable Hydrogels. Biomacromolecules 2015. In Press.