Dual responsive physical networks from asymmetric biosynthetic triblock copolymers

The aim of the project is to develop biosynthetically produced amino acid polymers which are composed of three distinct blocks A-C-B, each with a separate function. A is a first self-assembling block capable of ‘recognizing’ (upon a trigger) other A blocks; C is an inert, random coil-like connector, and B is a second self-assembling block. A and B have to be chosen such that they do not cross-assemble. With these molecules it should be possible to fabricate hydrogels in which direct ‘loops’ are excluded. We exploited genetic engineering to design proper genes encoding asymmetric triblock protein polymer and fermentation to produce monodisperse protein polymers. There different asymmetric triblock protein polymers were produced and characterized. The first molecule, silk-elastin hybrid molecule (SCE), was inspired by natural silk and elastin. The silk-like block (S) forms a pH-sensitive beta-roll (beta-sheet like) structure that further stacks into long fibrils. The elastin-like block(E) has thermo-responsive properties; above the lower critical solution temperature (LCST), it forms aggregates. We find that polymers that have both silk and elastin-like domains show temperature dependent fibril formation. At high temperature, the elastin blocks irreversibly induce bundling and aggregation of fibrils. The presence of the elastin-like block also changes the kinetics of fibril formation. Whereas silk-like protein without elastin forms monodisperse fibrils, the presence of elastin results in polydisperse fibrils due to homogenous nucleation. The self-assembly of silk-elastin hybrid molecule is further analysed in the presence of NaCl. We find that the thermo-responsive behaviors of elastin-like block are strongly dependent on salt concentration. At high salt concentration, the aggregation transition is much more pronounced. At high pH, where the S block does not self-assemble, the polymer forms micellar aggregates upon heating in the presence of NaCl. At low temperature, lowering the pH leads to fibril formation. When both blocks are induced to self-assemble, the final structure reveals a pathway-dependence. Heating the solution of fibrils formed at low temperature results in fibril aggregates which do not dissociate upon cooling. The pH-triggered fibril formation of preheated protein solutions leads to the formation of large objects, which likely cause sedimentation. The structural difference is also demonstrated clearly in the morphology of gels formed at high protein concentration: whereas the gel formed in the first pathway (first lower the pH, then increase the temperature) is transparent, the gel formed in the latter pathway (first increase the temperature, then lower the pH) is milky and has a higher elastic modulus. The second type of asymmetric triblock copolymer (TR4H or TR4K) has a collagen-like, triple-helix-forming motif at one end, and a poly cationic block at the other. The collagen-like end-block T consists of 9 (PGP) repeats and forms thermo-responsive triple helices upon cooling. Such helices are reversibly disrupted when the temperature is raised above the melting temperature. The other end-block has 6 positively charged amino acids (histidine-H or lysine-K) and forms micelles when a negatively charged polymer is added. The charge-driven complexation of this block depends on its degree of deprotonation, which is determined by the pKa and the pH. The additives used in this study are a flexible polyanion (polystyrene sulfonate, PSS) and a semi-flexible polyanion (xanthan). We find micelle-to-network transition of the triblock TR4H in complexation with PSS. First, the self-assembly of each end-block is studied separately. As expected, the collagen-like block reversibly forms triple helices upon cooling. The cationic H block forms charge-driven complexes upon adding PSS, leading to micelles with an aggregation number that depends on ionic strength. At high concentration, the micellar TR4H/PSS solutions form a viscoelastic gel upon cooling, which melts at high temperature, indicating the formation of helical junctions between the micelles. Liquid-liquid phase separation is observed when the concentration is below the gelation point (around 90 g/L). This leads to a dilute phase on top of a concentrated gel phase. The phase separation is driven by the attraction between charge-driven micelles caused by the triple helices. It disappears when the solution is heated or when the ionic strength is increased. The charge-driven complexation of TR4K with xanthan, a negatively charged polysaccharide is also studied. At high temperature and at very low xanthan concentration, the TR4K binds to the xanthan backbone via the charged block K, leading to charge-driven bottle brushes, as indicated by a significant increase in light scattering intensity due to the increased mass. This interaction is dependent on the pH, due to protonation of the cationic K block. The xanthan/TR4K complex shows thermo-sensitivity due to the helical interaction of the collagen-like blocks. At a xanthan concentration around the overlap concentration (~7 g/L), the presence of the triblock results in an increase in elastic modulus of xanthan gels. At high temperature, the elastic modulus increases by 3 times after adding the triblock. As triple helices do not form, this must be due to changes in the entanglement of the bottle brushes. Also the non-linear rheology of the xanthan/TR4K gels differs significantly from that of xanthan alone. At low temperatures when the helical junctions are formed, the elastic modulus increases even further approximately two times compared with the corresponding value at high temperature. This is ascribed to the formation of crosslinks induced by the proteins between the xanthan molecules. The triblock protein modifies the properties of the xanthan hydrogels in three ways: (1) a significant increase in storage modulus, (2) thermo-sensitivity and (3) a two-step strain softening, where the first step is probably due to unbinding of the proteins from the xanthan backbones. The third molecule is an asymmetric triblock copolymer (TR4T-Cys), which has two triple helix forming end-blocks (T), with a cysteine residue (Cys) added to one of these. Under oxidizing conditions, the cysteine residues can form disulfide bonds between two polymers whereas reducing conditions restore the thiol groups. Since cysteine can form only one S-S bridge, intramolecular loops are prevented. The presence of S-S bonds significantly enhances the thermal stability of the triple helical network. This results in the appearance of two melting temperatures, of which the higher one is due to the S-S stabilized triple helices. The elastic modulus of the physical gels in the presence of S-S bonds is almost 2 times higher than that of the physical gels in the absence of S-S bonds. The relaxation time also triples under oxidizing conditions, which indicates that triple helical knots are also kinetically stabilized by S-S bonds. In summary, the design of S-C-S (S: functional end-block, C: connector) network-forming components might meet the increasing demands of high performance biomaterials that must be able to build a physical gel under narrowly defined conditions. Such class of telechelic polymer might display various interesting dynamic behaviors including shear banding, self-assembly, rheochaos, and phase-separation. Another aspect is the functionality of the end-block which self-assembles upon triggering. However, connectors often return to the same nodes, resulting in loop formation. Loop formation is a structural imperfection that weakens network connectivity and lowers the material’s elasticity. The asymmetric triblock with two different end-blocks is designed in order to: (1) prevent unimolecular loops and improve mechanical properties (2) achieve multi-responsiveness: this allows us to observe different assembling pathways. In this work, with respect to (1), we indeed observed the decrease in loop formation in physical gels formed by TR4T-Cys due to the formation of S-S bridges. With respect to (2), we indeed obtained multi-responsive hydrogels with all three asymmetric triblock proteins. However, we have only scratched the surface as understanding kinetics of self-assembly and pathway dependent processes. Further investigations are needed to get more insights into how to manipulate various parameters in controlling the final structures. </p

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Disulfide bond-stabilized physical gels of an asymmetric collagen-inspired telechelic protein polymer

We designed and produced an asymmetric collagen-inspired telechelic protein polymer with end blocks that can form triple helices of different thermal stabilities. Both end blocks consist of a motif that can form triple helices at low temperature, but one of these blocks carries an additional cysteine residue at the end. The cysteine residues can form disulfide bridges under oxidizing conditions, leading to dimer formation. This effectively stabilizes the triple helices, resulting in a double melting peak in differential scanning calorimetry: one corresponding to helices without disulfide bridges and one at significantly higher temperature, corresponding to stabilized helices. Under reducing conditions, the disulfide bridges are broken and the molecule behaves similarly to the symmetric variant. We find that these disulfide bridges also lead to an increase of the elastic modulus of the helical polymer network, probably because the number of helices in the system increases and also the disulfide bridges can crosslink different triple helical nodes

Disulfide bond-stabilized physical gels of an asymmetric collagen-inspired telechelic protein polymer

We designed and produced an asymmetric collagen-inspired telechelic protein polymer with end blocks that can form triple helices of different thermal stabilities. Both end blocks consist of a motif that can form triple helices at low temperature, but one of these blocks carries an additional cysteine residue at the end. The cysteine residues can form disulfide bridges under oxidizing conditions, leading to dimer formation. This effectively stabilizes the triple helices, resulting in a double melting peak in differential scanning calorimetry: one corresponding to helices without disulfide bridges and one at significantly higher temperature, corresponding to stabilized helices. Under reducing conditions, the disulfide bridges are broken and the molecule behaves similarly to the symmetric variant. We find that these disulfide bridges also lead to an increase of the elastic modulus of the helical polymer network, probably because the number of helices in the system increases and also the disulfide bridges can crosslink different triple helical nodes

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