Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

A Cancer Nanovaccine for Co-Delivery of Peptide Neoantigens and Optimized Combinations of STING and TLR4 Agonists.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment and led to complete and durable responses, but only for a minority of patients. Resistance to ICB can largely be attributed to insufficient number and/or function of antitumor CD8+ T cells in the tumor microenvironment. Neoantigen targeted cancer vaccines can activate and expand the antitumor T cell repertoire, but historically, clinical responses have been poor because immunity against peptide antigens is typically weak, resulting in insufficient activation of CD8+ cytotoxic T cells. Herein, we describe a nanoparticle vaccine platform that can overcome these barriers in several ways. First, the vaccine can be reproducibly formulated using a scalable confined impingement jet mixing method to coload a variety of physicochemically diverse peptide antigens and multiple vaccine adjuvants into pH-responsive, vesicular nanoparticles that are monodisperse and less than 100 nm in diameter. Using this approach, we encapsulated synergistically acting adjuvants, cGAMP and monophosphoryl lipid A (MPLA), into the nanocarrier to induce a robust and tailored innate immune response that increased peptide antigen immunogenicity. We found that incorporating both adjuvants into the nanovaccine synergistically enhanced expression of dendritic cell costimulatory markers, pro-inflammatory cytokine secretion, and peptide antigen cross-presentation. Additionally, the nanoparticle delivery increased lymph node accumulation and uptake of peptide antigen by dendritic cells in the draining lymph node. Consequently, nanoparticle codelivery of peptide antigen, cGAMP, and MPLA enhanced the antigen-specific CD8+ T cell response and delayed tumor growth in several mouse models. Finally, the nanoparticle platform improved the efficacy of ICB immunotherapy in a murine colon carcinoma model. This work establishes a versatile nanoparticle vaccine platform for codelivery of peptide neoantigens and synergistic adjuvants to enhance responses to cancer vaccines.

Engineering endosomolytic nanocarriers of diverse morphologies using confined impingement jet mixing.

The clinical translation of many biomolecular therapeutics has been hindered by undesirable pharmacokinetic (PK) properties, inadequate membrane permeability, poor endosomal escape and cytosolic delivery, and/or susceptibility to degradation. Overcoming these challenges merits the development of nanoscale drug carriers (nanocarriers) to improve the delivery of therapeutic cargo. Herein, we implement a flash nanoprecipitation (FNP) approach to produce nanocarriers of diverse vesicular morphologies by using various molecular weight PEG-bl-DEAEMA-co-BMA (PEG-DB) polymers. We demonstrated that FNP can produce uniform (PDI < 0.1) particles after 5 impingements, and that by varying the copolymer hydrophilic mass fraction, FNP enables access to a diverse variety of nanoarchitectures including micelles, unilamellar vesicles (polymersomes), and multi-compartment vesicles (MCVs). We synthesized a library of 2 kDa PEG block copolymers, with DEAEMA-co-BMA second block molecular weights of 3, 6, 12, 15, 20, and 30 kDa. All formulations were both pH responsive, endosomolytic, and capable of loading and cytosolically delivering small negatively charged molecules - albeit to different degrees. Using a B16.F10 melanoma model, we showcased the therapeutic potential of a lead FNP formulated PEG-DB nanocarrier, encapsulating the cyclic dinucleotide (CDN) cGAMP to activate the stimulator of interferon genes (STING) pathway in a therapeutically relevant context. Collectively, these data demonstrate that an FNP process can be used to formulate pH-responsive nanocarriers of diverse morphologies using a PEG-DB polymer system. As FNP is an industrially scalable process, these data address the critical translational challenge of producing PEG-DB nanoparticles at scale. Furthermore, the diverse morphologies produced may specialize in the delivery of distinct biomolecular cargos for other therapeutic applications, implicating the therapeutic potential of this platform in an array of disease applications.

Kinetic Growth of Multicomponent Microcompartment Shells.

An important goal of systems and synthetic biology is to produce high value chemical species in large quantities. Microcompartments, which are protein nanoshells encapsulating catalytic enzyme cargo, could potentially function as tunable nanobioreactors inside and outside cells to generate these high value species. Modifying the morphology of microcompartments through genetic engineering of shell proteins is one viable strategy to tune cofactor and metabolite access to encapsulated enzymes. However, this is a difficult task without understanding how changing interactions between the many different types of shell proteins and enzymes affect microcompartment assembly and shape. Here, we use multiscale molecular dynamics and experimental data to describe assembly pathways available to microcompartments composed of multiple types of shell proteins with varied interactions. As the average interaction between the enzyme cargo and the multiple types of shell proteins is weakened, the shell assembly pathway transitions from (i) nucleating on the enzyme cargo to (ii) nucleating in the bulk and then binding the cargo as it grows to (iii) an empty shell. Atomistic simulations and experiments using the 1,2-propanediol utilization microcompartment system demonstrate that shell protein interactions are highly varied and consistent with our multicomponent, coarse-grained model. Furthermore, our results suggest that intrinsic bending angles control the size of these microcompartments. Overall, our simulations and experiments provide guidance to control microcomparmtent size and assembly by modulating the interactions between shell proteins.

A soil-inspired dynamically responsive chemical system for microbial modulation.

Interactions between the microbiota and their colonized environments mediate critical pathways from biogeochemical cycles to homeostasis in human health. Here we report a soil-inspired chemical system that consists of nanostructured minerals, starch granules and liquid metals. Fabricated via a bottom-up synthesis, the soil-inspired chemical system can enable chemical redistribution and modulation of microbial communities. We characterize the composite, confirming its structural similarity to the soil, with three-dimensional X-ray fluorescence and ptychographic tomography and electron microscopy imaging. We also demonstrate that post-synthetic modifications formed by laser irradiation led to chemical heterogeneities from the atomic to the macroscopic level. The soil-inspired material possesses chemical, optical and mechanical responsiveness to yield write-erase functions in electrical performance. The composite can also enhance microbial culture/biofilm growth and biofuel production in vitro. Finally, we show that the soil-inspired system enriches gut bacteria diversity, rectifies tetracycline-induced gut microbiome dysbiosis and ameliorates dextran sulfate sodium-induced rodent colitis symptoms within in vivo rodent models.

Dual-Responsive Glycopolymers for Intracellular Codelivery of Antigen and Lipophilic Adjuvants.

Traditional approaches to vaccines use whole organisms to trigger an immune response, but they do not typically generate robust cellular-mediated immunity and have various safety risks. Subunit vaccines composed of proteins and/or peptides represent an attractive and safe alternative to whole organism vaccines, but they are poorly immunogenic. Though there are biological reasons for the poor immunogenicity of proteins and peptides, one other key to their relative lack of immunogenicity could be attributed to the poor pharmacokinetic properties of exogenously delivered proteins and peptides. For instance, peptides often aggregate at the site of injection and are not stable in biological fluids, proteins and peptides are rapidly cleared from circulation, and both have poor cellular internalization and endosomal escape. Herein, we developed a delivery system to address the lack of protein immunogenicity by overcoming delivery barriers as well as codelivering immune-stimulating adjuvants. The glycopolymeric nanoparticles (glycoNPs) are composed of a dual-stimuli-responsive block glycopolymer, poly[2-(diisopropylamino)ethyl methacrylate]-b-poly[(pyridyl disulfide ethyl methacrylate)-co-(methacrylamidoglucopyranose)] (p[DPA-b-(PDSMA-co-MAG)]). This polymer facilitates protein conjugation and cytosolic release, the pH-responsive release of lipophilic adjuvants, and pH-dependent membrane disruption to ensure cytosolic delivery of antigens. We synthesized p[DPA-b-(PDSMA-co-MAG)] by reversible addition-fragmentation chain transfer (RAFT) polymerization, followed by the formation and physicochemical characterization of glycoNPs using the p[DPA-b-(PDSMA-co-MAG)] building blocks. These glycoNPs conjugated the model antigen ovalbumin (OVA) and released OVA in response to elevated glutathione levels. Moreover, the glycoNPs displayed pH-dependent drug release of the model hydrophobic drug Nile Red while also exhibiting pH-responsive endosomolytic behavior as indicated by a red blood cell hemolysis assay. GlycoNPs coloaded with OVA and the toll-like receptor 7/8 (TLR-7/8) agonist Resiquimod (R848) activated DC 2.4 dendritic cells (DCs) significantly more than free OVA and R848 and led to robust antigen presentation of the OVA epitope SIINFEKL on major histocompatibility complex I (MHC-I). In sum, the dual-stimuli-responsive glycopolymer introduced here overcomes major protein and peptide delivery barriers and could vastly improve the immunogenicity of protein-based vaccines.

ROS-Responsive Glycopolymeric Nanoparticles for Enhanced Drug Delivery to Macrophages.

Macrophages play a diverse, key role in many pathologies, including inflammatory diseases, cardiovascular diseases, and cancer. However, many therapeutic strategies targeting macrophages suffer from systemic off-target toxicity resulting in notoriously narrow therapeutic windows. To address this shortcoming, the development of poly(propylene sulfide)-b-poly(methacrylamidoglucopyranose) [PPS-b-PMAG] diblock copolymer-based nanoparticles (PMAG NPs) capable of targeting macrophages and releasing drug in the presence of reactive oxygen species (ROS) is reported. PMAG NPs have desirable physicochemical properties for systemic drug delivery, including slightly negative surface charge, ≈100 nm diameter, and hemo-compatibility. Additionally, due to the presence of PPS in the NP core, PMAG NPs release drug cargo preferentially in the presence of ROS. Importantly, PMAG NPs display high cytocompatibility and are taken up by macrophages in cell culture at a rate ≈18-fold higher than PEGMA NPs-NPs composed of PPS-b-poly(oligoethylene glycol methacrylate). Computational studies indicate that PMAG NPs likely bind with glucose transporters such as GLUT 1/3 on the macrophage cell surface to facilitate high levels of internalization. Collectively, this study introduces glycopolymeric NPs that are uniquely capable of both receptor-ligand targeting to macrophages and ROS-dependent drug release and that can be useful in many immunotherapeutic settings.

Electrostatic Control of Shape Selection and Nanoscale Structure in Chiral Molecular Assemblies.

How molecular chirality manifests at the nano- to macroscale has been a scientific puzzle since Louis Pasteur discovered biochirality. Chiral molecules assemble into meso-shapes such as twisted and helical ribbons, helicoidal scrolls (cochleates), or möbius strips (closed twisted ribbons). Here we analyze self-assembly for a series of amphiphiles, C n -K, consisting of an ionizable amino acid [lysine (K)] coupled to alkyl tails with n = 12, 14, or 16 carbons. This simple system allows us to probe the effects of electrostatic and van der Waals interactions in chiral assemblies. Small/wide-angle X-ray scattering (SAXS/WAXS) reveals that at low pH, where the headgroups are ionized (+1), C16-K forms high aspect ratio, planar crystalline bilayers. Molecular dynamics (MD) simulations reveal that tilted tails of the bilayer leaflets are interdigitated. SAXS shows that, with increasing salt concentration, C16-K molecules assemble into cochleates, whereas at elevated pH (reduced degree of ionization), helices are observed for all C n -K assemblies. The shape selection between helices and scrolls is explained by a membrane energetics model. The nano- to meso-scale structure of the chiral assemblies can be continuously controlled by solution ionic conditions. Overall, our study represents a step toward an electrostatics-based approach for shape selection and nanoscale structure control in chiral assemblies.

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology.

Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

Lipid Phase Separation in Vesicles Enhances TRAIL-Mediated Cytotoxicity.

Ligand spatial presentation and density play important roles in signaling pathways mediated by cell receptors and are critical parameters when designing protein-conjugated therapeutic nanoparticles. Here, we harness lipid phase separation to spatially control the protein presentation on lipid vesicles. We use this system to improve the cytotoxicity of TNF-related apoptosis inducing ligand (TRAIL), a therapeutic anticancer protein. Vesicles with phase-separated TRAIL presentation induce more cell death in Jurkat cancer cells than vesicles with uniformly presented TRAIL, and cytotoxicity is dependent on TRAIL density. We assess this relationship in other cancer cell lines and demonstrate that phase-separated vesicles with TRAIL only enhance cytotoxicity through one TRAIL receptor, DR5, while another TRAIL receptor, DR4, is less sensitive to TRAIL density. This work demonstrates a rapid and accessible method to control protein conjugation and density on vesicles that can be adopted to other nanoparticle systems to improve receptor signaling by nanoparticles.

Programming "Atomic Substitution" in Alloy Colloidal Crystals Using DNA.

Although examples of colloidal crystal analogues to metal alloys have been reported, general routes for preparing 3D analogues to random substitutional alloys do not exist. Here, we use the programmability of DNA (length and sequence) to match nanoparticle component sizes, define parent lattice symmetry and substitutional order, and achieve faceted crystal habits. We synthesized substitutional alloy colloidal crystals with either ordered or random arrangements of two components (Au and Fe3O4 nanoparticles) within an otherwise identical parent lattice and crystal habit, confirmed via scanning electron microscopy and small-angle X-ray scattering. Energy dispersive X-ray spectroscopy reveals information regarding composition and local order, while the magnetic properties of Fe3O4 nanoparticles can direct different structural outcomes for different alloys in an applied magnetic field. This work constitutes a platform for independently defining substitution within multicomponent colloidal crystals, a capability that will expand the scope of functional materials that can be realized through programmable assembly.

Acetaminophen Interactions with Phospholipid Vesicles Induced Changes in Morphology and Lipid Dynamics.

Acetaminophen (APAP) or paracetamol, despite its wide and common use for pain and fever symptoms, shows a variety of side effects, toxic effects, and overdose effects. The most common form of toxic effects of APAP is in the liver where phosphatidylcholine is the major component of the cell membrane with additional associated functionalities. Although this is the case, the effects of APAP on pure phospholipid membranes have been largely ignored. Here, we used 1,2-di-(octadecenoyl)-sn-glycero-3-phosphocholine (DOPC), a commonly found phospholipid in mammalian cell membranes, to synthesize large unilamellar vesicles to investigate how the incorporation of APAP changes the pure lipid vesicle structure, morphology, and fluidity at different concentrations. We used a combination of dynamic light scattering, small-angle neutron and X-ray scattering (SANS, SAXS), and cryo-TEM for structural characterization, and neutron spin-echo (NSE) spectroscopy to investigate the dynamics. We showed that the incorporation of APAP in the lipid bilayer significantly impacts the spherical phospholipid self-assembly in terms of its morphology and influences the lipid content in the bilayer, causing a decrease in bending rigidity. We observe a decrease in the number of lipids per vesicle by almost 28% (0.06 wt % APAP) and 19% (0.12 wt % APAP) compared to the pure DOPC (0 wt % APAP). Our results showed that the incorporation of APAP reduces the membrane rigidity by almost 50% and changes the spherical unilamellar vesicles into much more irregularly shaped vesicles. Although the bilayer structure did not show much change when observed by SAXS, NSE and cryo-TEM results showed the lipid dynamics change with the addition of APAP in the bilayer, which causes the overall decreased membrane rigidity. A strong effect on the lipid tail motion showed that the space explored by the lipid tails increases by a factor of 1.45 (for 0.06 wt % APAP) and 1.75 (for 0.12 wt % APAP) compared to DOPC without the drug.

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles.

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

A decellularized oocyte-derived scaffold provides a "sperm safe" to preserve mammalian spermatozoa.

BACKGROUND: Although only one spermatozoon is needed to create a zygote, a significant challenge is the storage and recovery of germ cells when sperm counts are extremely low. OBJECTIVES: We engineered an oocyte-derived biomaterial-the zona pellucida (ZP)-as a "sperm safe" for storing spermatozoon. The ZP is a glycoprotein matrix that surrounds the mammalian oocyte. MATERIALS AND METHODS: We made a hole in the ZPs using a Piezo drill and mechanically separated them from the oocyte cytoplasm. A subset of ZPs were further purified through decellularization. Using a modified ICSI approach, we injected sperm heads into purified ZPs and tested the efficacy of cryopreservation and recovery of spermatozoon as well as function. RESULTS: Between 1-6 sperm heads were injected into purified ZPs (average 2.7 ± 1.7 sperm heads/ZP), which were then cryopreserved. Upon thawing, an average of 2.5 ± 1.4 sperm heads/ZP were observed, and in 11 of 12 thawed "sperm safes," we recovered all spermatozoa. Decellularized "sperm safes" maintained their three-dimensional structure and had a denser matrix relative to untreated controls as assessed by scanning and transmitted electron microscopy. The efficacy of "sperm safe" derived spermatozoon was evaluated by ICSI. Spermatozoon stored in either untreated or decellularized "sperm safes" elicited egg activation-associated calcium transients and zinc sparks when injected into eggs. Of the resulting zygotes, >80% of them formed pronuclei irrespective of the sperm source. 26.8 ± 4.6% and 18.1 ± 7.0% of the pre-implantation embryos generated from spermatozoon recovered from untreated or decellularized "sperm safes" developed to the blastocyst stage, respectively. Although this development was lower than that using fresh spermatozoon (59.3 ± 19.3%) or conventionally frozen-thawed spermatozoon (28.4 ± 1.7%), these differences were not significant. DISCUSSION AND CONCLUSION: Purified ZPs represent a natural biomaterial for the efficient preservation and recovery of small sperm numbers.

Epidermal SR-A Complexes Are Lipid Raft Based and Promote Nucleic Acid Nanoparticle Uptake.

Scavenger receptors clear pathogens, transport lipid, and mediate polyanionic ligand uptake in macrophages, but their expression and role in the skin are poorly understood. Although the epidermal barrier typically excludes nucleic acid entry, topically applied, spherically arranged oligonucleotide nanoconjugates (spherical nucleic acids [SNAs]) penetrate mouse skin, three-dimensional (3D) skin equivalents, and human skin. We explored the mechanism of SNA uptake in normal human epidermal keratinocytes and 3D skin equivalents. Normal human epidermal keratinocytes and 3D raft treatment with SR-A inhibitors reduced SNA uptake by >80%. The human epidermis expresses SR-As SCARA3 and, to a lesser extent, MARCO. Simultaneous lentiviral knockdown of SCARA3 and MARCO reduced SNA uptake in normal human epidermal keratinocytes and 3D rafts after topical application, affirming a role for SR-As in SNA uptake and 3D raft penetration. Incubation of normal human epidermal keratinocytes at 4oC or with sodium azide prevented SNA uptake, suggesting active endocytosis. Endocytosis inhibitors, immunofluorescence, immunoprecipitation, and knockdown studies localized functional SR-As to FLOT-1-containing lipid rafts throughout the epidermis and CAV-1-containing rafts only in the upper epidermis. These studies suggest a central role for SR-A complexes in epidermal lipid rafts in mediating the uptake of nucleic acid‒laden nanoparticles.

Making the Most of your Electrons: Challenges and Opportunities in Characterizing Hybrid Interfaces with STEM.

Inspired by the unique architectures composed of hard and soft materials in natural and biological systems, synthetic hybrid structures and associated soft-hard interfaces have recently evoked significant interest. Soft matter is typically dominated by fluctuations even at room temperature, while hard matter (which often serves as the substrate or anchor for the soft component) is governed by rigid mechanical behavior. This dichotomy offers considerable opportunities to leverage the disparate properties offered by these components across a wide spectrum spanning from basic science to engineering insights with significant technological overtones. Such hybrid structures, which include polymer nanocomposites, DNA functionalized nanoparticle superlattices and metal organic frameworks to name a few, have delivered promising insights into the areas of catalysis, environmental remediation, optoelectronics, medicine, and beyond. The interfacial structure between these hard and soft phases exists across a variety of length scales and often strongly influence the functionality of hybrid systems. While scanning/transmission electron microscopy (S/TEM) has proven to be a valuable tool for acquiring intricate molecular and nanoscale details of these interfaces, the unusual nature of hybrid composites presents a suite of challenges that make assessing or establishing the classical structure-property relationships especially difficult. These include challenges associated with preparing electron-transparent samples and obtaining sufficient contrast to resolve the interface between dissimilar materials given the dose sensitivity of soft materials. We discuss each of these challenges and supplement a review of recent developments in the field with additional experimental investigations and simulations to present solutions for attaining a nano or atomic-level understanding of these interfaces. These solutions present a host of opportunities for investigating and understanding the role interfaces play in this unique class of functional materials.

Colloidal crystal engineering with metal-organic framework nanoparticles and DNA.

Colloidal crystal engineering with nucleic acid-modified nanoparticles is a powerful way for preparing 3D superlattices, which may be useful in many areas, including catalysis, sensing, and photonics. To date, the building blocks studied have been primarily based upon metals, metal oxides, chalcogenide semiconductors, and proteins. Here, we show that metal-organic framework nanoparticles (MOF NPs) densely functionalized with oligonucleotides can be programmed to crystallize into a diverse set of superlattices with well-defined crystal symmetries and compositions. Electron microscopy and small-angle X-ray scattering characterization confirm the formation of single-component MOF superlattices, binary MOF-Au single crystals, and two-dimensional MOF nanorod assemblies. Importantly, DNA-modified porphyrinic MOF nanorods (PCN-222) were assembled into 2D superlattices and found to be catalytically active for the photooxidation of 2-chloroethyl ethyl sulfide (CEES, a chemical warfare simulant of mustard gas). Taken together, these new materials and methods provide access to colloidal crystals that incorporate particles with the well-established designer properties of MOFs and, therefore, increase the scope of possibilities for colloidal crystal engineering with DNA.

Apparent size and morphology of bacterial microcompartments varies with technique.

Bacterial microcompartments (MCPs) are protein-based organelles that encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.

Colloidal Crystal "Alloys".

Colloidal crystal engineering with DNA has emerged as a powerful tool for precisely controlling the arrangement of nanoscale building blocks in three-dimensional superlattices, where nanoparticles densely modified with DNA can be viewed as "programmable atom equivalents" (PAEs). Although a wide variety of complementary DNA-modified nanoparticles, differentiated by size, shape, and composition, have been assembled into many "ionic" phases, the predictable formation of "alloy" phases remains elusive. Here, we describe the design of "colloidal crystal alloys" by combining gold PAEs of two different sizes (core diameters ranging from 5 to 40 nm) with complementary DNA-modified 2 nm gold nanoparticles (∼15 DNA strands/particle) that act as electron equivalents (EEs). Electron microscopy and small-angle X-ray scattering (SAXS) experiments reveal the formation of four classes of colloidal alloy equivalents: interstitial, substitutional, phase-separated, and intermetallic alloys. In these colloidal alloy phases, PAEs occupy lattice positions, while EEs stabilize the PAE lattice but do not occupy specific lattice sites. A set of chemical design guidelines emerge from this study, analogous to that of the Hume-Rothery rules, allowing for programmed synthesis of different alloy phases depending on PAE particle size ratio, DNA surface coverage, stoichiometric ratio, and thermal annealing pathways. Furthermore, we study the phase separation process via in situ SAXS experiments as well as ex situ electron microscopy, revealing the critical role of kinetics on the phase behavior in these systems.

The role of trace Ag in the synthesis of Au nanorods.

One of the more useful syntheses of single crystalline, uniform Au nanorods from Au spherical seeds relies on the addition of trace Ag ions, yet the role that Ag+ plays has remained both elusive and controversial, due in part to lack of knowledge of how the Ag distribution in the nanorod evolves over time. In this work, we fill in this knowledge gap by correlating the spatial distribution of Ag within Au nanorods with nanorod anisotropic growth through time-course X-ray absorption spectroscopy (XAFS)-derived atomic-level elemental coordination paired with electron microscopy for nanoscale morphological analysis. Using this method, a plausible pathway for the conversion of spherical seeds into Au nanorods is proposed. Evidence shows that the nanorod anisotropic growth is directly related to the Ag surface coverage. Anisotropy is induced early in the reaction when Ag first deposits onto the nanoparticle surface, but growth occurs more isotropically as the reaction progresses and Ag diffuses into the nanorod bulk. The results of this investigation and methods employed should be extendable to many anisotropic nanoparticle syntheses that make use of trace elemental species as shape-control additives.