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The lower critical solution temperature (LCST) of the thermo-responsive engineered elastin-like polypeptide (ELP) biopolymer is being exploited for the thermal targeted delivery of doxorubicin (Dox) to solid tumors. We examine the impact of Dox labeling on the thermodynamic and hydrodynamic behavior of an ELP drug carrier and how Dox influences the liquid-liquid phase separation (LLPS). Turbidity, dynamic light scattering (DLS), and differential scanning calorimetry measurements show that ELP undergoes a cooperative liquid-liquid phase separation from a soluble to insoluble coacervated state that is enhanced by Dox labeling. Circular dichroism measurements show that below the LCST ELP consists of both random coils and temperature-dependent β-turn structures. Labeling with Dox further enhances β-turn formation. DLS measurements reveal a significant increase in the hydrodynamic radius of ELP below the LCST consistent with weak self-association. Dox-labeled SynB1-ELP1 (Dox-ELP) has a significant increase in the hydrodynamic radius by DLS measurements that is consistent with stable oligomers and, at high Dox-ELP concentrations, micelle structures. Enhanced association by Dox-ELP is confirmed by sedimentation velocity analytical ultracentrifugation measurements. Both ELP self-association and the ELP inverse phase transition are entropically driven with positive changes in enthalpy and entropy. We show by turbidity and DLS that the ELP phase transition is monophasic, whereas mixtures of ELP and Dox-ELP are biphasic, with Dox-labeled ELP phase changing first and unlabeled ELP partitioning into the coacervate as the temperature is raised. DLS reveals a complex growth in droplet sizes consistent with coalescence and fusion of liquid droplets. Differential scanning calorimetry measurements show a -11 kcal/mol change in enthalpy for Dox-ELP coacervation relative to the unlabeled ELP, consistent with droplet formation being stabilized by favorable enthalpic interactions. We propose that the ELP phase change is initiated by ELP self-association, enhanced by increased Dox-ELP oligomer and micelle formation and stabilized by favorable enthalpic interactions in the liquid droplets., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

In this study, scanning electron microscopy (SEM) was used to observe the interaction between de-solvated SynB1-elastin-like polypeptide (SynB1-ELP) and silica at a temperature above ELP's lower critical solution temperature (LCST). ELP was seen to initially wet the surface of the silica before rearranging to form narrowly distributed spherical particles. After formation, the ELP particles dynamically rearranged to increase and subsequently decrease in size until 24 h at which time they collapsed. SEM and Energy Dispersive X-ray Spectroscopy revealed that the formation of a thin layer of salt from the PBS solution preceded the initial wetting of ELP on silica, which was shown to play a role in the continuous rearrangement of ELP. FT-IR revealed that the salt, in combination with the hydrophilic silica, trapped water that provided a repulsive surface to the hydrophobic ELP and forced the ELP to continuously minimize its surface area until the water evaporated. This behavior shows that ELP's thermo-responsive nature coupled with its hydrophobicity can be used to create ELP particles and surfaces that can reorganize with minimal water present., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Elastin-like proteins (ELPs) are known to undergo liquid-liquid phase separation reversibly above a concentration-dependent transition temperature. Previous studies suggested that, as temperature increases, ELPs experience an increased propensity for type II β-turns. However, how the ELPs behave below the phase transition temperature itself is still elusive. Here, we investigate the importance of β-turn formation during the early stages of ELP self-association. We examined the behavior of two ELPs, a 150-repeat construct that had been investigated previously (ELP[V 5 G 3 A 2 -150] as well as a new 40-repeat construct (ELP40) suitable for nuclear magnetic resonance measurements. Structural analysis of ELP40 reveals a disordered conformation, and chemical shifts throughout the sequence are insensitive to changes in temperature over 20°C. However, a low population of β-turn conformation cannot be ruled out based on chemical shifts alone. To examine the structural consequences of β-turns in ELPs, a series of structural ensembles of ELP[V 5 G 3 A 2 -150] were generated, incorporating differing amounts of β-turn bias throughout the chain. To mimic the early stages of the phase change, two monomers were paired, assuming preferential interaction at β-turn regions. This approach was justified by the observation that buried hydrophobic turns are commonly observed to interact in the Protein Data Bank. After dimerization, the ensemble-averaged hydrodynamic properties were calculated for each degree of β-turn bias, and the results were compared with analytical ultracentrifugation experiments at various temperatures. We find that the temperature dependence of the sedimentation coefficient (s 20,w o ) can be reproduced by increasing the β-turn content in the structural ensemble. This analysis allows us to estimate the presence of β-turns and weak associations under experimental conditions. Because disordered proteins frequently exhibit weak biases in secondary structure propensity, these experimentally-driven ensemble calculations may complement existing methods for modeling disordered proteins generally., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Elastin-like polypeptides (ELPs) are large, nonpolar polypeptides under investigation as components of a novel drug delivery system. ELPs are soluble at low temperatures, but they desolvate and aggregate above a transition temperature (TT). This aggregation is being utilized for targeting systemically delivered ELP-drug conjugates to heated tumors. We previously examined the structural, thermodynamic, and hydrodynamic properties of ELP[V5G3A2-150] to understand its behavior as a therapeutic agent. In this study, we investigate the effect that adding basic cell-penetrating peptides (CPPs) to ELP[V5G3A2-150] has on the polypeptide's solubility, structure, and aggregation properties. CPPs are known to enhance the uptake of ELP into cultured cells in vitro and into tumor tissue in vivo. Interestingly, the asymmetric addition of basic residues decreased the solubility of ELP[V5G3A2-150], although below the TT we still observed a low level of self-association that increased with temperature. The ΔH of the aggregation process correlates with solubility, suggesting that the basic CPPs stabilize the aggregated state. This is potentially beneficial as the decreased solubility will increase the fraction aggregated and enhance drug delivery efficacy at a heated tumor. Otherwise, the basic CPPs did not significantly alter the biophysical properties of ELP. All constructs were monomeric at low temperatures but self-associate with increasing temperature through an indefinite isodesmic association. This self-association was coupled to a structural transition to type II β-turns. All constructs reversibly aggregated in an endothermic reaction, consistent with a reaction driven by the release of water.

The therapeutic potential of elastin-like polypeptide (ELP) conjugated to therapeutic compounds is currently being investigated as an approach to target drugs to solid tumors. ELPs are hydrophobic polymers that are soluble at low temperatures and cooperatively aggregate above a transition temperature (TT), allowing for thermal targeting of covalently attached drugs. They have been shown to cooperatively transition from a disordered structure to a repeating type II β-turn structure, forming a β-spiral above the TT. Here we present biophysical measurements of the structural, thermodynamic, and hydrodynamic properties of a specific ELP being investigated for drug delivery, ELP[V5G3A2-150]. We examine the biophysical properties below and above the TT to understand and predict the therapeutic potential of ELP-drug conjugates. We observed that below the TT, ELP[V5G3A2-150] is soluble, with an extended conformation consisting of both random coil and heterogeneous β structures. Sedimentation velocity experiments indicate that ELP[V5G3A2-150] undergoes weak self-association with increasing temperature, and above the TT the hydrophobic effect drives aggregation entropically. These experiments also reveal a previously unreported temperature-dependent critical concentration (Cc) that resembles a solubility constant. Labeling ELP[V5G3A2-150] with fluorescein lowers the TT by 3.5°C at 20 μM, whereas ELP[V5G3A2-150] dissolution in physiological media (fetal bovine serum) increases the TT by ∼2.2°C., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)