Photoreactive elastin-like proteins for use as versatile bioactive materials and surface coatings

Abstract

Photocrosslinkable, protein-engineered biomaterials combine a rapid, controllable, cytocompatible crosslinking method with a modular design strategy to create a new family of bioactive materials. These materials have a wide range of biomedical applications, including the development of bioactive implant coatings, drug delivery vehicles, and tissue engineering scaffolds. We present the successful functionalization of a bioactive elastin-like protein with photoreactive diazirine moieties. Scalable synthesis is achieved using a standard recombinant protein expression host followed by site-specific modification of lysine residues with a heterobifunctional N-hydroxysuccinimide ester-diazirine crosslinker. The resulting biomaterial is demonstrated to be processable by spin coating, drop casting, soft lithographic patterning, and mold casting to fabricate a variety of two- and three-dimensional photocrosslinked biomaterials with length scales spanning the nanometer to millimeter range. Protein thin films proved to be highly stable over a three-week period. Cell-adhesive functional domains incorporated into the engineered protein materials were shown to remain active post-photo-processing. Human adipose-derived stem cells achieved faster rates of cell adhesion and larger spread areas on thin films of the engineered protein compared to control substrates. The ease and scalability of material production, processing versatility, and modular bioactive functionality make this recombinantly engineered protein an ideal candidate for the development of novel biomaterial coatings, films, and scaffolds.

Engineered protein coatings to improve the osseointegration of dental and orthopaedic implants

“…[33] Briefly, the NHS-diazirine was dissolved in dimethyl sulfoxide (0.5–1 g/mL) and mixed with a solution of ELP (50 mg/mL in phosphate buffered saline (PBS)) to a final stoichiometric ratio of 1:1 functional groups. After reaction, the solution was dialyzed, lyophilized, and stored at 4 °C.…”

“…Alternatively, recombinant protein based coatings are attractive as they retain the bioactivity of naturally derived materials, can be manufactured reproducibly, and are highly customizable. [32, 33] …”

“…The ELP is modified with a photoreactive diazirine moiety to allow for UV-mediated crosslinking to form stable films. [33] Previous analysis of our photocrosslinkable ELP thin films focused on in vitro cytocompatibility tests with human adipose-derived stem cells for general use in regenerative medicine therapies. [33] In this work, we identify the carbene insertion mechanism as a novel strategy to create covalently crosslinked, mechanically stable protein thin films on metallic implants for orthopaedic and dental applications.…”

“…[33] Previous analysis of our photocrosslinkable ELP thin films focused on in vitro cytocompatibility tests with human adipose-derived stem cells for general use in regenerative medicine therapies. [33] In this work, we identify the carbene insertion mechanism as a novel strategy to create covalently crosslinked, mechanically stable protein thin films on metallic implants for orthopaedic and dental applications. In particular, we demonstrate the versatility of ELP thin films by using both spin and dip coating to form stable coatings on common implant materials (cp-Ti and Ti6Al4V) in a variety of geometries (discs, screws, and rods).…”

Precise Protein Photolithography (P3): High Performance Biopatterning Using Silk Fibroin Light Chain as the Resist

“…To date, several techniques, such as Electron beam (E‐beam) writing,9, 10 imprinting,11, 12 molding,13, 14 electrospinning,15 embossing,16, 17 inkjet printing,18 and photolithography (P 3 )19, 20 have enabled the development of a variety of material formats, including hydrogels, fibers, particles, and films using fibroin or sericin as well as their blends with other materials 3, 21, 22. Photolithography, in particular, remains one of the most appealing techniques for scalable biomanufacturing as it is Complementary Metal Oxice Semiconductor (CMOS)‐compatible and can rapidly fabricate high fidelity micro/nanopatterns in parallel—in contrast, scanning‐probe lithography and electron beam lithography for biomanufacturing use serial manufacturing techniques 23, 24, 25, 26, 27…”