Novel hydrogel-forming elastin-like recombinamers for biomedical applications

Resumen

There is an increasing interest in developing advanced biomaterials with improved biocompatibility and functionality that might find uses in the field of biomedicine, for example in tissue engineering and regenerative medicine (TERM). Nowadays, recombinant polypeptides or polymers are one of the most prominent types of biomaterials, due to their nature. They are obtained through recombinant DNA technology, which allows the controlled biosynthesis of tailored polypeptides that may be designed to include combinations of polymeric amino acid sequences and/or bioactive domains. Within this type of biomaterials we can find elastin-like polypeptides (ELPs), which have also been recently termed elastin-like recombinamers (ELRs), according to their recombinant origin. These ELRs are composed of repetitions of the VPGXG (Val-Pro-Gly-X-Gly) pentapeptide, in which X (guest residue) can be any amino acid expect L-Proline. This composition confers the ELRs a smart behaviour of thermoresponsiveness defined by the so-called Inverse Temperature Transition (ITT) occurring above the Transition Temperature (Tt), which implies a phase transition of the dissolved ELR when this Tt is reached. Therefore, this thermal response is of great interest, since it has been shown to promote the formation of different structures, such as hydrogels that mimic the extracellular matrix, at the physiological temperature, whereas an ELR solution can be easily handled (e.g. injected in vivo) below the Tt. Furthermore, the Tt can be modulated depending on the polarity of the side chain of the amino acid chosen as guest residue. Moreover, if this residue contains functional goups, it can also be used for further chemical modifications in order to achieve, for instance, chemically (covalently) cross-linked hydrogels. Lately, different types of ELRs have shown potential applications in TERM. However, the general biocompatibility of these ELRs and of the hydrogels based on them has not been extensively studied to date. Thus, it is one of the aims of this Thesis to evaluate whether two types of hydrogels based on ELRs (physically or chemically cross-linked) are biocompatible or not by use of wide-ranging methods, hence being suitable for TERM applications. For this purpose, the in vitro cytocompatibility was studied by culturing endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase-expressing human mesenchymal stem cells (Luc-hMSCs) were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR-based hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR-based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL-1β, IL-4, IL-6 and IL 10 concentrations were measured by ELISA, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for TERM-related applications. Once the biocompatibility of ELR-based hydrogels was confirmed, novel ELRs were specifically designed and bioproduced to promote optimal bone regeneration, taking into account the increasing morbidity of bone fractures and defects due to changes in the age pyramid, and the limitations in the use of auto-, allo-, and xenografts. In order to assess this hypothesis, an ELR containing RGD (Arg-Gly-Asp) cell-adhesion sequences and another one fused to the osteogenic bone morphogenetic protein-2 (BMP-2) were used to form injectable physically cross-linked hydrogels. Moreover, elastase-sensitive domains were included in both ELR molecules, thereby conferring biodegradation as a result of enzymatic cleavage and avoiding the need for scaffold removal after bone regeneration. Both ELRs and their combination showed excellent in vitro cytocompatibility, and the culture of cells on RGD-containing ELRs resulted in optimal cell adhesion. In addition, hydrogels based on a mixture of both ELRs were implanted in a pilot study involving a femoral bone injury model in New Zealand White rabbits, showing complete regeneration in six out of seven cases, with the other showing partial closure of the defect. Moreover, bone neo-formation was confirmed using different techniques, such as radiography, computed tomography and histology. Therefore, this hydrogel system therefore displays significant potential in the regeneration of bone defects, promoting self-regeneration by the surrounding tissue with no involvement of stem cells or osteogenic factors other than BMP-2, which is released in a controlled manner by elastase-mediated cleavage from the ELR backbone. On the other hand, it was also proposed that the fusion of fluorescent proteins (FPs) to ELRs that include silk-like domains (thus termed silk-elastin-like recombinamers or SELRs), which further stabilize the ELR-based hydrogels, would confer fluorescence to the hydrogels, hence improving their traceability if used to produce biomedical devices for potential TERM applications. In this Thesis, we fused two different fluorescent proteins (FPs), i.e. the green Aequorea coerulescens EGFP (AcEGFP) and the near-infrared eqFP650, to a SELR able to form irreversible hydrogels through physical cross-linking. These recombinamers showed an emission of fluorescence similar to the single FPs, and they were capable of forming hydrogels with different stiffness (G’ = 60-4000 Pa), by varying the concentration of the SELR-FPs. These results support the hypothesis, suggesting that a combination of these SELRs with other SELRs including different bioactivities, such as cell adhesion, may provide a biomimetic scaffold that is also liable to be tracked in vivo by non-invasive fluorescence measurements, which is a starting point for further work in this regard. In addition, the absorption spectrum of SELR-eqFP650 showed a peak greatly overlapping the emission spectrum of the SELR-AcEGFP. New possibilities arose from this finding, since this overlap could enable Förster resonance energy transfer (FRET) upon the interaction between two SELR molecules, each one containing a different FP. FRET has been defined to take place at a distance of less than 10 nm between the donor (in this case SELR-AcEGFP) and the acceptor (SELR-eqFP650), the latter inducing the quenching of the donor. The interaction between both SELR-FPs (i.e. reducing the distance below 10 nm) might be driven by the self-assembly of the elastin-like domains above the Tt, and by the stacking of silk domains at any temperature. This effect was studied by different methods and a FRET efficiency of 0.06-0.2 was observed, depending on the technique used for its calculation. Therefore, biosensing applications may derive by taking advantage of the FRET occurring between both SELR-FPs through the development of ratiometric biosensors by including amino acid sequences that bind to different targets using genetic engineering methods. In summary, the work showed in this Thesis provides new insights about hydrogel-forming ELRs designed to be used in biomedical applications. Specifically, it describes the physico-chemical characterization of several novel ELRs, and confirms the biocompatibility of two types of hydrogels based on these ELRs. Furthermore, bone regeneration has shown to be optimized by the use of ELR-based hydrogels. On the other hand, basic characterization of the fluorescence emitted by SELR-FPs provide evidence about the feasibility of constructing biomedical devices that include them, in order to improve their traceability in vivo. Moreover, the finding of a FRET interaction sets the basis for further use as ratiometric biosensors upon improvement of the SELR functionality through incorporation of target-binding domains by recombinant DNA technology.