Exploration of Organic Nanomaterials with Liquid-Phase Transmission Electron Microscopy.

ConspectusOrganic, soft materials with solution-phase nanoscale structures, such as emulsions, hydrogels, and thermally responsive materials, are inherently difficult to directly image via dry state and cryogenic-transmission electron microscopy (TEM). Therefore, we lack a routine microscopy method with sufficient resolution that can, in tandem with scattering techniques, probe the morphology and dynamics of these and many related systems. These challenges motivate liquid cell (LC) TEM method development, aimed at making the technique generally available and routine. To date, the field has been and continues to be dominantly focused on analyzing solution-phase inorganic materials. These mostly metallic nanoparticles have been studied at electron fluxes that can allow for high-resolution imaging, in the range of hundreds to thousands of e- Å-2 s-1. Despite excellent contrast, in these cases, one often contends with knock-on damage, direct radiolysis, and sensitization of the solvent by virtue of enhanced secondary electron production by the impinging electron beam. With an interest in soft materials, we face both related and distinct challenges, especially in achieving a high-enough contrast within solvated liquid cells. Additionally, we must be aware of artifacts associated with high-flux imaging conditions in terms of direct radiolysis of the solvent and the sensitive materials themselves. Regardless, with care, it has become possible to gain real insight into both static and dynamic organic nanomaterials in solution. This is due, in large part, to key advances that have been made, including improved sample preparation protocols, image capture technologies, and image analysis, which have allowed LCTEM to have utility. To enable solvated soft matter characterization by LCTEM, a generalizable multimodal workflow was developed by leveraging both experimental and theoretical precedents from across the LCTEM field and adjacent works concerned with solution radiolysis and nanoparticle tracking analyses. This workflow consists of (1) modeling electron beam-solvent interactions, (2) studying electron beam-sample interactions via LCTEM coupled with post-mortem analysis, (3) the construction of "damage plots" displaying sample integrity under varied imaging and sample conditions, (4) optimized LCTEM imaging, (5) image processing, and (6) correlative analysis via X-ray or light scattering. In this Account, we present this outlook and the challenges we continue to overcome in the direct imaging of dynamic solvated nanoscale soft materials.

Upper critical solution temperature polymer assemblies via variable temperature liquid phase transmission electron microscopy and liquid resonant soft X-ray scattering.

Here, we study the upper critical solution temperature triggered phase transition of thermally responsive poly(ethylene glycol)-block-poly(ethylene glycol) methyl ether acrylate-co-poly(ethylene glycol) phenyl ether acrylate-block-polystyrene nanoassemblies in isopropanol. To gain mechanistic insight into the organic solution-phase dynamics of the upper critical solution temperature polymer, we leverage variable temperature liquid-cell transmission electron microscopy correlated with variable temperature liquid resonant soft X-ray scattering. Heating above the upper critical solution temperature triggers a reduction in particle size and a morphological transition from a spherical core shell particle with a complex, multiphase core to a micelle with a uniform core and Gaussian polymer chains attached to the surface. These correlated solution phase methods, coupled with mass spectral validation and modeling, provide unique insight into these thermoresponsive materials. Moreover, we detail a generalizable workflow for studying complex, solution-phase nanomaterials via correlative methods.

Inflammation-Responsive Micellar Nanoparticles from Degradable Polyphosphoramidates for Targeted Delivery to Myocardial Infarction.

Nanoparticles that undergo a localized morphology change to target areas of inflammation have been previously developed but are limited by their lack of biodegradability. In this paper, we describe a low-ring-strain cyclic olefin monomer, 1,3-dimethyl-2-phenoxy-1,3,4,7-tetrahydro-1,3,2-diazaphosphepine 2-oxide (MePTDO), that rapidly polymerizes via ring-opening metathesis polymerization at room temperature to generate well-defined degradable polyphosphoramidates with high monomer conversion (>84%). Efficient MePTDO copolymerizations with norbornene-based monomers are demonstrated, including a norbornenyl monomer functionalized with a peptide substrate for inflammation-associated matrix metalloproteinases (MMPs). The resulting amphiphilic peptide brush copolymers self-assembled in aqueous solution to generate micellar nanoparticles (30 nm in diameter) which exhibit excellent cyto- and hemocompatibility and undergo MMP-induced assembly into micron-scale aggregates. As MMPs are upregulated in the heart postmyocardial infarction (MI), the MMP-responsive micelles were applied to target and accumulate in the infarcted heart following intravenous administration in a rat model of MI. These particles displayed a distinct biodistribution and clearance pattern in comparison to nondegradable analogues. Specifically, accumulation at the site of MI competed with elimination predominantly through the kidney rather than the liver. Together, these results suggest this as a promising new biodegradable platform for inflammation targeted delivery.

Biomimetic pheomelanin to unravel the electronic, molecular and supramolecular structure of the natural product.

Herein, we investigate synthetic routes to a close mimic of natural pheomelanin. Three different oxidative polymerization routes were attempted to generate synthetic pheomelanin, each giving rise to structurally dissimilar materials. Among them, the route employing 5-cysteinyl-dihydroxyphenylalanine (5-CD) as a monomer was verified as a close analogue of extracted pheomelanin from humans and birds. The resulting biomimetic and natural pheomelanins were compared via various techniques, including solid-state Nuclear Magnetic Resonance (ssNMR) and Electron Paramagnetic Resonance (EPR). This synthetic pheomelanin closely mimics the structure of natural pheomelanin as determined by parallel characterization of pheomelanin extracted from multiple biological sources. With a good synthetic biomimetic material in hand, we describe cation-π interactions as an important driving force for pheomelanogenesis, further advancing our fundamental understanding of this important biological pigment.

Gradient Copolymer Synthesis through Self-Assembly.

Polymerization-induced self-assembly (PISA) is typically performed to produce polymer nanoparticles featuring specific assembly morphologies. Herein, we demonstrate the use of PISA as a synthetic tool to direct gradient copolymer synthesis. Specifically, we leverage hydrophobicity-induced reaction selectivity and the rate acceleration typically associated with polymer compartmentalization upon assembly during PISA to bias reaction selectivity. In the chain extension of a poly(ethylene glycol) macrochain transfer agent, the selectivity of diacetone acrylamide (DAAm) and N,N-dimethylacrylamide (DMA), two monomers with near-identical reactivity in water, can be modulated in situ such that DAAm is preferentially incorporated over DMA upon self-assembly. By increasing the feed ratio of DAAm, monomer differentiation can be further biased toward DAAm due to the locus of polymerization becoming increasingly hydrophobic. This change in selectivity affords the autonomous generation of DAAm-DMA gradient sequences, otherwise inaccessible without outside intervention. Finally, a mild hydrolysis protocol can then be employed to harvest DAAm-DMA sequences, yielding compositionally unique gradient copolymers.

Self-Assembly of Tunable Intrinsically Disordered Peptide Amphiphiles.

Intrinsically disordered peptide amphiphiles (IDPAs) present a novel class of synthetic conjugates that consist of short hydrophilic polypeptides anchored to hydrocarbon chains. These hybrid polymer-lipid block constructs spontaneously self-assemble into dispersed nanoscopic aggregates or ordered mesophases in aqueous solution due to hydrophobic interactions. Yet, the possible sequence variations and their influence on the self-assembly structures are vast and have hardly been explored. Here, we measure the nanoscopic self-assembled structures of four IDPA systems that differ by their amino acid sequence. We show that permutations in the charge pattern along the sequence remarkably alter the headgroup conformation and consequently alter the pH-triggered phase transitions between spherical, cylindrical micelles and hexagonal condensed phases. We demonstrate that even a single amino acid mutation is sufficient to tune structural transitions in the condensed IDPA mesophases, while peptide conformations remain unfolded and disordered. Furthermore, alteration of the peptide sequence can render IDPAs to become susceptible to enzymatic cleavage and induce enzymatically activated phase transitions. These results hold great potential for embedding multiple functionalities into lipid nanoparticle delivery systems by incorporating IDPAs with the desired properties.

Thermoresponsive polymer assemblies via variable temperature liquid-phase transmission electron microscopy and small angle X-ray scattering.

Herein, phase transitions of a class of thermally-responsive polymers, namely a homopolymer, diblock, and triblock copolymer, were studied to gain mechanistic insight into nanoscale assembly dynamics via variable temperature liquid-cell transmission electron microscopy (VT-LCTEM) correlated with variable temperature small angle X-ray scattering (VT-SAXS). We study thermoresponsive poly(diethylene glycol methyl ether methacrylate) (PDEGMA)-based block copolymers and mitigate sample damage by screening electron flux and solvent conditions during LCTEM and by evaluating polymer survival via post-mortem matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). Our multimodal approach, utilizing VT-LCTEM with MS validation and VT-SAXS, is generalizable across polymeric systems and can be used to directly image solvated nanoscale structures and thermally-induced transitions. Our strategy of correlating VT-SAXS with VT-LCTEM provided direct insight into transient nanoscale intermediates formed during the thermally-triggered morphological transformation of a PDEGMA-based triblock. Notably, we observed the temperature-triggered formation and slow relaxation of core-shell particles with complex microphase separation in the core by both VT-SAXS and VT-LCTEM.

Dipeptide Nanostructure Assembly and Dynamics via in Situ Liquid-Phase Electron Microscopy.

In this paper, we report the in situ growth of FF nanotubes examined via liquid-cell transmission electron microscopy (LCTEM). This direct, high spatial, and temporal resolution imaging approach allowed us to observe the growth of peptide-based nanofibrillar structures through directional elongation. Furthermore, the radial growth profile of FF nanotubes through the addition of monomers perpendicular to the tube axis has been observed in real-time with sufficient resolution to directly observe the increase in diameter. Our study demonstrates that the kinetics, dynamics, structure formation, and assembly mechanism of these supramolecular assemblies can be directly monitored using LCTEM. The performance of the peptides and the assemblies they form can be verified and evaluated using post-mortem techniques including time-of-flight secondary ion mass spectrometry (ToF-SIMS).

Glycopolymer Microarrays with Sub-Femtomolar Avidity for Glycan Binding Proteins Prepared by Grafted-To/Grafted-From Photopolymerizations.

We report a novel glycan array architecture that binds the mannose-specific glycan binding protein, concanavalin A (ConA), with sub-femtomolar avidity. A new radical photopolymerization developed specifically for this application combines the grafted-from thiol-(meth)acrylate polymerization with thiol-ene chemistry to graft glycans to the growing polymer brushes. The propagation of the brushes was studied by carrying out this grafted-to/grafted-from radical photopolymerization (GTGFRP) at >400 different conditions using hypersurface photolithography, a printing strategy that substantially accelerates reaction discovery and optimization on surfaces. The effect of brush height and the grafting density of mannosides on the binding of ConA to the brushes was studied systematically, and we found that multivalent and cooperative binding account for the unprecedented sensitivity of the GTGFRP brushes. This study further demonstrates the ease with which new chemistry can be tailored for an application as a result of the advantages of hypersurface photolithography.

Orthogonal Images Concealed Within a Responsive 6-Dimensional Hypersurface.

A photochemical printer, equipped with a digital micromirror device (DMD), leads to the rapid elucidation of the kinetics of the surface-initiated atom-transfer radical photopolymerization of N,N-dimethylacrylamide (DMA) and N-isopropylacrylamide (NIPAM) monomers. This effort reveals conditions where polymer brushes of identical heights can be grown from each monomer. With these data, hidden images are created that appear upon heating the substrate above the lower critical solution temperature (LCST) of polyNIPAM. By introducing a third monomer, methacryloxyethyl thiocarbamoyl rhodamine B, a second, orthogonal image appears upon UV-irradiation. With these studies, it is shown how a new photochemical printer accelerates discovery, creates arbitrary patterns, and addresses long-standing problems in brush polymer and surface chemistry. With this technology in hand a new method is demonstrated to encrypt data within hypersurfaces.

Synthetic Porous Melanin.

Commonly known as a skin pigment, melanin has a vital role in UV radiation protection, primarily acting as a radical scavenger. However, a lesser known natural property of melanin, observed in some melanized organisms, is its capacity to adsorb toxins, including metals and organic molecules. Inspired by this, we set out to generate a synthetic porous melanin that would pave the way to enhancing the natural adsorbent properties of melanin and melanin-like materials. Here, we developed a method for the synthesis of porous polydopamine-based melanin utilizing a mesoporous silica (MS) nanoparticle template and characterized its physical properties. Through the oxidative polymerization of dopamine, followed by the etching of silica, we generated synthetic porous melanin (SPM) with the highest measured surface area of any known polydopamine-based material. The prepared SPM was effective for the uptake of various gases and organophosphate toxins, with the material exhibiting high selectivity for CO2 over CH4 and high potential for ammonia capture. Given the demonstrated advantages provided by synthetic porous melanin and melanin's role as an adsorbent in nature, we anticipate the discovery of porous analogues in biological systems.

Enhancing and Mitigating Radiolytic Damage to Soft Matter in Aqueous Phase Liquid-Cell Transmission Electron Microscopy in the Presence of Gold Nanoparticle Sensitizers or Isopropanol Scavengers.

In this work, we describe the radiolytic environment experienced by a polymer in water during liquid-cell transmission electron microscopy (LCTEM). We examined the radiolytic environment of aqueous solutions of poly(ethylene glycol) (PEG, 2400 g/mol) in the presence of sensitizing gold nanoparticles (GNPs, 100 nm) or radical scavenging isopropanol (IPA). To quantify polymer damage, we employed post-mortem analysis via matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). This approach confirms IPA (1-10% w/v) can significantly mitigate radiolysis-induced damage to polymers in water, while GNPs significantly enhance damage. We couple LCTEM experiments with simulations to provide a generalizable strategy for assessing radiolysis mitigation or enhancement. This study highlights the caution required for LCTEM experiments on inorganic nanoparticles where solution phase properties of surrounding organic materials or the solvent itself are under investigation. Furthermore, we anticipate an increased use of scavengers for LCTEM studies of all kinds.

100th Anniversary of Macromolecular Science Viewpoint: Polymeric Materials by In Situ Liquid-Phase Transmission Electron Microscopy.

A century ago, Hermann Staudinger proposed the macromolecular theory of polymers, and now, as we enter the second century of polymer science, we face a different set of opportunities and challenges for the development of functional soft matter. Indeed, many fundamental questions remain open, relating to physical structures and mechanisms of phase transformations at the molecular and nanoscale. In this Viewpoint, we describe efforts to develop a dynamic, in situ microscopy tool suited to the study of polymeric materials at the nanoscale that allows for direct observation of discrete structures and processes in solution, as a complement to light, neutron, and X-ray scattering methods. Liquid-phase transmission electron microscopy (LPTEM) is a nascent in situ imaging technique for characterizing and examining solvated nanomaterials in real time. Though still under development, LPTEM has been shown to be capable of several modes of imaging: (1) imaging static solvated materials analogous to cryo-TEM, (2) videography of nanomaterials in motion, (3) observing solutions or nanomaterials undergoing physical and chemical transformations, including synthesis, assembly, and phase transitions, and (4) observing electron beam-induced chemical-materials processes. Herein, we describe opportunities and limitations of LPTEM for polymer science. We review the basic experimental platform of LPTEM and describe the origin of electron beam effects that go hand in hand with the imaging process. These electron beam effects cause perturbation and damage to the sample and solvent that can manifest as artefacts in images and videos. We describe sample-specific experimental guidelines and outline approaches to mitigate, characterize, and quantify beam damaging effects. Altogether, we seek to provide an overview of this nascent field in the context of its potential to contribute to the advancement of polymer science.

In Situ Ni2+ Stain for Liposome Imaging by Liquid-Cell Transmission Electron Microscopy.

Solvated soft matter, both biological and synthetic, can now be imaged in liquids using liquid-cell transmission electron microscopy (LCTEM). However, such systems are usually composed solely of organic molecules (low Z elements) producing low contrast in TEM, especially within thick liquid films. We aimed to visualize liposomes by LCTEM rather than requiring cryogenic TEM (cryoTEM). This is achieved here by imaging in the presence of aqueous metal salt solutions. The increase in scattering cross-section by the cation gives a staining effect that develops in situ, which could be captured by real space TEM and verified by in situ energy dispersive x-ray spectroscopy (EDS). We identified beam-induced staining as a time-dependent process that enhances contrast to otherwise low contrast materials. We describe the development of this imaging method and identify conditions leading to exceptionally low electron doses for morphology visualization of unilamellar vesicles before beam-induced damage propagates.

Polymer brush hypersurface photolithography.

Polymer brush patterns have a central role in established and emerging research disciplines, from microarrays and smart surfaces to tissue engineering. The properties of these patterned surfaces are dependent on monomer composition, polymer height, and brush distribution across the surface. No current lithographic method, however, is capable of adjusting each of these variables independently and with micrometer-scale resolution. Here we report a technique termed Polymer Brush Hypersurface Photolithography, which produces polymeric pixels by combining a digital micromirror device (DMD), an air-free reaction chamber, and microfluidics to independently control monomer composition and polymer height of each pixel. The printer capabilities are demonstrated by preparing patterns from combinatorial polymer and block copolymer brushes. Images from polymeric pixels are created using the light reflected from a DMD to photochemically initiate atom-transfer radical polymerization from initiators immobilized on Si/SiO2 wafers. Patterning is combined with high-throughput analysis of grafted-from polymerization kinetics, accelerating reaction discovery, and optimization of polymer coatings.