Revealing nanoscale structure and interfaces of protein and polymer condensates via cryo-electron microscopy.

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.

Scattering Elimination in 2D IR Immune from Detector Artifacts.

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.

Observing the Dynamics of an Electrochemically Driven Active Material with Liquid Electron Microscopy.

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.

Hybrid Photoiniferter and Ring-Opening Polymerization Yields One-Pot Anisotropic Nanorods.

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.

Liquid-liquid phase separation induced auto-confinement.

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.

CryoEM reveals the complex self-assembly of a chemically driven disulfide hydrogel.

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.

Correlating electrochemical stimulus to structural change in liquid electron microscopy videos using the structural dissimilarity metric.

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.

Characterizing the Ligand Shell Morphology of PEG-Coated ZnO Nanocrystals Using FRET Spectroscopy.

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 (R0), 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.

Understanding and controlling the nucleation and growth of metal-organic frameworks.

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.

Waste-Free Fully Electrically Fueled Dissipative Self-Assembly System.

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.

Facile Synthesis of Multifunctional Bioreducible Polymers for mRNA Delivery.

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.

Toward Imaging Defect-Mediated Energy Transfer between Single Nanocrystal Donors and Single Molecule Acceptors.

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.

Gaining Structural Control by Modification of Polymerization Rate in Ring-Opening Polymerization-Induced Crystallization-Driven Self-Assembly.

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.

Role of Molecular Modification and Protein Folding in the Nucleation and Growth of Protein-Metal-Organic Frameworks.

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.

Discrete Libraries of Amphiphilic Poly(ethylene glycol) Graft Copolymers: Synthesis, Assembly, and Bioactivity.

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.

Cyclodextrin metal-organic framework-based protein biocomposites.

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.

Electrically Fueled Active Supramolecular Materials.

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.

Mapping and Controlling Liquid Layer Thickness in Liquid-Phase (Scanning) Transmission Electron Microscopy.

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 (SiNx ) 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.