Introduction: Manipulation, spatio-temporal control, and hierarchical order of proteins are major goals for the next generation of functional biomaterials. However, the capacity to accurately control self-assembly of these building-blocks across scales with significant spatial and temporal control is still limited. Here we present a protein/peptide self-assembling system that exhibits hierarchical architecture, controlled assembly capabilities, adhesion to surfaces, self-healing, and the capacity to undergo morphogenesis into complex geometries with high spatio-temporal control. We demonstrate the potential of the system for fabricating bioactive and biomimetic scaffolds for tissue engineering.
Materials and Methods: System formation. ELP (0.1 mM) and PA (8.7 mM) were dissolved in MilliQ water. A PA drop was then injected inside a larger ELP drop.
Scanning electron microscopy (SEM). Membranes were fixed in 2.5% glutaraldehyde. After dehydration (ethanol), samples underwent critical point drying, and were sputter-coated with palladium/gold (30 nm).
Single-angle X-ray scattering (SAXS). Measurements were performed using the SAXSLAB GANESHA 300-XL system. The scattering curve was fitted to an appropriate model by a least-squares method (n=45). SAXS experiments on solutions were performed at the BM29 beamline at the European Synchrotron Radiation Facility (energy of 12.5 keV for a wavelength of 0.998 A-1).
Cell culture. Membranes were cross-linked with Genipin (0.15 mM), washed with PBS and sterilised with UV before cell seeding. Cell attachment was assessed seeding 50,000 human endothelial cells cells (hUVECs) onto the membranes. Cells were cultured for 4 h before fixation for SEM. Similarly, 50,000 mouse adipose-derived stem cells (mADSCs) were seeded onto the tube and cell metabolic activity (AlamarBlueTM), proliferation (Picogreen®) and morphology (SEM) after 1, 7, 14 and 21 days.
Results and Discussion: When a PA solution is immersed in a large volume of ELP solution above the ELP’s Tt, a dynamic interfacial membrane develops[1] (Fig. 1a). Within the first minute of formation and on contact with any surface, the membrane spontaneously, yet controllably, adheres, focally opens and seals to the surface forming a tubular structure with a multi-layered microarchitecture (Fig. 1b). The system can be manipulated with spatiotemporal control and be maintained in non-equilibrium for substantial periods of time: a simple 1D extension promotes lateral growth (Fig. 1c) and additional manipulation generates more complex morphogenesis into a network of tubes (Fig. 1d,e). The system can repeatedly self-heal large-scale ruptures (Fig. 1f). SAXS measurements of the molecules in solution and membranes demonstrated that the opening of the ELP molecules by PA molecules is critical for the formation of the distinctive multilayered architecture and for the controlled dynamic behaviours observed in the system. Culture of endothelial and adipose-derived stem cells on membranes were conducted to demonstrate the functionality and biocompatibility of the system (Fig. 1g,h).
Conclusion: We report a way of assembling organic molecules into robust and dynamic tubular networks by directed self-assembly without the use of predefined moulds. The phenomena reported here may provide a novel nanofabrication platform for developing a new kind of hybrid protein/peptide nanomaterials of high complexity and functionality for applications in fields such as tissue engineering and drug discovery.
European Research Council Starting Grant (STROFUNSCAFF); European Commission under FP7 and H2020 programs ((NMP3-LA-2011-263363, HEALTH-F4-2011-278557, PITN-GA-2012-317304, MSCA-ITN-2014- ETN- 642687, 642687 H2020-NMP-2014-646075); Ministry of Economy and Competitiveness (Spain) (MAT2012-38043-C02-01, MAT2013-41723-R, MAT2013- 42473-R); Junta de Castilla y Leon (VA244U13, VA313U14); Portuguese Foundation for Science and Technology, grants PTDC/EBB-BIO/114523/2009 and SFRH/ BD/44977/2008; Bilateral Program Portugal– Spain Integrated Actions 2011 (E-50/11); Marie Curie Career Integration Grant 618335; European Synchrotron Research Facility for access to synchrotron beamline BM29 and P. Pernot for support during the experiments; C. López (Centres Científics i Tecnològics University of Barcelona); C. Semino (Institut Químic de Sarrià); E. Rebollo (Advanced Fluorescence Microscopy Unit in the Molecular Biology Institute of Barcelona); J. P. Aguilar; R. Doodkorte; A. Amzour; Technical staff of the Material Characterization Laboratory (Queen Mary, University of London); Nanovision Laboratory (Queen Mary, University of London)
References:
[1] Inostroza-Brito, et al. Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein–peptide system. Nature Chemistry (2015) doi:10.1038/nchem.2349