Bulk network architectures from ELP-POP mixtures

The T cp of POPs and ELPs are tunable by the identity and mole fraction of the guest residue (X) in the VPGXG repeat unit, and the T cp s of POPs are further tunable by the mole fraction of embedded oligoalanine helices. We therefore used different ratios of alanine (A) and valine (V) in the guest residue position in ELPs and POP and the helical content of POPs to span a range of T cp s of ELPs and POPs from 20 °C to 50 °C (Supplementary Figs. 1–2 and Supplementary Table 1). This range of T cp s allows us to use two distinct types of POP-ELP mixtures: (1) one in which the POP is designed to transition at a lower temperature than the ELP upon heating the mixture, and (2) another in which the POP is designed to transition at a higher temperature than the ELP. Within POP-ELP mixtures, the two IDP populations are not fully miscible, with distinct aggregation events observed for both populations.

To demonstrate the structural consequence of phase separation in system 1, we used a mixture of POP(V)-25% (where ‘V’ designates the guest residue amino acid and 25% designates the fraction of oligoalanine) with ELP(V 4 A 1 ). Above the T cp -heating of POP(V)-25%, the IDP forms a stable, porous network as expected. Continued heating to above the T cp of the ELP causes ELP coacervates to form and grow until they are able to interact with the preformed POP network. Upon contact with the network, they become immobile, forming ‘fruits’ of ELP on a POP network ‘vine’ in solution as illustrated in the schematic (Fig. 1di) and confocal microscopy sections (Fig. 1ei–ii). While the size of the ELP globules is somewhat varied, the average volume of the globules is directly correlated with the concentration of ELP in solution without altering the POP network structure (Supplementary Fig. 3). The system can also be cooled below the ELP T cp —but above the T cp -cooling of the POP—and reheated with no change in the average globule size (Supplementary Fig. 3).

System 2, with ELP(V) designed to coacervate before POP(V 1 A 4 )-25%, forms an entirely different type of structure. Heating above the T cp of the ELP forms polymer coacervate droplets, as expected. Upon raising the temperature above the T cp -heating of the POPs, however, the POPs do not form a microporous network but instead wet with the outer edges of ELP coacervate droplets and form a physically crosslinked interconnected porous shell, such that the entire structure consists of spherical ELP coacervate droplets encased in a lattice-like shell of the POP coacervate as illustrated (Fig. 1dii) and shown in confocal microscopy sections (Fig. 1eiii–iv). Due to the hysteretic nature of POPs and their significantly lower T cp -cooling than T cp -heating, subsequent cooling of the system to T < T cp of the ELP but T > T cp -cooling of the POP dissolves the ELP cores into the aqueous phase through the pores of the POP shell, and creates an interconnected network of hollow protein shells (Supplementary Fig. 4). These types of two-protein systems are a simple way to create a drug eluting scaffold using a single injectable system. To demonstrate proof-of-concept of this approach, we used fluorescence molecular tomography (FMT) to monitor the release of ELP(V 4 A 1 ) co-injected with POP(V)-25% in the subcutaneous flank of mice (Supplementary Fig. 5). The ELP “fruits” that hang from the POP “vine” slowly dissolve and are secreted out of the POP scaffold over 10 days without affecting the size of the POP scaffold. The release kinetics are further tunable with ELP concentration without altering the concentration of the co-injected POP.

Production of atypical microparticle architectures

Given the limited available architectures for biomaterial microparticles and the ease of formation for these atypical POP-ELP architectures in bulk, we next sought to translate these structures to microscale droplets. To do so, we used a microfluidic emulsion droplet generator in an X-junction design46 (Fig. 2a) capable of producing highly monodisperse water-in-oil emulsion droplets (Fig. 2b). The ELP and POP components in PBS were premixed and the entire device was kept at 4 °C during droplet generation to ensure uniform distribution of the soluble IDPs within each droplet. We controllably triggered subsequent ELP and POP phase transitions within the microdroplets by heating and cooling to generate a range of coacervate microstructures. We first examined the structures formed solely by POP(V)-25% in microdroplets, and found that the POP produces stable structures with microarchitectures similar to those observed in bulk (Fig. 2c–e and Supplementary Fig. 6), forming fractal-like porous microparticles with high void volume above the T cp -heating. Continued heating and cooling above the aggregation temperature leads to nonlinear shrinking and swelling of the microparticles (Supplementary Fig. 7). Heated particles shrink/swell by as much as 20% between 20 and 50 °C, and the process is fully reversible. POP(V)-12.5% can also be used, forming identical microparticles with only a slightly higher T cp -heating than the 25% POP (Supplementary Fig. 8). All particles remain stable when cooled into the meta-stable hysteretic temperature range, and subsequent cooling below the T cp -cooling of the POP results in complete dissolution of the microstructure.

Fig. 2: Microdroplet architectures. a Depiction of the microfluidic device used to generate microparticles. b Image analysis of droplets (n = 125 droplets) reveal a high degree of monodispersity. c Partial phase diagram for POP(V)-25% illustrating the different discrete states possible during a heating and cooling cycle. d Fluorescence images of POP(V)-25% (500 µM) microdroplets during a heat-cool cycle through the states shown in (c). The metastable hysteretic range prevents dissolution of the particles until the solution temperature is lowered below the T cp -cooling. e Confocal images (25 µm stack) of the same particles in the metastable hysteretic state—state 3. f Partial phase diagram for mixtures of POP(V)-25% (200 µM) + ELP(V 4 A 1 ) (200 µM) depicting the different discrete states possible during a heat-cool cycle of this system in which the POP aggregates at a temperature below the ELP. g Fluorescent images of each state of the cycle demonstrating the formation of the fruits-on-a-vine architecture in state 3. h Confocal images of state 3 clearly depicting the ELP “fruits”. i Partial phase diagram of ELP(V) (500 µM) + POP(V 1 A 4 )-25% (100 µM) depicting the states available during a heat-cool cycle. j Fluorescent images of each state of the cycle. Due to the hysteretic nature of the POP and its lower T cp -cooling than the T cp of the ELP, the ELP dissolves first upon cooling, diffusing out and leaving a network of hollow POP shells. k Confocal images of core-shell networks formed in state 3. Source data are provided as a Source Data file. Full size image

We next included ELP as a second polymer component within the aqueous phase of our microfluidic set-up to recapitulate the unique architectures seen in bulk within a confined microenvironment with the end goal of creating microparticles with unique morphologies and internal microstructures. Mixtures of POP(V)-25% and ELP(V 4 A 1 ), in which the POP phase separates at a lower temperature than the ELP, were thermally programmed into the five distinct states seen in the overlaid ELP and POP phase diagram (Fig. 2f–h and Supplementary Fig. 6): (1) ELP and POP are soluble; (2) upon heating to a temperature > T cp -heating of the POP, the POP phase separates and forms a porous microparticle; (3) upon continued heating to T > T cp of the ELP, the ELP coacervates into immiscible globules that wet the POP network; (4) upon cooling below the T cp of the ELP, the ELP dissolves; (5) and further cooling to T < T cp -cooling of the POP cooling also re-dissolves the POP.

In contrast, mixtures of ELP(V) + POP(V 1 A 4 )-25% in which the ELP coacervates at a lower temperature than the POP (T cp ELP < T cp -heating POP), can be cycled through core-shell and hollow shell networks (Fig. 2i–k and Supplementary Fig. 6) as follows: (1) both IDPs are fully dissolved; (2) as the temperature is raised such that T > T cp of the ELP, the ELP coacervates into aqueous-immiscible droplets; (3) raising the temperature to T > T cp -heating of the POP triggers the phase separation of the POP, leading to the formation of a conformal porous POP shell on the ELP core; (4) upon cooling to T < T cp of the ELP but T > T cp -cooling of the POP, the ELP dissolves resulting in a network of hollow POP shells; (5) finally, as the temperature is lowered below the T cp -cooling of the POP, the POP re-dissolves, fully restoring the system to its original state of a mixture of soluble ELP and POP. Like the porous POP microparticle networks, the hollow POP shells also swell and shrink ~20% in size when heated and cooled after formation (Supplementary Fig. 7). After reaching state 4, where the ELP has dissolved out from within the POP shells into the aqueous phase of the droplets, leaving behind intact porous POP shells, if the temperature is then raised above the T cp of the ELP, the ELPs will re-coacervate, forming aqueous-immiscible ELP globules that wet the outside of the hollow POP shells (Supplementary Fig. 9).

Incorporation of unnatural amino acids (UAAs) for UV crosslinking

To augment their stability, we next devised a method to crosslink the microstructures without the need of extrinsic crosslinking agents, and without the formation of potentially toxic byproducts. 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) light49. 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-gels47,50, a small library of UV crosslinkable xPOPs was created and their thermal and microarchitecture properties were characterized (Supplementary Fig. 10). xPOPs are identical to POPs in their sequence, with the exception that pAzF residues are equally spaced throughout the polymer at 1 pAzF per 100 residues. While the T cp -heating and T cp -cooling of the xPOPs are slightly depressed relative to the parent POP due to the hydrophobicity of pAzF, the xPOP microemulsions undergo an identical coacervation process, forming porous microparticles. The xPOPs photochemically react after only short UV exposure, requiring <10 s of exposure time to fully crosslink. Network architecture and void volume in bulk are unaffected by crosslinking (Supplementary Fig. 10). When crosslinked above their T cp -heating, subsequent cooling to below T cp -cooling does not resolubilize the microparticles, unlike their non-crosslinked counterparts (Supplementary Fig. 11).

xPOPs can also be readily mixed with ELPs to stabilize microparticle architectures. Mixtures of xPOP(V)-25% and ELP(V 4 A 1 ) form a fruits-on-a-vine architecture when heated above the IDPs’ aggregation temperatures similar to that previously observed for a POP and ELP mixture (Supplementary Fig. 12). Subsequent exposure to UV light crosslinks the POP, allowing the ELP to solubilize, but preventing the POP from solubilizing even when cooled well below its T cp -cooling. If the system is re-heated, ELP globules reform, and this process can be repeatedly cycled without altering the POP microparticle architecture. Cooling and re-heating does not alter ELP globule properties, and the average “fruit” size remains unchanged. Compared to bulk mixtures, the ELP “fruits” formed in microparticles are similar in shape but slightly larger in size (p < 0.05, Student’s t-test, n = 50).

Using heating rate to control microarchitectures

Within the polymer sequence framework presented in this manuscript, the order in which the two components—ELP and POP—phase separate controls the type of architecture that is formed, rather than the specific sequences of the chosen POP and ELP. For example, mixtures of ELP(V) + POP(V 1 A 4 )-12.5% (Supplementary Fig. 13) and ELP(V) + POP(V 1 A 1 )-25% (Supplementary Fig. 9) both form core-shell structures similar to ELP(V) + POP(V 1 A 4 )-25% (Fig. 3) despite the differences in all three POP sequences. However, these structures are not wholly identical. 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 non-hysteretic ELP and hysteretic POP and our previous work on dual emulsion ELPs46,47. 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. In the ELP-POP system, increasing the temperature range of thermal hysteresis—the T cp gap—also increases the amount of time that ELP is given to coalesce at a constant thermal ramp rate, prior to entrapment by POPs. Given the sequence homology between the disordered components of POPs and ELPs, POPs prefer to aggregate around the ELPs, and once even a very small layer of POP has formed around the ELP, the ELP coacervate droplets can no longer continue to coalesce.

Fig. 3: Controlling hollow shell architecture. a Size distribution of xPOP shells formed after heating a mixture of ELP(V) (1 mM) + xPOP(V 1 A 4 )-12.5% (100 µM) at different constant rates (10–90% box and whiskers plot with median central line bounded by 25 and 75% quartiles, *p < 0.05 as determined by one-way ANOVA with Tukey’s post hoc test, n = 50 shells measured using 3D confocal image stacks for each rate). b Histogram of the size distribution of the xPOP shells shows a broad distribution of shell diameters at ramp rates > 1 °C, the development of a bimodal distribution at 1 °C/min, and emergence of a unimodal size at 0.5 °C/min. c Fluorescence microscopy images and corresponding cartoon of the hollow xPOP shell architectures that form at different heating rates. The system shifts from a network of interconnected hollow shells to a single hollow protein shell as the heating rate is slowed. d Hollow xPOP protein shell diameter increases with increased ELP concentration (10–90% box and whiskers plot with median central line bounded by 25 and 75% quartiles, *p < 0.01 as determined by two-tailed Students t-test, n = 50 droplets for each rate). e Linear regression analysis for a polydisperse mixture of ELP(V) (1 mM) + xPOP(V 1 A 4 )-12.5% (100 µM) comparing diameter of the water-in-oil emulsion droplets with the diameter of the xPOP shells contained within the droplets (n = 280 droplets). f Typical fluorescence images illustrating the linear correlation between droplet diameter and xPOP shell diameter and the ~0.5 scaling pre-factor that relates droplet diameter to xPOP shell diameter. Source data for are provided as a Source Data file. Full size image

These results suggested that the core-shell architecture can be controlled by heating ELP and POP mixtures at different rates. To investigate this, we used mixtures of ELP(V) and xPOP(V 1 A 4 )-12.5% to illustrate the spectrum of core-shell structures achievable (Fig. 3a–c and Supplementary Fig. 15). ELP + xPOP mixtures were heated at ramp rates of 0.5–20 °C/min in a thermocycler from 4 to 50 °C and were then UV-crosslinked at the final temperature. Samples were then cooled and transferred to a fluorescent microscope for imaging. At high ramp rates, the ELP is given limited time to coalesce, resulting in disperse ELP globules that become encapsulated by conformal porous shells of the POP, producing a large network of small hollow crosslinked shells. Slightly faster ramp rates produce similar networks, with slightly larger POP shells and some “network-like” arms likely due to (a) the absence of ELP and (b) insufficient time to interact with already aggregated POP. At 1 °C/min, a bimodal distribution emerges with an average of one large and one small shell per aqueous droplet. Notably, when the heating rate is slowed to 0.5 °C/min, a single hollow-spherical POP shell per droplet is formed. Reheating does not re-fill the shells (Supplementary Fig. 14), but it does cause aggregation of ELP within and outside of the hollow spheres.

Not only can the architecture of these POP shells be controlled, from a network or hollow protein shells to a single hollow protein shell, but we can also control their size (Fig. 3d–f and Supplementary Fig. 15). If the diameter of the aqueous droplet is kept constant, doubling the volume of ELP within each aqueous droplet, for example, also doubles the volume within a POP shell. The diameter of the droplet provides an even more convenient way of controlling size. By varying the speed of the aqueous phase during droplet generation, and therefore creating a polydisperse mixture of droplet sizes, we were able to linearly correlate the diameter of the resultant POP shell to the droplet diameter. With 1 mM ELP(V) as the core-forming component, the diameters of the POP shells were consistently half that of the particle diameter (Fig. 3e, f).

Microparticle extraction into an all-aqueous environment

For many downstream applications, POP microparticles must be extracted back into an aqueous environment from the emulsion. In addition to stabilizing the microstructures that are formed within water-in-oil emulsions, UV crosslinking through UAA incorporation allows extraction of these microstructures into an all aqueous phase (Fig. 4a). Using a simple de-emulsification process (see Methods), porous POP microparticles were successfully recovered into buffered saline (Fig. 4b, c). The particles are mechanically stable enough to retain their shape and porosity, albeit with a 40% reduction in their size following extraction (40.7 µm ± 1.8 µm pre-extraction, and 25.3 µm ± 3.4 µm post extraction, n = 50 particles each).

Fig. 4: Extraction into an aqueous environment. a Schematic of the process of extraction from water-in-oil to an all aqueous (buffered saline) environment. b Microscopy images of (i) unextracted (500 µM) and (ii) extracted xPOP(V)-25% porous microparticles and (iii) unextracted and (iv) extracted xPOP shells formed from mixtures of ELP(V) (1 mM) + xPOP (V 1 A 4 )-12.5% (100 µM) mixtures. The inset in iv demonstrates that hollow shell networks created with faster heating rates can also be extracted. c SEM of a xPOP(V)-25% microparticle showing the interconnected coacervate architecture that comprises the networked particle. d Cryo-SEM images of (i) the outer surface and (ii) a fractured xPOP (V 1 A 4 )-12.5% shell. The walls of each shell range from ~200–400 nm thick are composed of tightly packed nano-coacervates of xPOP. Full size image

To determine their mechanical stability, we used high frequency atomic force microscopy (AFM) to evaluate the Young’s modulus (E) (Supplementary Fig. 16) of extracted POP microparticles and controls—planar bulk gels of crosslinked ELP (xELP(V)) and a crosslinked POP with 25% helical content (xPOP(V)-25%). While the planar xELP(V) has an E of 0.12 ± 0.02 kPa, the additional stabilization conferred by physical crosslinking increases E of the bulk xPOP(V)-25% by almost one order of magnitude to 1.4 ± 0.3 kPa. Notably, the microparticles of the xPOP(V)-25% were more than an order of magnitude stiffer than the bulk material, with xPOP(V)-25% particles reaching a Young’s modulus of 20.9 ± 2.8 kPa, indicating that the ~40% compaction in the size of the microparticles that occurs post-extraction has an unexpectedly significant effect on its mechanical stiffness. However, the degree of helix incorporation did not have a statistically significant effect on the Young’s modulus of crosslinked microparticles, as the Young’s moduli of xPOP(V)-25% and xPOP(V)-12.5% were not statistically different, suggesting that any additional physical stabilization conferred by the increased helical content is overwhelmed by the effect of chemical crosslinking and their physical compaction upon extraction from the water-in-oil emulsion.