Nanoscopic Interfacial Hydrogel Viscoelasticity Revealed from Comparison of Macroscopic and Microscopic Rheology.

Deviations between macrorheological and particle-based microrheological measurements are often considered to be a nuisance and neglected. We study aqueous poly(ethylene oxide) (PEO) hydrogels for varying PEO concentrations and chain lengths that contain microscopic tracer particles and show that these deviations reveal the nanoscopic viscoelastic properties of the particle-hydrogel interface. Based on the transient Stokes equation, we first demonstrate that the deviations are not due to finite particle radius, compressibility, or surface-slip effects. Small-angle neutron scattering rules out hydrogel heterogeneities. Instead, we show that a generalized Stokes-Einstein relation, accounting for an interfacial shell around tracers with viscoelastic properties that deviate from bulk, consistently explains our macrorheological and microrheological measurements. The extracted shell diameter is comparable to the PEO end-to-end distance, indicating the importance of dangling chain ends. Our methodology reveals the nanoscopic interfacial rheology of hydrogels and is applicable to different kinds of viscoelastic fluids and particles.

Colloidal Chemistry in Water Treatment: The Effect of Ca2+ on the Interaction between Humic Acid and Poly(diallyldimethylammonium chloride) (PDADMAC).

The complexation of humic acid (HA), as a major component of natural organic matter (NOM) in raw water, with polycations is a key step in the water treatment process. At sufficiently high addition of a polycation, it leads to neutralization of the formed complexes and precipitation. In this work, we studied the effect of the presence of Ca2+ ions on this process, with poly(diallyldimethylammonium chloride) (PDADMAC) as a polycation. This was done by determining the phase behavior and characterizing the structures in solution by light scattering and small-angle neutron scattering (SANS). We observe that with increasing Ca2+ concentration, the phase boundaries of the precipitation region shift to a lower PDADMAC concentration, which coincides well with a shift of the ζ-potential of the aggregates in solution. Light scattering shows the formation of aggregates of a 120-150 nm radius, and SANS shows that Ca2+ addition promotes a compaction in the size range of 10-50 nm within these aggregates. This agrees well with the observation of more densely packed precipitates by confocal microscopy in the presence of Ca2+. Following the precipitation kinetics by turbidimetry shows a marked speeding up of the process already in the presence of rather small Ca2+ concentrations of 1 mg/L. It can be stated that the presence of Ca2+ during the complexation process of HA with a polycation has a marked effect on phase behavior and precipitation kinetics of the formed aggregates. In general, the presence of Ca2+ facilitates the process largely already at rather low concentrations, and this appears to be linked to a compaction of the formed structures in the mesoscopic size range of about 10-50 nm. These findings should be of significant importance for tailoring the flocculation process in water treatment, which is a highly important process in delivering drinking water of sufficient quality to humans.

How Chromophore Labels Shape the Structure and Dynamics of a Peptide Hydrogel.

Biocompatible and functionalizable hydrogels have a wide range of (potential) medicinal applications. The hydrogelation process, particularly for systems with very low polymer weight percentages (<1 wt %), remains poorly understood, making it challenging to predict the self-assembly of a given molecular building block into a hydrogel. This severely hinders the rational design of self-assembled hydrogels. In this study, we demonstrate the impact of an N-terminal group on the self-assembly and rheology of the peptide hydrogel hFF03 (hydrogelating, fibril forming peptide 03) using molecular dynamics simulations, oscillatory shear rheology, and circular dichroism spectroscopy. We find that the chromophore and even its specific regioisomers have a significant influence on the microscopic structure and dynamics of the self-assembled fibril, and on the macroscopic mechanical properties. This is because the chromophore influences the possible salt bridges, which form and stabilize the fibril formation. Furthermore, we find that the solvation shell fibrils by itself cannot explain the viscoelasticity of hFF03 hydrogels. Our atomistic model of the hFF03 fibril formation enables a more rational design of these hydrogels. In particular, altering the N-terminal chromophore emerges as a design strategy to tune the mechanic properties of these self-assembled peptide hydrogels.

Comprehensive Characterization of the Viscoelastic Properties of Bovine Submaxillary Mucin (BSM) Hydrogels and the Effect of Additives.

This study presents a comprehensive characterization of the viscoelastic and structural properties of bovine submaxillary mucin (BSM), which is widely used as a commercial source to conduct mucus-related research. We conducted concentration studies of BSM and examined the effects of various additives, NaCl, CaCl2, MgCl2, lysozyme, and DNA, on its rheological behavior. A notable connection between BSM concentration and viscoelastic properties was observed, particularly under varying ionic conditions. The rheological spectra could be well described by a fractional Kelvin-Voigt model with a minimum of model parameters. A detailed proteomics analysis provided insight into the protein, especially mucin composition within BSM, showing MUC19 as the main component. Cryo-scanning electron microscopy enabled the visualization of the porous BSM network structure. These investigations give us a more profound comprehension of the BSM properties, especially those pertaining to viscoelasticity, and how they are influenced by concentration and environmental conditions, aspects relevant to the field of mucus research.

Impact of glycan nature on structure and viscoelastic properties of glycopeptide hydrogels.

Mucus is a complex biological hydrogel that acts as a barrier for almost everything entering or exiting the body. It is therefore of emerging interest for biomedical and pharmaceutical applications. Besides water, the most abundant components are the large and densely glycosylated mucins, glycoproteins of up to 20 MDa and carbohydrate content of up to 80 wt%. Here, we designed and explored a library of glycosylated peptides to deconstruct the complexity of mucus. Using the well-characterized hFF03 coiled-coil system as a hydrogel-forming peptide scaffold, we systematically probed the contribution of single glycans to the secondary structure as well as the formation and viscoelastic properties of the resulting hydrogels. We show that glycan-decoration does not affect α-helix and coiled-coil formation while it alters gel stiffness. By using oscillatory macrorheology, dynamic light scattering microrheology, and fluorescence lifetime-based nanorheology, we characterized the glycopeptide materials over several length scales. Molecular simulations revealed that the glycosylated linker may extend into the solvent, but more frequently interacts with the peptide, thereby likely modifying the stability of the self-assembled fibers. This systematic study highlights the interplay between glycan structure and hydrogel properties and may guide the development of synthetic mucus mimetics.

Longitudinal effects of elexacaftor/tezacaftor/ivacaftor on sputum viscoelastic properties, airway infection and inflammation in patients with cystic fibrosis.

BACKGROUND: Recent studies demonstrated that the triple combination cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) improves lung function and reduces pulmonary exacerbations in cystic fibrosis (CF) patients with at least one F508del allele. However, effects of ETI on downstream consequences of CFTR dysfunction, i.e. abnormal viscoelastic properties of airway mucus, chronic airway infection and inflammation have not been studied. The aim of this study was to determine the longitudinal effects of ETI on airway mucus rheology, microbiome and inflammation in CF patients with one or two F508del alleles aged ≥12 years throughout the first 12 months of therapy. METHODS: In this prospective observational study, we assessed sputum rheology, the microbiome, inflammation markers and proteome before and 1, 3 and 12 months after initiation of ETI. RESULTS: In total, 79 patients with CF and at least one F508del allele and 10 healthy controls were enrolled in this study. ETI improved the elastic modulus and viscous modulus of CF sputum at 3 and 12 months after initiation (all p<0.01). Furthermore, ETI decreased the relative abundance of Pseudomonas aeruginosa in CF sputum at 3 months and increased the microbiome α-diversity at all time points. In addition, ETI reduced interleukin-8 at 3 months (p<0.05) and free neutrophil elastase activity at all time points (all p<0.001), and shifted the CF sputum proteome towards healthy. CONCLUSIONS: Our data demonstrate that restoration of CFTR function by ETI improves sputum viscoelastic properties, chronic airway infection and inflammation in CF patients with at least one F508del allele over the first 12 months of therapy; however, levels close to healthy were not reached.

Microscopic Analysis of the Water/Glycerol/EO30PS System in Bulk and on a Solid Substrate.

Surfactant systems are often employed in cosmetic formulations where they dry on skin as a surface, thereby becoming increasingly concentrated systems. To better understand this drying process, we focused on the difference of self-assembled structures of the water/glycerol/polyoxyethylene (30) phytosteryl ether (EO30PS) system in bulk and on a solid substrate because the interaction between the substrate and the surfactant may have a substantial effect on the self-assembly, which may be related to the bulk structure but in detail may also differ strongly from the bulk situation. In bulk, small-angle neutron scattering (SANS) experiments showed that with increasing loss of water, the degree of ordering increases but changes of the aggregate structure are rather small. The results indicate that ellipsoidal micelles of EO30PS are densely packed and simply become more ordered in bulk during the drying process. On the other hand, neutron reflectometry revealed that EO30PS molecules adsorb onto a Si surface in the form of bilayers and analysis indicates that at a high concentration (c = 20 wt %), there are on average two bilayers (a double bilayer) on the Si substrate. The adsorbed membrane structure of EO30PS is rather thin with respect to its hydrophobic part, indicating tilted molecules, containing only some solvent, and being not highly ordered. These experimental results then allow for a much deeper understanding of the structural properties of practical formulations as they are applied, for instance, in cosmetic lotions.

Modifying the Properties of Microemulsion Droplets by Addition of Thermoresponsive BAB* Copolymers.

Oil-in-water (O/W) microemulsions (ME) typically feature a low viscosity and exhibit ordinary viscosity reduction as a function of temperature. However, for certain applications, avoiding or even reverting the temperature trend might be required. This can be conceived by adding thermoresponsive (TR) block copolymers that induce network formation as the temperature increases. Accordingly, various ME-polymer mixtures were studied for which three different block copolymer architectures of BAB*-, B2AB*-, and B(AB*)2-types were employed. Here, "B" represents a permanently hydrophobic, "A" a permanently hydrophilic, and "B*" a TR block. For the TR-block, three different poly(acrylamide)s, namely poly(N-n-propylacrylamide) (pNPAm), poly(N,N-diethylacrylamide) (pDEAm), and poly(N-isopropylacrylamide) (pNiPAm), were used, which all exhibit a lower critical solution temperature. For a well-selected ME concentration, these block copolymers lead to a viscosity enhancement with increasing temperature. At a polymer concentration of about 22 g L-1, the most pronounced enhancement was observed for the pNPAm-based systems with factors up to 3, 5, and 8 for BAB*, B2AB*, and B(AB*)2, respectively. This phenomenon is caused by the formation of a transitory network mediated by TR-blocks, as evidenced by the direct correlation between the attraction strength and the viscosity enhancement. For applications requiring a high hydrophobic payload, which is attained via ME droplets, this kind of tailored temperature-dependent viscosity control of surfactant systems should therefore be advantageous.

Using Microemulsions: Formulation Based on Knowledge of Their Mesostructure.

Microemulsions, as thermodynamically stable mixtures of oil, water, and surfactant, are known and have been studied for more than 70 years. However, even today there are still quite a number of unclear aspects, and more recent research work has modified and extended our picture. This review gives a short overview of how the understanding of microemulsions has developed, the current view on their properties and structural features, and in particular, how they are related to applications. We also discuss more recent developments regarding nonclassical microemulsions such as surfactant-free (ultraflexible) microemulsions or ones containing uncommon solvents or amphiphiles (like antagonistic salts). These new findings challenge to some extent our previous understanding of microemulsions, which therefore has to be extended to look at the different types of microemulsions in a unified way. In particular, the flexibility of the amphiphilic film is the key property to classify different microemulsion types and their properties in this review. Such a classification of microemulsions requires a thorough determination of their structural properties, and therefore, the experimental methods to determine microemulsion structure and dynamics are reviewed briefly, with a particular emphasis on recent developments in the field of direct imaging by means of electron microscopy. Based on this classification of microemulsions, we then discuss their applications, where the application demands have to be met by the properties of the microemulsion, which in turn are controlled by the flexibility of their amphiphilic interface. Another frequently important aspect for applications is the control of the rheological properties. Normally, microemulsions are low viscous and therefore enhancing viscosity has to be achieved by either having high concentrations (often not wished for) or additives, which do not significantly interfere with the microemulsion. Accordingly, this review gives a comprehensive account of the properties of microemulsions, including most recent developments and bringing them together from a united viewpoint, with an emphasis on how this affects the way of formulating microemulsions for a given application with desired properties.

Structure of microemulsions in the continuous phase channel.

We have studied the microemulsion and lamellar phases of two of the most commonly described systems based on nonionic C12E5 and ionic AOT surfactants. We show that C12E5 is best described by the symmetric disordered open connected lamellar model (DOC-lamellar), contrary to the more commonly employed standard flexible model. In the case of AOT, the bicontinuous microemulsion structure is best described by the standard flexible model at high temperatures. Around room temperature, connected cylinders in a molten cubic crystal phase are the only description which corresponds to the data. In the lamellar phase, around one third of the available surface area is lost in fluctuations and defects. Comparing structurally predictive models with results from conductivity measurements show that salt adsorption in the hydrated ethoxy groups is dominant for C12E5 (nonionic). For AOT, our conductivity measurements clarify the role of tortuosity versus cation absorption.

High unimer concentration and asymmetric bilayer observed in small unilamellar vesicles of mixed surfactants TDMAO/LiPFO.

The mixed surfactant system of tetradecyldimethylamine oxide (TDMAO) and lithium perfluorooctanoate (LiPFO) is known to spontaneously self-assemble into well-defined small unilamellar vesicles. For a quantitative analysis of small-angle x-ray scattering on this model system, we complemented the measurements with densitometry, conductimetry, and contrast-variation small-angle neutron scattering. The analysis points to two main findings: first, the vesicles formed to contain a much higher mole fraction (0.61-0.64) of TDMAO than the bulk sample (0.43) and predicted by Regular Solution Theory (RST, 0.46). In consequence, the unimer concentration of LiPFO is more than 5 times higher than predicted by RST. Second, the vesicle bilayer is asymmetric with a higher fraction of LiPFO on the outside. These findings on a model system should be of broader relevance for the understanding of similar mixed surfactant vesicle systems and thereby also be of importance for their use in a number of applications.

Polyelectrolyte-Surfactant Complexes As a Formulation Tool for Drug Delivery.

Aqueous polyelectrolyte-surfactant complexes (PESCs) are very rich with respect to their properties and the structures formed by them. By design they normally contain hydrophobic micellar surfactant aggregates complexed by long polyelectrolyte chains, thereby combining the formation of small hydrophobic domains given by the surfactant with large-scale structuring due to the presence of the polyelectrolyte chain. In addition, they contain highly polar regions of surfactant head groups in contact with polyelectrolyte, forming a shell around the micellar aggregates, which often also possesses a certain hydrophobic character. Accordingly, the ability for solubilization of water-insoluble compounds of different sorts is particularly versatile in PESCs. Their solubilization sites with very different polarities and hydrophobic characters make them very flexible in adapting to the requirements of a given drug molecule. This renders them attractive for potential applications in drug delivery. In addition, modification of the rheological properties via self-assembly and network formation can be very important in PESC applications. In the following, we discuss the structures of PESCs and their properties, with a focus on the solubilization properties. Subsequently, examples are described where PESCs have been employed in the context of drug solubilization and delivery. These comprise examples with individual aggregates, cross-linked hydrogels, and ones taking advantage of the high solubilization capacity of microemulsions.

Thermoresponsive Self-Assembly of Twofold Fluorescently Labeled Block Copolymers in Aqueous Solution and Microemulsions.

A nonionic double hydrophilic block copolymer with a long permanently hydrophilic and a small thermoresponsive block is synthesized by reversible addition-fragmentation chain-transfer polymerization (RAFT). By employing a specifically designed chain-transfer agent, the polymer is functionalized with complementary end groups which are suited for Förster resonance energy transfer (FRET). The end group attached to the permanently hydrophilic block of poly(N,N-dimethylacrylamide) pDMAm is designed as a permanently hydrophobic segment ("sticker") comprising a long alkyl chain and the 4-aminonaphthalimide fluorophore. The other end attached to the thermoresponsive block of poly(N-isopropylacrylamide) pNiPAm incorporates a coumarin fluorophore. The temperature-dependent self-assembly of the twofold fluorescently labeled copolymer is studied in pure aqueous solution as well as in an o/w microemulsion by several techniques including turbidimetry, dynamic light scattering (DLS), and fluorescence spectroscopy. It is compared to the behaviors of the analogous twofold-labeled pDMAm and pNiPAm homopolymer references. The findings indicate that the block copolymer behaves as a polymeric surfactant at low temperatures, with one relatively small hydrophobic end block and an extended hydrophilic chain forming "hairy micelles". At elevated temperatures above the LCST phase transition of the pNiPAm block, however, the copolymer behaves as an associative telechelic polymer with two nonsymmetrical hydrophobic end blocks, which do not mix. Thus, instead of a network of bridged "flower micelles", large dynamic aggregates are formed. These are connected alternatingly by the original micellar cores as well as by clusters of the collapsed pNiPAm blocks. This type of structure is even more favored in the o/w microemulsion than in pure aqueous solution, as the microemulsion droplets constitute an attractive anchoring point for the hydrophobic dodecyl sticker but not for the collapsed pNiPAm chains.

Self-Assembled Single-Stranded DNA Nano-Networks in Solution and at Surfaces.

We studied the directed self-assembly of two types of complementary single-stranded DNA (ssDNA) strands [i.e., poly(dA) and poly(dT)] into more complex, organized, and percolating networks in dilute solutions and at surfaces. Understanding ssDNA self-assembly into 2D networks on surfaces is important for the use of such networks in the fabrication of well-defined nanotechnological devices, as, for instance, required in nanoelectronics or for biosensing. To control the formation of 2D networks on surfaces, it is important to know whether DNA assemblies are formed already in dilute solutions or only during the drying/immobilization process at the surface, where the concentration automatically increases. Fluorescence cross-correlation spectroscopy clearly shows the presence of larger DNA complexes in mixed poly(dA) and poly(dT) solutions already at very low DNA concentrations (<1 nM), that is, well below the overlap concentration. Here, we describe for the first time such supramolecular complexes in solution and how their structure depends on the ssDNA length and concentration and ionic strength. Hence, future attempts to control such networks should also focus on network precursors in solution and not only on their immobilization on surfaces.

Particle Diffusivity and Free-Energy Profiles in Hydrogels from Time-Resolved Penetration Data.

A combined experimental and theoretical method to simultaneously determine diffusivity and free-energy profiles of particles that penetrate into inhomogeneous hydrogel systems is presented. As the only input, arbitrarily normalized concentration profiles from fluorescence intensity data of labeled tracer particles for different penetration times are needed. The method is applied to dextran molecules of varying size that penetrate into hydrogels of polyethylene-glycol chains with different lengths that are covalently cross-linked by hyperbranched polyglycerol hubs. Extracted dextran bulk diffusivities agree well with fluorescence correlation spectroscopy data obtained separately. Empirical scaling laws for dextran diffusivities and free energies inside the hydrogel are identified as a function of the dextran mass. An elastic free-volume model that includes dextran as well as polyethylene-glycol linker flexibility quantitively describes the repulsive dextran-hydrogel interaction free energy, which is of steric origin, and furthermore suggests that the hydrogel mesh-size distribution is rather broad and particle penetration is dominated by large hydrogel pores. Particle penetration into hydrogels for steric particle-hydrogel interactions is thus suggested to be governed by an elastic size-filtering mechanism that involves the tail of the hydrogel pore-size distribution.

Surfactants - Compounds for inactivation of SARS-CoV-2 and other enveloped viruses.

We provide here a general view on the interactions of surfactants with viruses, with a particular emphasis on how such interactions can be controlled and employed for inhibiting the infectivity of enveloped viruses, including coronaviruses. The aim is to provide to interested scientists from different fields, including chemistry, physics, biochemistry, and medicine, an overview of the basic properties of surfactants and (corona)viruses, which are relevant to understanding the interactions between the two. Various types of interactions between surfactant and virus are important, and they act on different components of a virus such as the lipid envelope, membrane (envelope) proteins and nucleocapsid proteins. Accordingly, this cannot be a detailed account of all relevant aspects but instead a summary that bridges between the different disciplines. We describe concepts and cover a selection of the relevant literature as an incentive for diving deeper into the relevant material. Our focus is on more recent developments around the COVID-19 pandemic caused by SARS-CoV-2, applications of surfactants against the virus, and on the potential future use of surfactants for pandemic relief. We also cover the most important aspects of the historical development of using surfactants in combatting virus infections. We conclude that surfactants are already playing very important roles in various directions of defence against viruses, either directly, as in disinfection, or as carrier components of drug delivery systems for prophylaxis or treatment. By designing tailor-made surfactants, and consequently, advanced formulations, one can expect more and more effective use of surfactants, either directly as antiviral compounds or as part of more complex formulations.

Directed Assembly of Multi-Walled Nanotubes and Nanoribbons of Amino Acid Amphiphiles Using a Layer-by-Layer Approach.

Monodisperse unilamellar nanotubes (NTs) and nanoribbons (NRs) were transformed to multilamellar NRs and NTs in a well-defined fashion. This was done by using a step-wise approach in which self-assembled cationic amino acid amphiphile (AAA) formed the initial NTs or NRs, and added polyanion produced an intermediate coating. Successive addition of cationic AAA formed a covering AAA layer, and by repeating this layer-by-layer (LBL) procedure, multi-walled nanotubes (mwNTs) and nanoribbons were formed. This process was structurally investigated by combining small-angle neutron scattering (SANS) and cryogenic-transmission electron microscopy (cryo-TEM), confirming the multilamellar structure and the precise layer spacing. In this way the controlled formation of multi-walled suprastructures was demonstrated in a simple and reproducible fashion, which allowed to control the charge on the surface of these 1D aggregates. This pathway to 1D colloidal materials is interesting for applications in life science and creating well-defined building blocks in nanotechnology.

Structural Characterization of Nonionic Surfactant Micelles with CO2/Ethylene Oxide Head Groups and Their Temperature Dependence.

Using CO2 as a resource in the production of materials is a viable alternative to conventional, petroleum-based raw materials and therefore offers great potential for more sustainable chemistry. This study presents a detailed structural characterization of aggregates of nonionic dodecyl surfactants with different amounts of CO2 substituting ethylene oxide (EO) in the head group. The micellar structure was characterized as a function of concentration and temperature by dynamic and static light scattering and, in further detail, by small-angle neutron scattering (SANS). The influence of the CO2 unit in the hydrophilic EO group is systematically compared to the incorporation of propylene oxide (PO) and propiolactone (PL). The surfactants with carbonate groups in their head groups form ellipsoidal micelles in an aqueous solution similar to conventional nonionic surfactants, becoming bigger with increasing CO2 content. In contrast, the incorporation of PO units hardly alters the behavior, while the incorporation of a PL unit has an effect comparable to the CO2 unit. The analysis of the SANS data shows decreasing hydration with increasing CO2 and PL content. By increasing the temperature, a typical sphere-rod transition is observed, where CO2 surfactants show a much higher elongation with increasing temperature, which is correlated with the reduced cloud point and a lower extent of head group hydration. Our findings demonstrate that CO2-containing surface-active compounds are an interesting, potentially "greener" alternative to conventional nonionic surfactants.

Adsorption Kinetics of Oppositely Charged Hard and Soft Nanoparticles with Phospholipid Membranes.

Nanoparticles (NPs) have great potential for biological applications as typically they exhibit strongly size-dependent properties. Specifically, the interaction of NPs with phospholipid membranes is significantly relevant to nanomedicine and the related field of nanotoxicology. Therefore, the investigation of interactions of NPs with model membranes is not only fundamentally important but also practically valuable to understand interactions of NPs with more complex cell membranes. Here, we report on the interaction of anionic vesicles of different charge densities and cationic SiO2 NPs, either covered by a bare surface functionalized with amino moieties (-NH2) or covered by poly[2-(dimethylamino) ethyl methacrylate]. We studied the kinetics of binding of NPs to the vesicle surface by time-resolved scattering experiments. A key result of the study is that binding is favored in the presence of electrostatic attraction, but the polymer layer decreases the binding rate drastically.

Microphase Separation of Resilin-like and Elastin-like Diblock Copolypeptides in Concentrated Solutions.

Diblock copolymers are valued for their ability to form thin films with nanoscale features that typically reflect those of their microphase-separated structures in concentrated solution. Here, we show that such self-assembled structures can be easily formed with diblock copolymers composed of thermally responsive polypeptides, such as resilin-like polypeptides (RLP) and elastin-like polypeptides (ELP), by exploiting the inverse temperature transition behavior of ELPs in aqueous media. Specifically, we examine the self-assembly of a series of RLP-b-ELP diblock copolypeptides in concentrated aqueous solution (30 and 50 wt %) by small-angle X-ray scattering (SAXS). By systematically varying RLP block length and temperature (10-45 °C), we observed microphase separation into hexagonally packed cylinders and lamellae. By analyzing the observed order-order transitions (OOT) and order-disorder transitions (ODT), we determined that self-assembly in this system is primarily driven by polymer-solvent interactions. While these thermally responsive polymers showed clear ODTs and OOTs at certain temperatures, temperature only had a weak effect on the spacing of the resulting nanostructures. In contrast, we found that nanostructure spacing was far more sensitive to RLP block length. Finally, we used atomic force microscopy (AFM) to demonstrate that spin casting RLP-b-ELP diblock copolypeptides also produce nanostructured thin films with spacings that correlate with those in concentrated solution.