Self-Organized ECM-Mimetic Model Based on an Amphiphilic Multiblock Silk-Elastin-Like Corecombinamer with a Concomitant Dual Physical Gelation Process

Abstract

ABSTRACT. Although significant progress has been made in the area of injectable hydrogels for biomedical applications and model cell niches, further improvements are still needed, especially in terms of mechanical performance, stability and biomimicry of the native fibrillar architecture found in the extracellular matrix (ECM). This work focuses on the design and production of a silk-elastin-based injectable multiblock co-recombinamer that spontaneously forms a stable physical nanofibrillar hydrogel under physiological conditions. That differs from previously reported silk-elastin-like polymers on a major content and predominance of the elastin-like part, as well as a more complex structure and behavior of such part of the molecule, which is aimed to obtain well defined hydrogels.Rheological and DSC experiments showed that this system displays a coordinated and concomitant dual gelation mechanism. In a first stage, a rapid, thermally driven gelation of the co-recombinamer solution takes place once the system reaches body temperature due to the thermal responsiveness of the elastinlike (EL) parts and the amphiphilic multiblock design of the co-recombinamer. A bridged micellar 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 structure is the dominant microscopic feature of this stage, as demonstrated by AFM and TEM.Completion of the initial stage triggers the second, which comprises a stabilization, reinforcement, and microstructuring of the gel. FTIR analysis shows that these events involve the formation of β-sheets around the silk motifs. The emergence of such β-sheet structures leads to the spontaneous selforganization of the gel into the final fibrous structure. Despite the absence of biological cues, here we set the basis of the minimal structure that is able to display such a set of physical properties and undergo microscopic transformation from a solution to a fibrous hydrogel. The results point to the potential of this system as a basis for the development of injectable fibrillar biomaterial platforms towards a fully functional, biomimetic, artificial extracellular matrix and cell niches.

Biocompatibility of two model elastin‐like recombinamer‐based hydrogels formed through physical or chemical cross‐linking for various applications in tissue engineering and regenerative medicine

“…This result highlights that both types of ELR hydrogels are very stable when implanted subdermally in vivo , even when the hydrogel network is only stabilised via non‐covalent interactions, which is the case of the SELR. This is in good agreement with previous findings for this type of recombinamer in vitro (Fernandez‐Colino et al, ) and suggests that additional biodegradation domains should be included in the amino acid sequence of the ELRs if a temporary implant is needed. However, the good stability over time observed here is relevant for both tissue‐engineering applications and for the development of drug‐delivery systems because the formation of a stable system could be crucial for achieving the long‐lasting effects required in many of these applications.…”

“…The resulting ELR‐cell suspensions were then injected subcutaneously in mice and hydrogels were formed instantaneously, as reported previously in vitro (Fernandez‐Colino et al, ; Gonzalez de Torre et al, ), and confirmed by the observation of a protuberance under the skin of the animals, which was not seen when PBS alone was injected. This is also in good agreement with previous in vitro studies regarding the gelation kinetics of SELRs (Fernandez‐Colino et al, ) and ELR‐CFCGs (Gonzalez de Torre et al, ). In the first case, a rapid gelation process was observed by rheological measurements upon increasing the temperature above the Tt, whereas for chemically cross‐linked hydrogels, these were formed in a few seconds upon mixing ELR‐azide and ELR‐cyclooctine solutions, as observed macroscopically.…”

“…The experimental molecular weight was found to be similar to the estimated value (120.4 vs. 121.0 kDa), as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization time‐of‐flight (Figure S1). Furthermore, the Tt was found to be 16.8 °C in PBS (Figure S2), which is 2.4 °C higher than the Tt reported for (EIS) 2 , that is, the non‐RGD‐containing SELR, in the same solvent (Fernandez‐Colino et al, ). This result is consistent with those reported in other studies, which predict an increase in the Tt when hydrophilic (poly)peptides are fused to the ELR sequence (Christensen, Hassouneh, Trabbic‐Carlson, & Chilkoti, ), which is the case of the RGD motif comprising several polar amino acids.…”

Modular protein domains: an engineering approach toward functional biomaterials

“…ELPs exhibit lower critical solution temperature (LCST) behavior; they are soluble below the LCST and form a coacervate, or a dense polymer-rich liquid phase, above the LCST. The Rodríguez-Cabello group has utilized the LCST behavior of ELPs to facilitate hydrogel formation of ELP-fusion proteins [9,10]. In addition, spherical structures of ELPs have been triggered by the thermo-responsive behavior [11].…”

Nanotechnological Approaches to Therapeutic Delivery Using Elastin-Like Recombinamers

“…Such properties are maintained when GAGAGS motifs are engineering along an ELR molecule, giving rise to so-called SELR (silk-elastin like recombinamer). Many SELR-based hydrogels have been reported in the literature [94][95][96][97] and some of them have been successfully explored for drug-delivery purposes. [98][99][100][101] Indeed, the utility of SELRs has been explored in both the field of drug delivery and for the delivery of viral vectors.…”