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Liquid-liquid phase separation (LLPS) is a ubiquitous demixing phenomenon observed in various molecular solutions, including in polymer and protein solutions. Demixing of solutions results in condensed, phase separated droplets which exhibit a range of liquid-like properties driven by transient intermolecular interactions. Understanding the organization within these condensates is crucial for deciphering their material properties and functions. This study explores the distinct nanoscale networks and interfaces in the condensate samples using a modified cryo-electron microscopy (cryo-EM) method. The method involves initiating condensate formation on electron microscopy grids to limit droplet growth as large droplet sizes are not ideal for cryo-EM imaging. The versatility of this method is demonstrated by imaging three different classes of condensates. We further investigate the condensate structures using cryo-electron tomography which provides 3D reconstructions, uncovering porous internal structures, unique core-shell morphologies, and inhomogeneities within the nanoscale organization of protein condensates. Comparison with dry-state transmission electron microscopy emphasizes the importance of preserving the hydrated structure of condensates for accurate structural analysis. We correlate the internal structure of protein condensates with their amino acid sequences and material properties by performing viscosity measurements that support that more viscous condensates exhibit denser internal assemblies. Our findings contribute to a comprehensive understanding of nanoscale condensate structure and its material properties. Our approach here provides a versatile tool for exploring various phase-separated systems and their nanoscale structures for future studies.

Highly scattering samples, such as polymer droplets or solid-state powders, are difficult to study via coherent two-dimensional infrared (2D IR) spectroscopy. Previously, researchers have employed (quasi-) phase cycling, local-oscillator chopping, and polarization control to reduce scattering, but the latter method poses a limit on polarization-dependent measurements. Here, we present a method for Scattering Elimination Immune from Detector Artifacts (SEIFDA) in pump-probe 2D IR experiments. Our method extends the negative probe delay method of removing scattering from pump-probe spectroscopy to 2D experiments. SEIFDA works well for all polarizations when combined with the optimized noise reduction scheme to remove additive and multiplicative noise. We demonstrate that our method can be employed with any polarization scheme and reliably lowers the scattering at parallel polarization to comparable levels to the conventional 8-frame phase cycling with probe chopping (8FPCPC) at perpendicular polarization. Our system can acquire artifact free spectra in parallel polarization when the signal intensity is as little as 5% of the intensity of the interference between the pump pulses scattered into the detector. It reduces the time required to characterize the scattering term by at least 50% over 8FPCPC. Through detailed analysis of detector nonlinearity, we show that the performance of 8FPCPC can be improved by incorporating nonlinear correction factors, but it is still worse than that of SEIFDA. Application of SEIFDA to study the encapsulation of Nile red in polymer droplets demonstrates that this method will be very useful for probing highly scattering systems.

Polymerization-induced self-assembly (PISA) has emerged as a scalable one-pot technique to prepare block copolymer (BCP) nanoparticles. Recently, a PISA process, that results in poly(l-lactide)-b-poly(ethylene glycol) BCP nanoparticles coined ring-opening polymerization (ROP)-induced crystallization-driven self-assembly (ROPI-CDSA), was developed. The resulting nanorods demonstrate a strong propensity for aggregation, resulting in the formation of 2D sheets and 3D networks. This article reports the synthesis of poly(N,N-dimethyl acrylamide)-b-poly(l)-lactide BCP nanoparticles by ROPI-CDSA, utilizing a two-step, one-pot approach. A dual-functionalized photoiniferter is first used for controlled radical polymerization of the acrylamido-based monomer, and the resulting polymer serves as a macroinitiator for organocatalyzed ROP to form the solvophobic polyester block. The resulting nanorods are highly stable and display anisotropy at higher molecular weights (>12k Da) and concentrations (>20% solids) than the previous report. This development expands the chemical scope of ROPI-CDSA BCPs and provides readily accessible nanorods made with biocompatible materials., (© 2024 Wiley‐VCH GmbH.)

Electrochemical liquid electron microscopy has revolutionized our understanding of nanomaterial dynamics by allowing for direct observation of their electrochemical production. This technique, primarily applied to inorganic materials, is now being used to explore the self-assembly dynamics of active molecular materials. Our study examines these dynamics across various scales, from the nanoscale behavior of individual fibers to the micrometer-scale hierarchical evolution of fiber clusters. To isolate the influences of the electron beam and electrical potential on material behavior, we conducted thorough beam-sample interaction analyses. Our findings reveal that the dynamics of these active materials at the nanoscale are shaped by their proximity to the electrode and the applied electrical current. By integrating electron microscopy observations with reaction-diffusion simulations, we uncover that local structures and their formation history play a crucial role in determining assembly rates. This suggests that the emergence of nonequilibrium structures can locally accelerate further structural development, offering insights into the behavior of active materials under electrochemical conditions.

In-situ liquid cell transmission electron microscopy (LCTEM) with electrical biasing capabilities has emerged as an invaluable tool for directly imaging electrode processes with high temporal and spatial resolution. However, accurately quantifying structural changes that occur on the electrode and subsequently correlating them to the applied stimulus remains challenging. Here, we present structural dissimilarity (DSSIM) analysis as segmentation-free video processing algorithm for locally detecting and quantifying structural change occurring in LCTEM videos. In this study, DSSIM analysis is applied to two in-situ LCTEM videos to demonstrate how to implement this algorithm and interpret the results. We show DSSIM analysis can be used as a visualization tool for qualitative data analysis by highlighting structural changes which are easily missed when viewing the raw data. Furthermore, we demonstrate how DSSIM analysis can serve as a quantitative metric and efficiently convert 3-dimensional microscopy videos to 1-dimenional plots which makes it easy to interpret and compare events occurring at different timepoints in a video. In the analyses presented here, DSSIM is used to directly correlate the magnitude and temporal scale of structural change to the features of the applied electrical bias. ImageJ, Python, and MATLAB programs, including a user-friendly interface and accompanying documentation, are published alongside this manuscript to make DSSIM analysis easily accessible to the scientific community., Competing Interests: Declaration of Competing Interest Y. Pivak and H. Sun work for DENSsolutions B.V., a company manufacturing and marketing in-situ TEM systems. The remaining authors have no conflicts to declare., (Copyright © 2023. Published by Elsevier B.V.)

Confinement allows macromolecules and biomacromolecules to attain arrangements typically unachievable through conventional self-assembly processes. In the field of block copolymers, confinement has been achieved by preparing thin films and controlled solvent evaporation through the use of emulsions. A significant advantage of the confinement-driven self-assembly process is its ability to enable block copolymers to form particles with complex internal morphologies, which would otherwise be inaccessible. Here, we show that liquid-liquid phase separation (LLPS) can induce confinement during the self-assembly of a model block copolymer system. Since this confinement is driven by the block copolymers' tendency to undergo LLPS, we define this confinement type as auto-confinement. This study adds to the growing understanding of how LLPS influences block copolymer self-assembly and provides a new method to achieve confinement driven self-assembly.

Liquid cell transmission electron microscopy is a powerful tool for visualizing nanoparticle (NP) assemblies in liquid environments with nanometer resolution. However, it remains a challenge to control the NP concentration in the high aspect ratio liquid enclosure where the diffusion of dispersed NPs is affected by the exposed surface of the liquid cell walls. Here, we introduce a semi-empirical model based on the 1D diffusion equation, to predict the NP loading time as they pass through the nanochannel into the imaging volume of the liquid cell. We show that loading of NPs into the imaging volume of the liquid cell may take several days if NPs are prone to attach to the surface of the mm-long nanochannel when using an industry-standard flat microchip. As a means to facilitate mass transport via diffusion, we tested a liquid cell incorporating a microchannel geometry resulting in a NP loading time in the order minutes that allowed us to observe the formation of a randomly oriented self-assembled monolayer in situ using scanning transmission electron microscopy., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Niels de Jonge has patent #US20120120226A1 issued to Niels de Jonge., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Inspired by the adaptability of biological materials, a variety of synthetic, chemically driven self-assembly processes have been developed that result in the transient formation of supramolecular structures. These structures form through two simultaneous reactions, forward and backward, which generate and consume a molecule that undergoes self-assembly. The dynamics of these assembly processes have been shown to differ from conventional thermodynamically stable molecular assemblies. However, the evolution of nanoscale morphologies in chemically driven self-assembly and how they compare to conventional assemblies has not been resolved. Here, we use a chemically driven redox system to separately carry out the forward and backward reactions. We analyze the forward and backward reactions both sequentially and synchronously with time-resolved cryogenic transmission electron microscopy (cryoEM). Quantitative image analysis shows that the synchronous process is more complex and heterogeneous than the sequential process. Our key finding is that a thermodynamically unstable stacked nanorod phase, briefly observed in the backward reaction, is sustained for ∼6 hours in the synchronous process. Kinetic Monte Carlo modeling show that the synchronous process is driven by multiple cycles of assembly and disassembly. The collective data suggest that chemically driven self-assembly can create sustained morphologies not seen in thermodynamically stable assemblies by kinetically stabilizing transient intermediates. This finding provides plausible design principles to develop and optimize supramolecular materials with novel properties., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Poly(ethylene glycol) (PEG) ligands can inhibit proteins and other biomolecules from adhering to underlying surfaces, making them excellent surface ligands for nanocrystal (NC)-based drug carriers. Quantifying the PEG ligand shell morphology is important because its structure determines the permeability of biomolecules through the shell to the NC surface. However, few in situ analytical tools can reveal whether the PEG ligands form either an impenetrable barrier or a porous coating surrounding the NC. Here, we present a Förster resonance energy transfer (FRET) spectroscopy-based approach that can assess the permeability of molecules through PEG-coated ZnO NCs. In this approach, ZnO NCs serve as FRET donors, and freely diffusing molecules in the bulk solution are FRET acceptors. We synthesized a series of variable chain length PEG-silane-coated ZnO NCs such that the longest chain length ligands far exceed the Förster radius ( R 0 ), where the energy transfer (EnT) efficiency is 50%. We quantified the EnT efficiency as a function of the ligand chain length using time-resolved photoluminescence lifetime (TRPL) spectroscopy within the framework of FRET theory. Unexpectedly, the longest PEG-silane ligand showed equivalent EnT efficiency as that of bare, hydroxyl-passivated ZnO NCs. These results indicate that the "rigid shell" model fails and the PEG ligand shell morphology is more likely porous or in a patchy "mushroom state", consistent with transmission electron microscopy data. While the spectroscopic measurements and data analysis procedures discussed herein cannot directly visualize the ligand shell morphology in real space, the in situ spectroscopy approach can provide researchers with valuable information regarding the permeability of species through the ligand shell under practical biological conditions.

Metal-organic frameworks offer a diverse landscape of building blocks to design high performance materials for implications in almost every major industry. With this diversity stems complex crystallization mechanisms with various pathways and intermediates. Crystallization studies have been key to the advancement of countless biological and synthetic systems, with MOFs being no exception. This review provides an overview of the current theories and fundamental chemistry used to decipher MOF crystallization. We then discuss how intrinsic and extrinsic synthetic parameters can be used as tools to modulate the crystallization pathway to produce MOF crystals with finely tuned physical and chemical properties. Experimental and computational methods are provided to guide the probing of MOF crystal formation on the molecular and bulk scale. Lastly, we summarize the recent major advances in the field and our outlook on the exciting future of MOF crystallization.

Defect-mediated energy transfer is an energy transfer process between midgap electronic states in a semiconductor nanocrystal (NC) and molecular acceptors, such as fluorescent dye molecules. Super-resolution fluorescence microscopy represents an exciting technique for pinpointing the nanoscale positions of lattice defect sites in, for example, a micrometer-sized particle or thin film sample by spatially resolving the location of the acceptor dye molecules with nanometer resolution. Toward this goal, our group performed ensemble-level, time-resolved fluorescence spectroscopy measurements of ZnO NC/Alexafluor 555 (A555) mixtures and calculated that the emissive defect sites are located, on average, 0.5 nm from the NC surface [Nilsson Z. N.; J. Chem. Phys.2021, 154 ( (5), ), 054704]. However, ensemble-level measurements cannot spatially resolve the defect sites on single particles, nor can they distinguish between surface-adsorbed dye molecules that participate in the energy transfer (EnT) process from those that do not. In this work, we compared the photoluminescence intensity trajectories of 789 isolated, single ZnO NC donors to those of 73 non-specifically bound and five specifically bound ZnO NC/A555 pairs, where the donor and acceptor centroid positions were separated by a distance that was smaller than our localization precision (40 nm). We observed minor fluorescence intensity fluctuations in the donor and defect channels instead of clear anticorrelated intensity fluctuations, which could be explained by (1) the presence of multiple emissive defect sites per NC, (2) donor-acceptor separation distances slightly larger than the Förster radius ( R 0 = 3.1 nm; defined as the distance at which EnT is 50% efficient), and/or (3) poor dipole-dipole coupling. The single molecule imaging methodology we developed, an alternating ultraviolet-visible excitation sequence combined with multicolor photon detection, successfully distinguishes specifically bound and non-specifically bound NC/dye pairs and can be applied to study a wide range of hybrid NC/dye energy transfer systems., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Co-published by Nanjing University and American Chemical Society.)

Bioreducible polymeric mRNA carriers are an emerging family of vectors for gene delivery and vaccine development. A few bioreducible systems have been generated through aqueous-phase ring-opening polymerization of lipoic acid derivatives, however this methodology limits hydrophobic group incorporation and functionality into resulting polymers. Herein, a poly(active ester)disulfide polymer is synthesized that can undergo facile aminolysis with amine-containing substrates under stoichiometric control and mild reaction conditions to yield a library of multifunctional polydisulfide polymers. Functionalized polydisulfide polymer species form stable mRNA-polymer nanoparticles for intracellular delivery of mRNAs in vitro. Alkyl-functionalized polydisulfide-RNA nanoparticles demonstrate rapid cellular uptake and excellent biodegradability when delivering EGFP and OVA mRNAs to cells in vitro. This streamlined polydisulfide synthesis provides a new facile methodology for accessing multifunctional bioreducible polymers as biomaterials for RNA delivery and other applications., (© 2022 Wiley-VCH GmbH.)

The importance and prevalence of energy-fueled active materials in living systems have inspired the design of synthetic active materials using various fuels. However, several major limitations of current designs remain to be addressed, such as the accumulation of chemical wastes during the process, unsustainable active behavior, and the lack of precise spatiotemporal control. Here, we demonstrate a fully electrically fueled (e-fueled) active self-assembly material that can overcome the aforementioned limitations. Using an electrochemical setup with dual electrocatalysts, the anodic oxidation of one electrocatalyst (ferrocyanide, [Fe(CN) 6 ] 4- ) creates a positive fuel to activate the self-assembly, while simultaneously, the cathodic reduction of the other electrocatalyst (methyl viologen, [MV] 2+ ) generates a negative fuel triggering fiber disassembly. Due to the fully catalytic nature for the reaction networks, this fully e-fueled active material system does not generate any chemical waste, can sustain active behavior for an extended period when the electrical potential is maintained, and provides spatiotemporal control.

Polymerization-induced self-assembly (PISA) has become an important one pot method for the preparation of well-defined block copolymer nanoparticles. In PISA, morphology is typically controlled by changing molecular architecture and polymer concentration. However, several computational and experimental studies have suggested that changes in polymerization rate can lead to morphological differences. Here, we demonstrate that catalyst selection can be used to control morphology independent of polymer structure and concentration in ring-opening polymerization-induced crystallization-driven self-assembly (ROPI-CDSA). Slower rates of polymerization give rise to slower rates of self-assembly, resulting in denser lamellae and more 3D structures when compared to faster rates of polymerization. Our explanation for this is that the fast samples transiently exist in a nonequilibrium state as self-assembly starts at a higher solvophobic block length when compared to the slow polymerization. We expect that subsequent examples of rate variation in PISA will allow for greater control over morphological outcome., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Materials are needed to increase the stability and half-life of therapeutic proteins during delivery. These materials should be biocompatible and biodegradable. Here, we demonstrate that enzymes and immunoproteins can be encapsulated inside cyclodextrin based metal-organic frameworks using potassium as the metal node. The release profile can be controlled with the solubility of the cyclodextrin linker. The activity of the proteins after release is determined using catalytic and in vitro assays. The results show that cyclodextrin metal-organic framework-based protein biocomposites are a promising class of materials to deliver therapeutic proteins.

Poly(ethylene glycol) (PEG) is an important and widely used polymer in biological and pharmaceutical applications for minimizing nonspecific binding while improving blood circulation for therapeutic/imaging agents. However, commercial PEG samples are polydisperse, which hampers detailed studies on chain length-dependent properties and potentially increases antibody responses in pharmaceutical applications. Here, we report a practical and scalable method to prepare libraries of discrete PEG analogues with a branched, nonlinear structure. These lipid-PEG derivatives have a monodisperse backbone with side chains containing a discrete number of ethylene glycol units (3 or 4) and unique functionalizable chain ends. Significantly, the branched, nonlinear structure is shown to allow for efficient nanoparticle assembly while reducing anti-PEG antibody recognition when compared to commercial polydisperse linear systems, such as DMG-PEG2000. By enabling the scalable synthesis of a broad library of graft copolymers, fundamental self-assembly properties can be understood and shown to directly correlate with the total number of PEG units, nature of the chain ends, and overall backbone length. These results illustrate the advantages of discrete macromolecules when compared to traditional disperse materials.

Metal-organic frameworks (MOFs) are a class of porous nanomaterials that have been extensively studied as enzyme immobilization substrates. During in situ immobilization, MOF nucleation is driven by biomolecules with low isoelectric points. Investigation of how biomolecules control MOF self-assembly mechanisms on the molecular level is key to designing nanomaterials with desired physical and chemical properties. Here, we demonstrate how molecular modifications of bovine serum albumin (BSA) with fluorescein isothiocyanate (FITC) can affect MOF crystal size, morphology, and encapsulation efficiency. Final crystal properties are characterized using scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), fluorescent microscopy, and fluorescence spectroscopy. To probe MOF self-assembly, in situ experiments were performed using cryogenic transmission electron microscopy (cryo-TEM) and X-ray diffraction (XRD). Biophysical characterization of BSA and FITC-BSA was performed using ζ potential, mass spectrometry, circular dichroism studies, fluorescence spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The combined data reveal that protein folding and stability within amorphous precursors are contributing factors in the rate, extent, and mechanism of crystallization. Thus, our results suggest molecular modifications as promising methods for fine-tuning protein@MOFs' nucleation and growth., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Fuel-driven dissipative self-assemblies play essential roles in living systems, contributing both to their complex, dynamic structures and emergent functions. Several dissipative supramolecular materials have been created using chemicals or light as fuel. However, electrical energy, one of the most common energy sources, has remained unexplored for such purposes. Here, we demonstrate a new platform for creating active supramolecular materials using electrically fueled dissipative self-assembly. Through an electrochemical redox reaction network, a transient and highly active supramolecular assembly is achieved with rapid kinetics, directionality, and precise spatiotemporal control. As electronic signals are the default information carriers in modern technology, the described approach offers a potential opportunity to integrate active materials into electronic devices for bioelectronic applications.

Liquid-phase transmission electron microscopy (LP-TEM) enables the real-time visualization of nanoscale dynamics in solution. This technique has been used to study the formation and transformation mechanisms of organic and inorganic nanomaterials. Here, we study the formation of block-copolymer-supported bilayers using LP-TEM. We observe two formation pathways that involve either liquid droplets or vesicles as intermediates toward supported bilayers. Quantitative image analysis methods are used to characterize vesicle spread rates and show the origin of defect formation in supported bilayers. Our results suggest that bilayer assembly methods that proceed via liquid droplet intermediates should be beneficial for forming pristine supported bilayers. Furthermore, supported bilayers inside the liquid cells may be used to image membrane interactions with proteins and nanoparticles in the future.

Molecular self-assembly is pervasive in the formation of living and synthetic materials. Knowledge gained from research into the principles of molecular self-assembly drives innovation in the biological, chemical, and materials sciences. Self-assembly processes span a wide range of temporal and spatial domains and are often unintuitive and complex. Studying such complex processes requires an arsenal of analytical and computational tools. Within this arsenal, the transmission electron microscope stands out for its unique ability to visualize and quantify self-assembly structures and processes. This review describes the contribution that the transmission electron microscope has made to the field of molecular self-assembly. An emphasis is placed on which TEM methods are applicable to different structures and processes and how TEM can be used in combination with other experimental or computational methods. Finally, we provide an outlook on the current challenges to, and opportunities for, increasing the impact that the transmission electron microscope can have on molecular self-assembly.

The antibiotic teixobactin targets bacterial cell walls. Previous research has proposed that the active form of teixobactin is a nano-/micron-sized supramolecular assembly. Here, we use cryogenic transmission electron microscopy to show that at 1 mg/mL, teixobactin forms sheet-like assemblies that selectively act upon the cell wall. At 4 μg/mL, teixobactin is active, and aggregates are formed either transiently or sparingly at the cell surface., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Electron microscopy (EM) of materials undergoing chemical reactions provides knowledge of the underlying mechanisms. However, the mechanisms are often complex and cannot be fully resolved using a single method. Here, we present a distributed electron microscopy method for studying complex reactions. The method combines information from multiple stages of the reaction and from multiple EM methods, including liquid phase EM (LP-EM), cryogenic EM (cryo-EM), and cryo-electron tomography (cryo-ET). We demonstrate this method by studying the desilication mechanism of zeolite crystals. Collectively, our data reveal that the reaction proceeds via a two-step anisotropic etching process and that the defects in curved surfaces and between the subunits in the crystal control the desilication kinetics by directing mass transport.

Liquid-Phase (Scanning) Transmission Electron Microscopy (LP-(S)TEM) has become an essential technique to monitor nanoscale materials processes in liquids in real-time. Due to the pressure difference between the liquid and the microscope vacuum, bending of the silicon nitride (SiN x ) membrane windows generally occurs. This causes a spatially varying liquid layer thickness that makes interpretation of LP-(S)TEM results difficult due to a locally varying achievable resolution and diffusion limitations. To mediate these difficulties, it is shown: 1) how to quantitatively map liquid layer thickness for any liquid at less than 0.01 e - Å -2 total dose; 2) how to dynamically modulate the liquid thickness by tuning the internal pressure in the liquid cell, co-determined by the Laplace pressure and the external pressure. It is demonstrated that reproducible inward bulging of the window membranes can be realized, leading to an ultra-thin liquid layer in the central window area for high-resolution imaging. Furthermore, it is shown that the liquid thickness can be dynamically altered in a programmed way, thereby potentially overcoming the diffusion limitations towards achieving bulk solution conditions. The presented approaches provide essential ways to measure and dynamically adjust liquid thickness in LP-(S)TEM experiments, enabling new experiment designs and better control of solution chemistry., (© 2021 The Authors. Small Methods published by Wiley-VCH GmbH.)

Periodic nano- or microscale structures are used to control light, energy and mass transportation. Colloidal organization is the most versatile method used to control nano- and microscale order, and employs either the enthalpy-driven self-assembly of particles at a low concentration or the entropy-driven packing of particles at a high concentration. Nonetheless, it cannot yet provide the spontaneous three-dimensional organization of multicomponent particles at a high concentration. Here we combined these two concepts into a single strategy to achieve hierarchical multicomponent materials. We tuned the electrostatic attraction between polymer and silica nanoparticles to create dynamic supracolloids whose components, on drying, reorganize by entropy into three-dimensional structured materials. Cryogenic electron tomography reveals the kinetic pathways, whereas Monte Carlo simulations combined with a kinetic model provide design rules to form the supracolloids and control the kinetic pathways. This approach may be useful to fabricate hierarchical hybrid materials for distinct technological applications.

In highly selective solvents, block copolymers (BCPs) form association colloids, while in solvents with poor selectivity, they exhibit a temperature-controlled (de)mixing behavior. Herein, it is shown that a temperature-responsive self-assembly behavior emerges in solvent mixtures of intermediate selectivity. A biocompatible poly-ethylene(oxide)- block -poly-ε-caprolactone (PEO-PCL) BCP is used as a model system. The polymer is dissolved in solvent mixtures containing water (a strongly selective solvent for PEO) and ethanol (a poorly selective solvent for PEO) to tune the solvency conditions. Using synchrotron X-ray scattering, cryogenic transmission electron microscopy, and scanning probe microscopy, it is shown that a rich temperature-responsive behavior can be achieved in certain solvent mixtures. Crystallization of the PCL block enriches the phase behavior of the BCP by promoting sphere-to-cylinder morphology transitions at low temperatures. Increasing the water fraction in the solvent causes a suppression of the sphere-to-cylinder morphology transition. These results open up the possibility to induce temperature-responsive properties on demand in a wide range of BCP systems., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Polymer self-assembly is used to form nanomaterials with a wide range of structures. While self-assembly of polymers in bulk has been thoroughly explored, the same process in solution remains widely used but partially unresolved, due to the formation of structures which are often kinetically trapped. In this paper we report kinetic state diagrams of polystyrene-b-poly(ethylene oxide) block copolymer in water by changing the solvent-switch assembly conditions. We study 36 different conditions for a single block copolymer, exploring three parameters: polymer concentration, temperature and rate addition of selective solvent. The data shows that polymer concentration plays an important role in determining which morphologies are accessible within a given set of experimental parameters and provides evidence that vesicles can evolve into particles with complex internal structures, supportive of recent mechanistic studies. Most importantly, the data shows a complex relationship between all parameters and the resulting kinetically trapped morphologies indicating that combined in situ and ex situ studies are required to gain a fundamental understanding of kinetically controlled block copolymer assembly processes.

The self-assembly of block copolymers into 1D, 2D and 3D nano- and microstructures is of great interest for a wide range of applications. A key challenge in this field is obtaining independent control over molecular structure and hierarchical structure in all dimensions using scalable one-pot chemistry. Here we report on the ring opening polymerization-induced crystallization-driven self-assembly (ROPI-CDSA) of poly-L-lactide-block-polyethylene glycol block copolymers into 1D, 2D and 3D nanostructures. A key feature of ROPI-CDSA is that the polymerization time is much shorter than the self-assembly relaxation time, resulting in a non-equilibrium self-assembly process. The self-assembly mechanism is analyzed by cryo-transmission electron microscopy, wide-angle x-ray scattering, Fourier transform infrared spectroscopy, and turbidity studies. The analysis revealed that the self-assembly mechanism is dependent on both the polymer molecular structure and concentration. Knowledge of the self-assembly mechanism enabled the kinetic trapping of multiple hierarchical structures from a single block copolymer.

Innovations in liquid-phase electron microscopy (LP-EM) have made it possible to perform experiments at the optimized conditions needed to examine soft matter. The main obstacle is conducting experiments in such a way that electron beam radiation can be used to obtain answers for scientific questions without changing the structure and (bio)chemical processes in the sample due to the influence of the radiation. By overcoming these experimental difficulties at least partially, LP-EM has evolved into a new microscopy method with nanometer spatial resolution and sub-second temporal resolution for analysis of soft matter in materials science and biology. Both experimental design and applications of LP-EM for soft matter materials science and biological research are reviewed, and a perspective of possible future directions is given., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As many physical properties of polymers scale with molecular weight, the ability to achieve polymers of nearly inaccessibly high molecular weight provides an opportunity to probe the upper size limit of macromolecular phenomena. Yet many of the most stimulating macromolecular designs remain out of reach of current ultrahigh molecular weight (UHMW) polymer synthetic approaches. Herein, we show that UHMW polymers of diverse composition can be achieved by irradiation of thiocarbonylthio photoiniferters with long-wave ultraviolet or visible light in concentrated organic solution. This facile photopolymerization strategy is general to acrylic-, acrylamido-, methacrylic-, and styrenic-based monomers, enabling the synthesis of well-defined macromolecules with molecular weights in excess of 10 6 g/mol. The high chain-end fidelity afforded by photoiniferter polymerization conditions facilitated the design of UHMW amphiphilic block copolymers, which were found to self-assemble into micellar morphologies up to 200 nm in diameter.

Protein-metal-organic frameworks (p-MOFs) are a prototypical example of how synthetic biological hybrid systems can be used to develop next-generation materials. Controlling p-MOF formation enables the design of hybrid materials with enhanced biological activity and high stability. However, such control is yet to be fully realized due to an insufficient understanding of the governing nucleation and growth mechanisms in p-MOF systems. The structural evolution of p-MOFs was probed by cryo-transmission electron microscopy, revealing nonclassical pathways via dissolution-recrystallization of highly hydrated amorphous particles and solid-state transformation of a protein-rich amorphous phase. On the basis of these data, we propose a general description of p-MOF crystallization which is best characterized by particle aggregation and colloidal theory for future synthetic strategies.

Natural materials, such as collagen, can assemble with multiple levels of organization in solution. Achieving a similar degree of control over morphology, stability and hierarchical organization with equilibrium synthetic materials remains elusive. For the assembly of peptidic materials the process is controlled by a complex interplay between hydrophobic interactions, electrostatics and secondary structure formation. Consequently, fine tuning the thermodynamics and kinetics of assembly remains extremely challenging. Here, we synthesized a set of block co polypeptides with varying hydrophobicity and ability to form secondary structure. From this set we select a sequence with balanced interactions that results in the formation of high-aspect ratio thermodynamically favored nanotubes, stable between pH 2 and 12 and up to 80 °C. This stability permits their hierarchical assembly into bundled nanotube fibers by directing the pH and inducing complementary zwitterionic charge behavior. This block co-polypeptide design strategy, using defined sequences, provides a straightforward approach to creating complex hierarchical peptide-based assemblies with tunable interactions., (This journal is © The Royal Society of Chemistry 2019.)

Stimuli-responsive polymers are an efficient means of targeted therapy. Compared to conventional agents, they increase bioavailability and efficacy. In particular, polymer hydrogel nanoparticles (NPs) can be designed to respond when exposed to a specific environmental stimulus such as pH or temperature. However, targeting a specific metabolite as the trigger for stimuli response could further elevate selectivity and create a new class of bioresponsive materials. In this work we describe an N-isopropylacrylamide (NIPAm) NP that responds to a specific metabolite, characteristic of a hypoxic environment found in cancerous tumors. NIPAm NPs were synthesized by copolymerization with an oxamate derivative, a known inhibitor of lactate dehydrogenase (LDH). The oxamate-functionalized NPs (OxNP) efficiently sequestered LDH to produce an OxNP-protein complex. When exposed to elevated concentrations of lactic acid, a substrate of LDH and a metabolite characteristic of hypoxic tumor microenvironments, OxNP-LDH complexes swelled (65%). The OxNP-LDH complexes were not responsive to structurally related small molecules. This work demonstrates a proof of concept for tuning NP responsiveness by conjugation with a key protein to target a specific metabolite of disease.

In this paper, experiment and simulation were combined to provide a view of the molecular rearrangements underlying the equilibrium and nonequilibrium transitions occurring in stimuli-responsive block copolymer amphiphile self-assemblies. Three block copolymer amphiphiles were prepared, each consisting of a hydrophilic peptide brush, responsive to proteolytic enzymes, and containing one of three possible hydrophobic blocks: (1) poly(ethyl acrylate), (2) poly(styrene), or (3) poly(lauryl acrylate). When assembled, they generate three spherical micelles each responsive to the addition of the bacterial protease, thermolysin. We found core-block-dependent phase transitions in response to the hydrophilic block being truncated by the stimulus. In one example, we found an unexpected, well-defined, pathway-dependent spherical micelle to vesicle phase transition induced by enzymatic stimulus.

The solubilization of lyophobic compounds in block copolymer micelles has been extensively investigated but remains only partially understood. There is a need to understand the fundamental parameters that determine the spatial distribution of the solubilized compounds within the micelles. Controlling this feature is a key aspect in the design of drug delivery systems with tailored release properties. Using Scheutjens-Fleer self-consistent field (SF-SCF) computations, we found that solubilization is regulated by a complex interplay between enthalpic and entropic contributions and that the spatial distribution can be controlled by the concentration and solubility of the guest compound in the dispersion medium. Upon solubilization, a characteristic change in size and mass of the micelles is predicted. This can be used as a fingerprint to indirectly assess the spatial distribution. Based on these findings, we developed two experimental protocols to control and assess the spatial distribution of lyophobic compounds within block copolymer micelles.

The self-assembly of amphiphilic molecules in solution is a ubiquitous process in both natural and synthetic systems. The ability to effectively control the structure and properties of these systems is essential for tuning the quality of their functionality, yet the underlying mechanisms governing the transition from molecules to assemblies have not been fully resolved. Here we describe how amphiphilic self-assembly can be preceded by liquid-liquid phase separation. The assembly of a model block co-polymer system into vesicular structures was probed through a combination of liquid-phase electron microscopy, self-consistent field computations and Gibbs free energy calculations. This analysis shows the formation of polymer-rich liquid droplets that act as a precursor in the bottom-up formation of spherical micelles, which then evolve into vesicles. The liquid-liquid phase separation plays a role in determining the resulting vesicles' structural properties, such as their size and membrane thickness, and the onset of kinetic traps during self-assembly.

Spider venom contains a number of small peptides that can control the gating properties of a wide range of ion channels with high affinity and specificity. These ion channels are responsible for coordination and control of many bodily functions such as transducing signals into sensory functions, smooth muscle contractions as well as serving as sensors in volume regulation. Hence, these peptides have been the topic of many research efforts in hopes that they can be used as biomedical therapeutics. Several peptides are known to control the gating properties of ion channels by involving the lipid membrane. GsMTx4, originally isolated from the Chilean Rose tarantula (Grammostola rosea), is known to selectively inhibit mechanosensitive ion channels by partitioning into the lipid bilayer. To further understand this indirect gating mechanism, we investigated the interactions between native GsAF2, VsTx1 and a synthetic form of GsMTx4 with model DMPC lipid bilayers using 31 P solid-state NMR, 13 C CP-MAS NMR, NS-TEM and cryo-TEM. The results reveal that these inhibitor cystine knot peptides perforate the DMPC lipid vesicles similarly with some subtle differences and ultimately create small spherical vesicles and anisotropic cylindrical and discoidal vesicles at concentrations near 1.0-1.5 mol% peptide. The anisotropic components align with their long axes along the NMR static B 0 magnetic field, a property that should be useful in future NMR structural investigations of these systems. These findings move us forward in our understanding of how these peptides bind and interact with the lipid bilayer., (Copyright © 2018. Published by Elsevier B.V.)

The intrinsic properties of nanomaterials offer promise for technological revolutions in many fields, including transportation, soft robotics, and energy. Unfortunately, the exploitation of such properties in polymer nanocomposites is extremely challenging due to the lack of viable dispersion routes when the filler content is high. We usually face a dichotomy between the degree of nanofiller loading and the degree of dispersion (and, thus, performance) because dispersion quality decreases with loading. Here, we demonstrate a potentially scalable pressing-and-folding method (P & F), inspired by the art of croissant-making, to efficiently disperse ultrahigh loadings of nanofillers in polymer matrices. A desired nanofiller dispersion can be achieved simply by selecting a sufficient number of P & F cycles. Because of the fine microstructural control enabled by P & F, mechanical reinforcements close to the theoretical maximum and independent of nanofiller loading (up to 74 vol %) were obtained. We propose a universal model for the P & F dispersion process that is parametrized on an experimentally quantifiable " D factor". The model represents a general guideline for the optimization of nanocomposites with enhanced functionalities including sensing, heat management, and energy storage.

Postsynthetic strategies for modifying metal-organic frameworks (MOFs) have proven to be an incredibly powerful approach for expanding the scope and functionality of these materials. Previously, we reported on the postsynthetic exchange (PSE) of metal ions and ligands in the University of Oslo (UiO) series of MOFs. Detailed characterization by several analytical methods, most notably inductively coupled plasma mass spectrometry and transmission electron microscopy reveal that metal ion deposition on the surface of these MOFs occurs in the form of nanoscale metal oxides, rather than yielding exchanged metal sites within the MOFs, as was previously reported. By contrast, these combined analytical methods do confirm that ligand-based PSE can occur in these MOFs. These findings provide new insight into the postsynthetic manipulation of MOF materials, highlight the importance of rigorously characterizing these materials to correctly assign their composition and structure, and provide a new route to making hybrid solids with a MOF@metal oxide architecture.

Amphiphilic small molecules and polymers form commonplace nanoscale macromolecular compartments and bilayers, and as such are truly essential components in all cells and in many cellular processes. The nature of these architectures, including their formation, phase changes, and stimuli-response behaviors, is necessary for the most basic functions of life, and over the past half-century, these natural micellar structures have inspired a vast diversity of industrial products, from biomedicines to detergents, lubricants, and coatings. The importance of these materials and their ubiquity have made them the subject of intense investigation regarding their nanoscale dynamics with increasing interest in obtaining sufficient temporal and spatial resolution to directly observe nanoscale processes. However, the vast majority of experimental methods involve either bulk-averaging techniques including light, neutron, and X-ray scattering, or are static in nature including even the most advanced cryogenic transmission electron microscopy techniques. Here, we employ in situ liquid-cell transmission electron microscopy (LCTEM) to directly observe the evolution of individual amphiphilic block copolymer micellar nanoparticles in solution, in real time with nanometer spatial resolution. These observations, made on a proof-of-concept bioconjugate polymer amphiphile, revealed growth and evolution occurring by unimer addition processes and by particle-particle collision-and-fusion events. The experimental approach, combining direct LCTEM observation, quantitative analysis of LCTEM data, and correlated in silico simulations, provides a unique view of solvated soft matter nanoassemblies as they morph and evolve in time and space, enabling us to capture these phenomena in solution.

Metal-organic frameworks (MOFs) have emerged as a versatile platform for the rational design of multifunctional materials, combining large specific surface areas with flexible, periodic frameworks that can undergo reversible structural transitions, or "breathing", upon temperature and pressure changes, and through gas adsorption/desorption processes. Although MOF breathing can be inferred from the analysis of adsorption isotherms, direct observation of the structural transitions has been lacking, and the underlying processes of framework reorganization in individual MOF nanocrystals is largely unknown. In this study, we describe the characterization and elucidation of these processes through the combination of in situ environmental transmission electron microscopy (ETEM) and computer simulations. This combined approach enables the direct monitoring of the breathing behavior of individual MIL-53(Cr) nanocrystals upon reversible water adsorption and temperature changes. The ability to characterize structural changes in single nanocrystals and extract lattice level information through in silico correlation provides fundamental insights into the relationship between pore size/shape and host-guest interactions.

Morphology plays an essential role in chemistry through the segregation of atoms and/or molecules into different phases, delineated by interfaces. This is a general process in materials synthesis and exploited in many fields including colloid chemistry, heterogeneous catalysis, and functional molecular systems. To rationally design complex materials, we must understand and control morphology evolution. Toward this goal, we utilize cryogenic transmission electron microscopy (cryoTEM), which can track the structural evolution of materials in solution with nanometer spatial resolution and a temporal resolution of <1 s. In this Account, we review examples of our own research where direct observations by cryoTEM have been essential to understanding morphology evolution in macromolecular self-assembly, inorganic nucleation and growth, and the cooperative evolution of hybrid materials. These three different research areas are at the heart of our approach to materials chemistry where we take inspiration from the myriad examples of complex materials in Nature. Biological materials are formed using a limited number of chemical components and under ambient conditions, and their formation pathways were refined during biological evolution by enormous trial and error approaches to self-organization and biomineralization. By combining the information on what is possible in nature and by focusing on a limited number of chemical components, we aim to provide an essential insight into the role of structure evolution in materials synthesis. Bone, for example, is a hierarchical and hybrid material which is lightweight, yet strong and hard. It is formed by the hierarchical self-assembly of collagen into a macromolecular template with nano- and microscale structure. This template then directs the nucleation and growth of oriented, nanoscale calcium phosphate crystals to form the composite material. Fundamental insight into controlling these structuring processes will eventually allow us to design such complex materials with predetermined and potentially unique properties.

The passive leakage of small molecules across membranes is a major limitation of liposomal drug formulations. Here, we evaluate the leakage of 3 clinically used chemotherapeutic agents (cytarabine, methotrexate and vincristine) encapsulated in liposomes comprised of a synthetic, archaea-inspired, membrane-spanning tetraether lipid. Liposomes comprised of the pure tetraether lipid exhibited superior retention of both a neutrally and positively charged drug (up to an ∼9-fold decrease in the rate of drug leakage) compared to liposomes formed from a commercial diacyl lipid, while exhibiting a similar retention of a negatively charged drug that did not appreciably leak from either type of liposome. We also demonstrate that liposomes made of the archaea-inspired lipid can be used for the delivery of encapsulated small molecules into living cells.

We report the synthesis and study of fluorocarbon (FC) modified polyethylenimine (PEI) for the purpose of siRNA delivery. Low-molecular-weight PEI (Mn = 600) was functionalized with fluorocarbon epoxides of varying length. All FC-modified samples with greater than 2.0 equiv of FC epoxide per PEI induced potent gene silencing in vitro. Compared to hydrocarbon (HC) analogues, the FC vectors showed greater general silencing efficacy, higher cell uptake, and reduced association with serum components. Collectively, the data suggest that modification of polyamines with FCs is a promising approach for the discovery of novel vectors for siRNA delivery.

A Gd 3+ -coordinated polymerizable analogue of the MRI contrast agent Gd-DOTA was used to prepare amphiphilic block copolymers, with hydrophilic blocks composed entirely of the polymerized contrast agent. The resulting amphiphilic block copolymers assemble into nanoparticles (NPs) of spherical- or fibril-shape, each demonstrating enhanced relaxivity over Gd-DOTA. As an initial examination of their behavior in vivo , intraperitoneal (IP) injection of NPs into live mice was performed, showing long IP residence times, observed by MRI. Extended residence times for particles of well-defined morphology may represent a valuable design paradigm for treatment or diagnosis of peritoneal malignances.

Liquid cell transmission electron microscopy (LCTEM) provides a unique insight into the dynamics of nanomaterials in solution. Controlling the addition of multiple solutions to the liquid cell remains a key hurdle in our ability to increase throughput and to study processes dependent on solution mixing including chemical reactions. Here, we report that a piezo dispensing technique allows for mixing of multiple solutions directly within the viewing area. This technique permits deposition of 50 pL droplets of various aqueous solutions onto the liquid cell window, before assembly of the cell in a fully controlled manner. This proof-of-concept study highlights the great potential of picoliter dispensing in combination with LCTEM for observing nanoparticle mixing in the solution phase and the creation of chemical gradients.

Direct polymerization of an oxaliplatin analogue was used to reproducibly generate amphiphiles in one pot, which consistently and spontaneously self-assemble into well-defined nanoparticles (NPs). Despite inefficient drug leakage in cell-free assays, the NPs were observed to be as cytotoxic as free oxaliplatin in cell culture experiments. We investigated this phenomenon by super-resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). In combination, these techniques revealed NPs are taken up via endocytic pathways before intracellular release of their cytotoxic cargo. As with other drug-carrying nanomaterials, these systems have potential as cellular delivery vehicles. However, high-resolution methods to track nanocarriers and their cargo at the micro- and nanoscale have been underutilized in general, limiting our understanding of their interactions with cells and tissues. We contend this type of combined optical and isotopic imaging strategy represents a powerful and potentially generalizable methodology for cellular tracking of nanocarriers and their cargo.

Amphiphilic block copolymers can assemble into a variety of structures on the nanoscale in selective solvent. The micelle blending protocol offers a simple unique route to reproducibly produce polymer nanostructures. Here we expand this blending protocol to a range of polymer micelle systems and self-assembly routes. We found by exploring a range of variables that the systems must be able to reach global equilibrium at some point for the blending protocol to be successful. Our results demonstrate the kinetics requirements, specifically core block glass transition temperature, T g , and length of the block limiting the exchange rates, for the blending protocol which can then be applied to a wide range of polymer systems to access this simple protocol for polymer self-assembly.

Amphiphilic triblock copolymers containing Fe(III) -catecholate complexes formulated as spherical- or cylindrical-shaped micellar nanoparticles (SMN and CMN, respectively) are described as new T1-weighted agents with high relaxivity, low cytotoxicity, and long-term stability in biological fluids. Relaxivities of both SMN and CMN exceed those of established gadolinium chelates across a wide range of magnetic field strengths. Interestingly, shape-dependent behavior is observed in terms of the particles' interactions with HeLa cells, with CMN exhibiting enhanced uptake and contrast via magnetic resonance imaging (MRI) compared with SMN. These results suggest that control over soft nanoparticle shape will provide an avenue for optimization of particle-based contrast agents as biodiagnostics. The polycatechol nanoparticles are proposed as suitable for preclinical investigations into their viability as gadolinium-free, safe, and effective imaging agents for MRI contrast enhancement., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The composition and surface properties of atmospheric aerosol particles largely control their impact on climate by affecting their ability to uptake water, react heterogeneously, and nucleate ice in clouds. However, in the vacuum of a conventional electron microscope, the native surface and internal structure often undergo physicochemical rearrangement resulting in surfaces that are quite different from their atmospheric configurations. Herein, we report the development of cryogenic transmission electron microscopy where laboratory generated sea spray aerosol particles are flash frozen in their native state with iterative and controlled thermal and/or pressure exposures and then probed by electron microscopy. This unique approach allows for the detection of not only mixed salts, but also soft materials including whole hydrated bacteria, diatoms, virus particles, marine vesicles, as well as gel networks within hydrated salt droplets-all of which will have distinct biological, chemical, and physical processes. We anticipate this method will open up a new avenue of analysis for aerosol particles, not only for ocean-derived aerosols, but for those produced from other sources where there is interest in the transfer of organic or biological species from the biosphere to the atmosphere.