Versatile Capillary Cells for Handling Concentrated Samples in Analytical Ultracentrifugation.

In concentrated macromolecular dispersions, far-from-ideal intermolecular interactions determine the dispersion behaviors including phase transition, crystallization, and liquid-liquid phase separation. Here, we present a novel versatile capillary-cell design for analytical ultracentrifugation-sedimentation equilibrium (AUC-SE), ideal for studying samples at high concentrations. Current setups for such studies are difficult and unreliable to handle, leading to a low experimental success rate. The design presented here is easy to use, robust, and reusable for samples in both aqueous and organic solvents while requiring no special tools or chemical modification of AUC cells. The key and unique feature is the fabrication of liquid reservoirs directly on the bottom window of AUC cells, which can be easily realized by laser ablation or mechanical drilling. The channel length and optical path length are therefore tunable. The success rate for assembling this new cell is close to 100%. We demonstrate the practicality of this cell by studying: (1) the equation of state and second virial coefficients of concentrated gold nanoparticle dispersions in water and bovine serum albumin (BSA) as well as lysozyme solution in aqueous buffers, (2) the gelation phase transition of DNA and BSA solutions, and (3) liquid-liquid phase separation of concentrated BSA/polyethylene glycol (PEG) droplets.

A Synthetic Multivalent Lipopeptide Derived from Pam3CSK4 with Irreversible Influenza Inhibition and Immuno-Stimulating Effects.

The activation of the host adaptive immune system is crucial for eliminating viruses. However, influenza infection often suppresses the innate immune response that precedes adaptive immunity, and the adaptive immune responses are typically delayed. Dendritic cells, serving as professional antigen-presenting cells, have a vital role in initiating the adaptive immune response. In this study, an immuno-stimulating antiviral system (ISAS) is introduced, which is composed of the immuno-stimulating adjuvant lipopeptide Pam3CSK4 that acts as a scaffold onto which it is covalently bound 3 to 4 influenza-inhibiting peptides. The multivalent display of peptides on the scaffold leads to a potent inhibition against H1N1 (EC50  = 20 nM). Importantly, the resulting lipopeptide, Pam3FDA, shows an irreversible inhibition mechanism. The chemical modification of peptides on the scaffold maintains Pam3CSK4's ability to stimulate dendritic cell maturation, thereby rendering Pam3FDA a unique antiviral. This is attributed to its immune activation capability, which also acts in synergy to expedite viral elimination.

Cholesterol-Containing Liposomes Decorated With Au Nanoparticles as Minimal Tunable Fusion Machinery.

Membrane fusion is essential for the basal functionality of eukaryotic cells. In physiological conditions, fusion events are regulated by a wide range of specialized proteins, operating with finely tuned local lipid composition and ionic environment. Fusogenic proteins, assisted by membrane cholesterol and calcium ions, provide the mechanical energy necessary to achieve vesicle fusion in neuromediator release. Similar cooperative effects must be explored when considering synthetic approaches for controlled membrane fusion. We show that liposomes decorated with amphiphilic Au nanoparticles (AuLips) can act as minimal tunable fusion machinery. AuLips fusion is triggered by divalent ions, while the number of fusion events dramatically changes with, and can be finely tuned by, the liposome cholesterol content. We combine quartz-crystal-microbalance with dissipation monitoring (QCM-D), fluorescence assays, and small-angle X-ray scattering (SAXS) with molecular dynamics (MD) at coarse-grained (CG) resolution, revealing new mechanistic details on the fusogenic activity of amphiphilic Au nanoparticles (AuNPs) and demonstrating the ability of these synthetic nanomaterials to induce fusion regardless of the divalent ion used (Ca2+ or Mg2+ ). The results provide a novel contribution to developing new artificial fusogenic agents for next-generation biomedical applications that require tight control of the rate of fusion events (e.g., targeted drug delivery).

Simultaneous Electrophysiology and Imaging Reveal Changes in Lipid Membrane Thickness and Tension upon Uptake of Amphiphilic Gold Nanoparticles.

Amphiphilic gold core nanoparticles (AmNPs) striped with hydrophilic 11-mercapto-1-undecanesulfonate (MUS) and hydrophobic 1-octanethiol (OT) ligands are promising candidates for drug carriers that passively and nondisruptively enter cells. Yet, how they interact with cellular membranes is still only partially understood. Herein, we use electrophysiology and imaging to carefully assess changes in droplet interface bilayer lipid membranes (DIBs) incurred by striped AmNPs added via microinjection. We find that AmNPs spontaneously reduce the steady-state specific capacitance and contact angle of phosphatidylcholine DIBs by amounts dependent on the final NP concentration. These reductions, which are greater for NPs with a higher % OT ligands and membranes containing unsaturated lipids but negligible for MUS-only-coated NPs, reveal that AmNPs passively embed in the interior of the bilayer where they increase membrane thickness and lateral tension through disruption of lipid packing. These results demonstrate the enhanced evaluation of nano-bio interactions possible via electrophysiology and imaging of DIBs.

Calcium-triggered fusion of lipid membranes is enabled by amphiphilic nanoparticles.

Lipid membrane fusion is an essential process for a number of critical biological functions. The overall process is thermodynamically favorable but faces multiple kinetic barriers along the way. Inspired by nature's engineered proteins such as SNAP receptor [soluble N-ethylmale-imide-sensitive factor-attachment protein receptor (SNARE)] complexes or viral fusogenic proteins that actively promote the development of membrane proximity, nucleation of a stalk, and triggered expansion of the fusion pore, here we introduce a synthetic fusogen that can modulate membrane fusion and equivalently prime lipid membranes for calcium-triggered fusion. Our fusogen consists of a gold nanoparticle functionalized with an amphiphilic monolayer of alkanethiol ligands that had previously been shown to fuse with lipid bilayers. While previous efforts to develop synthetic fusogens have only replicated the initial steps of the fusion cascade, we use molecular simulations and complementary experimental techniques to demonstrate that these nanoparticles can induce the formation of a lipid stalk and also drive its expansion into a fusion pore upon the addition of excess calcium. These results have important implications in general understanding of stimuli-triggered fusion and the development of synthetic fusogens for biomedical applications.

Unraveling the complexity of amyloid polymorphism using gold nanoparticles and cryo-EM.

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer's disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

Polyanionic Amphiphilic Dendritic Polyglycerols as Broad-Spectrum Viral Inhibitors with a Virucidal Mechanism.

Heparin has been known to be a broad-spectrum inhibitor of viral infection for almost 70 years, and it has been used as a medication for almost 90 years due to its anticoagulant effect. This nontoxic biocompatible polymer efficiently binds to many types of viruses and prevents their attachment to cell membranes. However, the anticoagulant properties are limiting their use as an antiviral drug. Many heparin-like compounds have been developed throughout the years; however, the reversible nature of the virus inhibition mechanism has prevented their translation to the clinics. In vivo, such a mechanism requires the unrealistic maintenance of the concentration above the binding constant. Recently, we have shown that the addition of long hydrophobic linkers to heparin-like compounds renders the interaction irreversible while maintaining the low-toxicity and broad-spectrum activity. To date, such hydrophobic linkers have been used to create heparin-like gold nanoparticles and ß-cyclodextrins. The former achieves a nanomolar inhibition concentration on a non-biodegradable scaffold. The latter, on a fully biodegradable scaffold, shows only a micromolar inhibition concentration. Here, we report that the addition of hydrophobic linkers to a new type of multifunctional scaffold (dendritic polyglycerol, dPG) creates biocompatible compounds endowed with nanomolar activity. Furthermore, we present an in-depth analysis of the molecular design rules needed to achieve irreversible virus inhibition. The most active compound (dPG-5) showed nanomolar activity against herpes simplex virus 2 (HSV-2) and respiratory syncytial virus (RSV), giving a proof-of-principle for broad-spectrum while keeping low-toxicity. In addition, we demonstrate that the virucidal activity leads to the release of viral DNA upon the interaction between the virus and our polyanionic dendritic polymers. We believe that this paper will be a stepping stone toward the design of a new class of irreversible nontoxic broad-spectrum antivirals.

Determination and evaluation of the nonadditivity in wetting of molecularly heterogeneous surfaces.

The interface between water and folded proteins is very complex. Proteins have "patchy" solvent-accessible areas composed of domains of varying hydrophobicity. The textbook understanding is that these domains contribute additively to interfacial properties (Cassie's equation, CE). An ever-growing number of modeling papers question the validity of CE at molecular length scales, but there is no conclusive experiment to support this and no proposed new theoretical framework. Here, we study the wetting of model compounds with patchy surfaces differing solely in patchiness but not in composition. Were CE to be correct, these materials would have had the same solid-liquid work of adhesion (WSL ) and time-averaged structure of interfacial water. We find considerable differences in WSL , and sum-frequency generation measurements of the interfacial water structure show distinctively different spectral features. Molecular-dynamics simulations of water on patchy surfaces capture the observed behaviors and point toward significant nonadditivity in water density and average orientation. They show that a description of the molecular arrangement on the surface is needed to predict its wetting properties. We propose a predictive model that considers, for every molecule, the contributions of its first-nearest neighbors as a descriptor to determine the wetting properties of the surface. The model is validated by measurements of WSL in multiple solvents, where large differences are observed for solvents whose effective diameter is smaller than ∼6 Å. The experiments and theoretical model proposed here provide a starting point to develop a comprehensive understanding of complex biological interfaces as well as for the engineering of synthetic ones.

Chemical sensing with Au and Ag nanoparticles.

Noble metal nanoparticles (NPs) are ideal scaffolds for the fabrication of sensing devices because of their high surface-to-volume ratio combined with their unique optical and electrical properties which are extremely sensitive to changes in the environment. Such characteristics guarantee high sensitivity in sensing processes. Metal NPs can be decorated with ad hoc molecular building blocks which can act as receptors of specific analytes. By pursuing this strategy, and by taking full advantage of the specificity of supramolecular recognition events, highly selective sensing devices can be fabricated. Besides, noble metal NPs can also be a pivotal element for the fabrication of chemical nose/tongue sensors to target complex mixtures of analytes. This review highlights the most enlightening strategies developed during the last decade, towards the fabrication of chemical sensors with either optical or electrical readout combining high sensitivity and selectivity, along with fast response and full reversibility, with special attention to approaches that enable efficient environmental and health monitoring.

Quantifying the Solution Structure of Metal Nanoclusters Using Small-Angle Neutron Scattering.

Metal nanoclusters are a unique class of synthetic material, as their crystal structures can be resolved using X-ray diffraction, and their chemical formula can be precisely determinated from mass spectroscopy. However, a complete structure characterization by these two techniques is often a challenging task. Here, we utilize small-angle neutron scattering (SANS) to directly quantify the key structure parameters of a series of silver and gold nanoclusters in solution. The results not only correlate well to their crystallographic structures, but also allow the quantification of the counterions layer surrounding charged nanoclusters in solution. Furthermore, when combining with X-ray scattering, it is possible to estimate the molecular weight of both the metal core and the ligand shell of nanoclusters. This work offers an alternative characterization tool for nanoclusters without the requirement of crystallization or gas phase ionization.

Sulfonated Nanomaterials with Broad-Spectrum Antiviral Activity Extending beyond Heparan Sulfate-Dependent Viruses.

Viral infections are among the main causes of death worldwide, and we lack antivirals for the majority of viruses. Heparin-like sulfated or sulfonated compounds have been known for decades for their ability to prevent infection by heparan sulfate proteoglycan (HSPG)-dependent viruses but only in a reversible way. We have previously shown that gold nanoparticles and ß-cyclodextrins coated with mercapto-undecane sulfonic acid (MUS) inhibit HSPG-dependent viruses irreversibly while retaining the low-toxicity profile of most heparin-like compounds. In this work, we show that, in stark contrast to heparin, these compounds also inhibit different strains of influenza virus and vesicular stomatitis virus (VSV), which do not bind HSPG. The antiviral action is virucidal and irreversible for influenza A virus (H1N1), while for VSV, there is a reversible inhibition of viral attachment to the cell. These results further broaden the spectrum of activity of MUS-coated gold nanoparticles and ß-cyclodextrins.

The Clustering of mApoE Anti-Amyloidogenic Peptide on Nanoparticle Surface Does Not Alter Its Performance in Controlling Beta-Amyloid Aggregation.

The deposition of amyloid-ß (Aß) plaques in the brain is a significant pathological signature of Alzheimer's disease, correlating with synaptic dysfunction and neurodegeneration. Several compounds, peptides, or drugs have been designed to redirect or stop Aß aggregation. Among them, the trideca-peptide CWG-LRKLRKRLLR (mApoE), which is derived from the receptor binding sequence of apolipoprotein E, is effectively able to inhibit Aß aggregation and to promote fibril disaggregation. Taking advantage of Atomic Force Microscopy (AFM) imaging and fluorescence techniques, we investigate if the clustering of mApoE on gold nanoparticles (AuNP) surface may affect its performance in controlling Aß aggregation/disaggregation processes. The results showed that the ability of free mApoE to destroy preformed Aß fibrils or to hinder the Aß aggregation process is preserved after its clustering on AuNP. This allows the possibility to design multifunctional drug delivery systems with clustering of anti-amyloidogenic molecules on any NP surface without affecting their performance in controlling Aß aggregation processes.

Stable Ultraconcentrated and Ultradilute Colloids of CsPbX3 (X = Cl, Br) Nanocrystals Using Natural Lecithin as a Capping Ligand.

Attaining thermodynamic stability of colloids in a broad range of concentrations has long been a major thrust in the field of colloidal ligand-capped semiconductor nanocrystals (NCs). This challenge is particularly pressing for the novel NCs of cesium lead halide perovskites (CsPbX3; X = Cl, Br) owing to their highly dynamic and labile surfaces. Herein, we demonstrate that soy lecithin, a mass-produced natural phospholipid, serves as a tightly binding surface-capping ligand suited for a high-reaction yield synthesis of CsPbX3 NCs (6-10 nm) and allowing for long-term retention of the colloidal and structural integrity of CsPbX3 NCs in a broad range of concentrations-from a few ng/mL to >400 mg/mL (inorganic core mass). The high colloidal stability achieved with this long-chain zwitterionic ligand can be rationalized with the Alexander-De Gennes model that considers the increased particle-particle repulsion due to branched chains and ligand polydispersity. The versatility and immense practical utility of such colloids is showcased by the single NC spectroscopy on ultradilute samples and, conversely, by obtaining micrometer-thick, optically homogeneous dense NC films in a single spin-coating step from ultraconcentrated colloids.

Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism.

Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism.  These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.

An Atomistic Look into Bio-inspired Nanoparticles and their Molecular Interactions with Cells.

Nanoparticles (NPs) have sizes that approach those of pathogens and they can interact with the membranes of eukaryotic cells in an analogous fashion. Typically, NPs are taken up by the cell via the plasma membrane by receptor-mediated processes and subsequently interact with various endomembranes. Unlike pathogens, however, NPs lack the remarkable specificity gained during the evolutionary process and their design and optimization remains an expensive and time-consuming undertaking, especially considering the limited information available on their molecular interactions with cells. In this context, molecular dynamics (MD) simulations have emered as a promising strategy to investigate the mechanistic details of the interaction of NPs with mammalian or viral membranes. In particular, MD simulations have been extensively used to study the uptake process of NPs into the cell, focusing on membrane vesiculation, endocytic routes, or passive permeation processes. While such work is certainly relevant for understanding NP-cell interactions, it remains very difficult to determine the correspondence between generic models and the actual NP. Here, we review how chemically-specific MD simulations can provide rational guidelines towards further bio-inspired NP optimization.

Amorphous CaCO3: Influence of the Formation Time on Its Degree of Hydration and Stability.

Calcium carbonate (CaCO3) is one of the most abundant biominerals that is prevalent in rocks and often used as a structural material in marine animals. Many of these natural CaCO3-based materials display excellent mechanical properties that are difficult to reproduce by man-made counterparts. This difficulty arises from the incomplete understanding of the influence of processing conditions on the structure and composition of CaCO3. To gain a better understanding of the evolution of the structure and composition of amorphous CaCO3 (ACC) particles during early stages, we introduce a new, organic solvent-free method that quenches this process with a high temporal resolution. We produce ACC particles inside small airborne drops that are formed with a microfluidic spray-dryer. These drops dry within 100 ms to 10 s and thereby arrest the formation of CaCO3 particles on that time scale. Using the microfluidic spray-dryer, we demonstrate that the amount of mobile water contained in ACC particles increases with increasing formation time and hence with increasing particle size. As a result of the higher concentration of mobile water, larger particles are less stable against temperature-induced solid-state crystallization and electron beam-induced decomposition than smaller counterparts. The amount of mobile water contained in ACC can be substantially reduced, and hence their kinetic stability against solid-state transformations increased, if certain organic additives, such as poly(acrylic acid) (PAA), are incorporated. These insights might open up new opportunities to fabricate biomimetic CaCO3-based materials with tunable structures and hence with properties that can be adapted to the needs of specific applications.

Cyclodextrin Modulated Type I Collagen Self-Assembly to Engineer Biomimetic Cornea Implants.

Collagen-rich tissues in the cornea exhibit unique and highly organized extracellular matrix ultrastructures, which contribute to its high load-bearing capacity and light transmittance. Corneal collagen fibrils are controlled during development by small leucine-rich proteoglycans (SLRPs) that regulate the fibril diameter and spacing in order to achieve the unique optical transparency. Cyclodextrins (CDs) of varying size and chemical functionality for their ability to regulate collagen assembly during vitrification process are screened in order to create biosynthetic materials that mimic the native cornea structure. Addition of ßCD to collagen vitrigels produces materials with aligned fibers and lamellae similar to native cornea, resulting in mechanically robust and transparent materials. Biochemistry analysis revealed that CD interacts with hydrophobic amino acids in collagen to influence assembly and fibril organization. To translate the self-assembled collagen materials for cornea reconstruction, custom molds for gelation and vitrification are engineered to create ßCD/Col implants with curvature matching that of the cornea. Acellular ßCD/Col materials are implanted in a rabbit partial keratoplasty model with interrupted sutures. The implants demonstrate tissue integration and support re-epithelialization. Therefore, the addition of CD molecules regulates collagen self-assembly and provides a simple process to engineer corneal mimetic substitutes with advanced structural and functional properties.

Structure-Property Relationships of Amphiphilic Nanoparticles That Penetrate or Fuse Lipid Membranes.

The development of synthetic nanomaterials that could embed within, penetrate, or induce fusion between membranes without permanent disruption would have great significance for biomedical applications. Here we describe structure-function relationships of highly water-soluble gold nanoparticles comprised of an ∼1.5-5 nm diameter metal core coated by an amphiphilic organic ligand shell, which exhibit membrane embedding and fusion activity mediated by the surface ligands. Using an environment-sensitive dye anchored within the ligand shell as a sensor of membrane embedding, we demonstrate that particles with core sizes of ∼2-3 nm are capable of embedding within and penetrating fluid bilayers. At the nanoscale, these particles also promote spontaneous fusion of liposomes or spontaneously embed within intact liposomal vesicles. These studies provide nanoparticle design and selection principles that could be used in drug delivery applications, as membrane stains, or for the creation of novel organic/inorganic nanomaterial self-assemblies.

Characterization of Ligand Shell for Mixed-Ligand Coated Gold Nanoparticles.

Gold nanoparticles owe a large number of their properties to their ligand shell. Indeed, many researchers routinely use mixtures of ligand molecules for their nanoparticles to impart complex property sets. It has been shown that the morphology of ligand shells (e.g., Janus, random, stripelike) leads to specific properties. Examples include wettability, solubility, protein nonspecific adsorption, cell penetration, catalysis, and cation-capturing abilities. Yet, it remains a great challenge to evaluate such morphologies in even the most fundamental terms such as dimension and shape. In this Account, we review recent progress in characterization techniques applicable to gold nanoparticles with ligand shells composed of mixed ligands. We divide the characterization into three major categories, namely, microscopy, spectroscopy, and simulation. In microscopy, we review progresses in scanning tunneling microscopy (STM), atomic force microscopy (AFM), and scanning/transmission electron microscopy. In spectroscopy, we mainly highlight recent achievements in nuclear magnetic resonance (NMR), mass spectrometry (MS), small angle neutron scattering (SANS), electron spin resonance (EPR), and adsorption based spectroscopies. In simulation, we point out the latest results in understanding thermodynamic stability of ligand shell morphology and emphasize the role of computer simulation for helping interpretation of experimental data. We conclude with a perspective of future development.

Novel Sensing Strategies Based on Monolayer Protected Gold Nanoparticles for the Detection of Metal Ions and Small Molecules.

Monolayer protected gold nanoparticles (AuNPs) have a huge potential for the development of innovative sensing systems for the detection of metal ions and small molecules. The organic ligand shell, primarily utilized to stabilize the gold core, can be rationally designed to promote selective interactions with a desired analyte. In addition, the outstanding physical and optical properties of AuNPs can be exploited to obtain analytically useful signals upon analyte binding. In this account, we review recent advances in AuNP-based sensing systems emphasizing on the rational design of the ligand shell for detection of heavy metal ions and small molecules. We examine various strategies of detection by evaluating their improvements in terms of sensitivity, selectivity and applicability to real samples.