Photo‐Crosslinkable Unnatural Amino Acids Enable Facile Synthesis of Thermoresponsive Nano‐ to Microgels of Intrinsically Disordered Polypeptides

Abstract

Hydrogel particles are versatile materials that provide exquisite, tunable control over the sequestration and delivery of materials in pharmaceutics, tissue engineering, and photonics. The favorable properties of hydrogel particles depend largely on their size, and particles ranging from nanometers to micrometers are used in different applications. Previous studies have only successfully fabricated these particles in one specific size regime and required a variety of materials and fabrication methods. A simple yet powerful system is developed to easily tune the size of polypeptide-based, thermoresponsive hydrogel particles, from the nano- to microscale, using a single starting material. Particle size is controlled by the self-assembly and unique phase transition behavior of elastin-like polypeptides in bulk and within microfluidic-generated droplets. These particles are then stabilized through ultraviolet irradiation of a photo-crosslinkable unnatural amino acid (UAA) cotranslationally incorporated into the parent polypeptide. The thermoresponsive property of these particles provides an active mechanism for actuation and a dynamic responsive to the environment. This work represents a fundamental advance in the generation of crosslinked biomaterials, especially in the form of soft matter colloids, and is one of the first demonstrations of successful use of UAAs in generating a novel material.

Complex microparticle architectures from stimuli-responsive intrinsically disordered proteins

“…We determined that the smaller the gap in transition temperatures between the ELP and POP, the smaller the resulting individual core-shell structures that make up the network. This observation highlights a critical difference between the combination of nonhysteretic ELP and hysteretic POP and our previous work on dual emulsion ELPs 46,47 . Given sufficient time, two immiscible ELPs with different transition temperatures will phase separate from one another into identical structures regardless of when the second ELP transition is triggered.…”

“…To do so, we pursued multi-site UAA incorporation of para-azidophenylalanine (pAzF), which participates in a host of crosslinking reactions following exposure to ultraviolet (UV) light 49 . Following the recent successes in engineering E. coli strains optimized to site-specifically express UAAs with high fidelity and yield, as well as their use in fabricating thermally responsive micro-gels 47,50 , a small library of UV crosslinkable xPOPs was created and their thermal and microarchitecture properties were characterized ( Supplementary Fig. 10).…”

“…The plasmids were then co-transformed into c321.ΔA E. coli alongside a pEvol tRNA/ aaRS vector with two copies of pAcFRS.1.t1 synthetase. The C321.ΔA genome has previously been edited to remove all instances of the amber stop codon, and the tRNA/aaRS pair has been optimized to recognize the amber stop codon and incorporate para-azidophenylalanine 47,50 .…”

“…Using water-in-oil droplet microfluidics, we also recently demonstrated that the miscibility of different ELPs can be finely controlled to create spherical core-shell particles of ELP coacervates 46 . These coacervate droplets could be further crosslinked in situ by brief exposure to UV radiation by genetically encoding a UV-reactive non-canonical azidophenylalanine residue into the monoblock ELP 47 .…”

Engineering the Architecture of Elastin‐Like Polypeptides: From Unimers to Hierarchical Self‐Assembly

“…Costa et al. developed a bio‐orthogonal crosslinking approach by genetically encoding a photoreactive unnatural amino acid, para‐azidophenylalanine (pAzF), into an ELP at the X position of the VPGXG repeat ( Figure ) . They designed two pAzF‐containing ELPs: i) a monoblock photocrosslinkable ELP (PCE) consisting of (VPGVG) 80 with four regularly spaced pAzF residues, and ii) a self‐assembling photocrosslinkable diblock (PCD) with an N‐terminal hydrophobic ELP block containing four equispaced photocrosslinkable pAzF residues and a C‐terminal hydrophilic ELP block that did not contain any pAzF residues.…”