Multifunctional magnetically responsive biocomposites based on genetically engineered silk-elastin-like protein

Abstract

Magnetic nanocomposite films, comprised of a genetically engineered silk-elastin-like protein (SELP) and CoFe₂O₄ nanoparticles (CFO NPs) with concentrations varying between 5 and 20 wt%, were produced by solvent casting. The obtained materials were analysed regarding their morphology, physical-chemical, thermal, mechanical and magnetic properties. It was found that the magnetic NPs are homogenously distributed among the film and do not induce any significant alterations in their physical-chemical properties. Regarding the thermal properties, the onset degradation temperature of the SELP-59-A films was also not significantly altered by the inclusion of the NPs. Further, strongly bound water is present in the material, which decreases with increasing NPs concentration. Likewise, the mechanical properties of the films were affected by the presence of NPs. Finally, it was demonstrated that the magnetization saturation increases with increasing CFO NPs content, showing the magnetic responsivity of the materials and opening new perspectives in the development of a new generation of multifunctional biocomposites suitable for a wide range of applications, from sensors to tissue engineering.

Introduction

Recombinant protein-based polymers show high potential for the development of (bio)materials suitable for an assortment of different applications. These polymers can mimic natural protein structures and functions and can be designed de novo for the synthesis of customizable materials [1,2]. In opposition to chemically synthesized polymers, recombinant DNA technology enables the design of protein polymers with an absolute control over its composition, stereochemistry and length [3,4]. The ability to be fully customized with absolute control over sequence, which is key attribute for the design of advanced polymeric materials, turns recombinant protein-based polymers as ideal templates for the development of advanced materials, with the added advantage of production not dependent on natural or oil based resources. This highlights the potential of genetically engineered protein polymers as central players to revolutionize the use of polymers in materials science. Moreover, the versatility and control over protein polymer architecture provides an unmatchable range of design options to extend biological and mechanical properties. For instance, the repeating blocks of amino acids responsible for the distinct properties of natural proteins like collagen, elastin, resilin and silk are used as building blocks for the synthesis of multifunctional complex molecules [[5], [6], [7]]. Silk-elastin-like protein polymers (SELPs) are a family of genetically engineered protein block copolymers, tailored to combine in the same molecule the structural components of silk fibroin and elastin. SELPs typically consist of tandem repeats of silk-like GAGAGS amino acid blocks as the hard (crystallizable) domain, and elastin-like VPGXG amino acid blocks as the soft (amorphous) domain, where X in the elastin block is any amino acid except for proline [8]. The silk-like blocks tend to self-assemble into tightly packed beta-sheets to provide thermal and chemical stability as well as physical cross-linking sites [[9], [10], [11]]. The elastin-blocks offer stimuli-responsiveness by undergoing a reversible structural transition upon exposure to specific environmental stimuli, reduces the overall crystallinity of the SELP system by disrupting the silk-like blocks and increases its flexibility [[9], [10], [11], [12]].

SELP-59-A is a SELP copolymer in which the central glycine of the elastin-block VPGVG was altered to an L-alanine to obtain the sequence VPAVG [13]. This simple substitution has been shown to dramatically change the mechanical response of elastin-like recombinamers (ELRs) from elastic to plastic deformation [14,15], associated with an acute thermal hysteresis behaviour [6,16]. Among the SELP family, SELP-59-A has been produced and purified with high volumetric productivities [17] and fabricated into fibre mats [18], and free standing films [10], demonstrating versatility of processing and unique physical, mechanical and biological properties. Noteworthy, the alanine-containing SELP-59-A demonstrated to be non-cytotoxic to normal human skin fibroblasts (BJ-5ta) [18] and mouse myoblast (C2C12) cell lines [13] and, additionally, cytocompatible SELP-59-A composites with enhanced cell adhesion properties were obtained by blending with fibronectin type II to achieve superior biological performance [19].

Magnetic responsive materials have drawn a lot of interest due to their potential breakthrough applications in a wide range of application such as in the biomedical field (drug delivery, tissue engineering, biosensors), coatings, microfluidics and microelectronics. For the development of physically active platforms intended for (multi)functional advanced applications, silk is currently considered the biomaterial of choice due to its piezoelectricity [20], biocompatibility and biodegradability. Indeed, piezoelectric polymers such as poly(vinylidene fluoride) (PVDF) are increasingly being used as suitable materials for the development of applications relying in the electroactive, mainly piezo-, pyro- and ferroelectric, response [21,22]. Further, the incorporation of magnetostrictive nanoparticles onto the matrix of piezoelectric polymers allows the development of nanocomposites with magnetic, magnetomechanical and magnetoelectric properties suitable for applications ranging from sensors and actuators to cells stimulation in tissue engineering [23,24]. Regarding sensors development, different PVDF-based magnetoelectric (ME) heterostructures such as nanosheets [25] or laminates [26,27] with high magnetoelectric responses have been produced. The application of a magnetic field on these type of composites causes a magnetostrictive response of the magnetic phase, with the mechanical stress being transferred to the piezoelectric polymer and, therefore, developing an electrical response due to the piezoelectric coupling within the polymer phase [28].

In tissue engineering, this ME effect has been used through the application of mechanical and/or magnetic stimuli using a lab-made bioreactor [29] able to impart a controlled magnetic stimulation to scaffolds. It has been demonstrated that magnetically [20] and mechanically [30] stimulated electrical responses on the surface of the material promotes a proper microenvironment for the efficient growth and proliferation of pre-osteoblastic cells [31], as well as an effective osteogenic differentiation of human adipose stem cells [32]. In general, this purpose has been achieved mainly by using PVDF-based polymers. However, the lack of biodegradability of these compounds is a concern in tissue engineering applications.

Despite its increasing interest, recombinant protein polymers have been underutilized in materials science due to the required interdisciplinary synergy between materials science engineers and molecular biologists that implies a convergence of knowledge.

Motivated by the potential application of genetically engineered protein polymers in materials science, the unique characteristics of SELP-59-A and the gap in the development on new functional biocomposites here, we report the fabrication of a novel physically active biodegradable nanocomposite comprising the genetically engineered SELP-59-A incorporated with magnetostrictive cobalt ferrite (CoFe₂O₄, CFO) nanoparticles. These particles are characterized by high magnetization properties and high magnetostrictive response [[33], [34], [35]], and have been successfully used for the formulation of non-cytotoxic magnetic biocomposites [36]. Therefore, the formulation of SELP-59-A/CFO composites will generate a magnetically responsive bio-based material that can be further used for advanced applications, such as in sensors development and tissue engineering.