Amino Acids and Their Biological Derivatives Modulate Protein-Protein Interactions in an Additive Way.

Protein-protein interactions (PPIs) differ when measured in test tubes and cells due to the complexity of the intracellular environment. Free amino acids (AAs) and their derivatives constitute a significant fraction of the intracellular volume and mass. Recently, we have found that AAs have a generic property of rendering protein dispersions more stable by reducing the net attractive part of PPIs. Here, we study the effects on PPIs of different AA derivatives, AA mixtures, and short peptides. We find that all the tested AA derivatives modulate PPIs in solution as effectively as AAs. Furthermore, we show that the modulation effect is additive when AAs form mixtures or are bound into short peptides. Therefore, this study demonstrates the additive effects of a class of small molecules (i.e., AAs and their biological derivatives) on PPIs and provides insights into rationally designing biocompatible molecules for stabilizing protein interactions and consequently tuning protein functions.

Modulating Weak Protein-Protein Cross-Interactions by the Addition of Free Amino Acids at Millimolar Concentrations.

In this paper, we quantify weak protein-protein interactions in solution using cross-interaction chromatography (CIC) and surface plasmon resonance (SPR) and demonstrate that they can be modulated by the addition of millimolar concentrations of free amino acids. With CIC, we determined the second osmotic virial cross-interaction coefficient (B23) as a proxy for the interaction strength between two different proteins. We perform SPR experiments to establish the binding affinity between the same proteins. With CIC, we show that the amino acids proline, glutamine, and arginine render the protein cross-interactions more repulsive or equivalently less attractive. Specifically, we measured B23 between lysozyme (Lys) and bovine serum albumin (BSA) and between Lys and protein isolates (whey and canola). We find that B23 increases when amino acids are added to the solution even at millimolar concentrations, corresponding to protein/ligand stoichiometric ratios as low as 1:1. With SPR, we show that the binding affinity between proteins can change by 1 order of magnitude when 10 mM glutamine is added. In the case of Lys and one whey protein isolate (WPI), it changes from the mM to the M range, thus by 3 orders of magnitude. Interestingly, this efficient modulation of the protein cross-interactions does not alter the protein's secondary structure. The capacity of amino acids to modulate protein cross-interactions at mM concentrations is remarkable and may have an impact across fields in particular for specific applications in the food or pharmaceutical industries.

Broad-Spectrum Supramolecularly Reloadable Antimicrobial Coatings.

Antimicrobial surfaces limit the spread of infectious diseases. To date, there is no antimicrobial coating that has widespread use because of short-lived and limited spectrum efficacy, poor resistance to organic material, and/or cost. Here, we present a paint based on waterborne latex particles that is supramolecularly associated with quaternary ammonium compounds (QACs). The optimal supramolecular pairing was first determined by immobilizing selected ions on self-assembled monolayers exposing different groups. The QAC surface loading density was then increased by using polymer brushes. These concepts were adopted to develop inexpensive paints to be applied on many different surfaces. The paint could be employed for healthcare and food production applications. Its slow release of QAC allows for long-lasting antimicrobial action, even in the presence of organic material. Its efficacy lasts for more than 90 washes, and importantly, once lost, it can readily be restored by spraying an aqueous solution of the QAC. We mainly tested cetyltrimethylammonium as QAC as it is already used in consumer care products. Our antimicrobial paint is broad spectrum as it showed excellent antimicrobial efficiency against four bacteria and four viruses.

Benzene with Alkyl Chains Is a Universal Scaffold for Multivalent Virucidal Antivirals.

Most viruses start their invasion by binding to glycoproteins' moieties on the cell surface (heparan sulfate proteoglycans [HSPG] or sialic acid [SA]). Antivirals mimicking these moieties multivalently are known as broad-spectrum multivalent entry inhibitors (MEI). Due to their reversible mechanism, efficacy is lost when concentrations fall below an inhibitory threshold. To overcome this limitation, we modify MEIs with hydrophobic arms rendering the inhibitory mechanism irreversible, i.e., preventing the efficacy loss upon dilution. However, all our HSPG-mimicking MEIs only showed reversible inhibition against HSPG-binding SARS-CoV-2. Here, we present a systematic investigation of a series of small molecules, all containing a core and multiple hydrophobic arms terminated with HSPG-mimicking moieties. We identify the ones that have irreversible inhibition against all viruses including SARS-CoV-2 and discuss their design principles. We show efficacy in vivo against SARS-CoV-2 in a Syrian hamster model through both intranasal instillation and aerosol inhalation in a therapeutic setting (12 h postinfection). We also show the utility of the presented design rules in producing SA-mimicking MEIs with irreversible inhibition against SA-binding influenza viruses.

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.

Beyond the limiting gap length: peripheral nerve regeneration through implantable nerve guidance conduits.

Peripheral nerve damage results in the loss of sensorimotor and autonomic functions, which is a significant burden to patients. Furthermore, nerve injuries greater than the limiting gap length require surgical repair. Although autografts are the preferred clinical choice, their usage is impeded by their limited availability, dimensional mismatch, and the sacrifice of another functional donor nerve. Accordingly, nerve guidance conduits, which are tubular scaffolds engineered to provide a biomimetic environment for nerve regeneration, have emerged as alternatives to autografts. Consequently, a few nerve guidance conduits have received clinical approval for the repair of short-mid nerve gaps but failed to regenerate limiting gap damage, which represents the bottleneck of this technology. Thus, it is still necessary to optimize the morphology and constituent materials of conduits. This review summarizes the recent advances in nerve conduit technology. Several manufacturing techniques and conduit designs are discussed, with emphasis on the structural improvement of simple hollow tubes, additive manufacturing techniques, and decellularized grafts. The main objective of this review is to provide a critical overview of nerve guidance conduit technology to support regeneration in long nerve defects, promote future developments, and speed up its clinical translation as a reliable alternative to autografts.

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.

Ligand concentration determines antiviral efficacy of silica multivalent nanoparticles.

We have learned from the recent COVID-19 pandemic that the emergence of a new virus can quickly become a global health burden and kill millions of lives. Antiviral drugs are essential in our fight against viral diseases, but most of them are virus-specific and are prone to viral mutations. We have developed broad-spectrum antivirals based on multivalent nanoparticles grafted with ligands that mimic the target of viral attachment ligands (VALs). We have shown that when the ligand has a sufficiently long hydrophobic tail, the inhibition mechanism switches from reversible (virustatic) to irreversible (virucidal). Here, we investigate further how ligand density and particle size affect antiviral efficacy, both in terms of half-inhibitory concentration (IC50) and of reversible vs irreversible mechanism. We designed antiviral silica nanoparticles modified with 11-mercaptoundecane-1-sulfonic acid (MUS), a ligand that mimics heparan sulfate proteoglycans (HSPG) and we showed that these nanoparticles can be synthesized with different sizes (4-200 nm) and ligand grafting densities (0.59-10.70 /nm2). By testing these particles against herpes simplex virus type 2 (HSV-2), we show that within the size and density ranges studied, the antiviral IC50 is determined solely by equivalent ligand concentration. The nanoparticles are found to be virucidal at all sizes and densities studied.

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.

Reversible Microscale Assembly of Nanoparticles Driven by the Phase Transition of a Thermotropic Liquid Crystal.

The arrangement of nanoscale building blocks into patterns with microscale periodicity is challenging to achieve via self-assembly processes. Here, we report on the phase-transition-driven collective assembly of gold nanoparticles in a thermotropic liquid crystal. A temperature-induced transition from the isotropic to the nematic phase under anchoring-driven planar alignment leads to the assembly of individual nanometer-sized particles into arrays of micrometer-sized agglomerates, whose size and characteristic spacing can be tuned by varying the cooling rate. Phase field simulations coupling the conserved and nonconserved order parameters exhibit a similar evolution of the morphology as the experimental observations. This fully reversible process offers control over structural order on the microscopic level and is an interesting model system for the programmable and reconfigurable patterning of nanocomposites with access to micrometer-sized periodicities.

Recycling of Polymerase Chain Reaction (PCR) Kits.

Polymerase chain reaction (PCR) kits have been used as common diagnosing tools during the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, with daily worldwide usage in the millions. It is well known that at the beginning of the pandemic, there was a shortage of PCR kits. So far, the ecosystem of a PCR kit is linear use; that is, kits are produced, used once, and disposed of as biolab waste. Here, we show that to mitigate the risk of future shortages, it is possible to envision recyclable PCR kits based on a more sustainable use of nucleic acid resources. A PCR kit is mainly composed of primers, nucleotides, and enzymes. In the case of a positive test, the free nucleotides are polymerized onto the primers to form longer DNA strands. Our approach depolymerizes such strands, keeping the primers and regenerating the nucleotides, i.e., returning the nucleic acid materials to the original state. The polymerized long DNA strands are hydrolyzed into nucleotide monophosphates that are then phosphorylated into triphosphates using a method that is developed from a recent publication. We used oligonucleotides with a 3'-terminal phosphorothioate (PS) backbone modification as nonhydrolyzable PCR primers, which are able to undergo the recycling process unchanged. The nuclease resistance of oligonucleotides with a ribose sugar modification was also evaluated, which showed worse recycling efficiency than PS-modified oligonucleotides. We successfully recycled both PCR primers and nucleotide monomers (∼75% yield). We demonstrate that the method allows for the direct reuse of PCR kits. We also show that the recycled primers can be isolated and then added to endpoint or quantitative PCR. This recycling approach provides a new path for circularly reusing nucleic acid materials in PCR kits.

Development of an inhalable antiviral powder formulation against respiratory syncytial virus.

Respiratory viruses including the respiratory syncytial virus (RSV) aggravate the global burden of virus-inflicted morbidity and mortality. Entry inhibitors are a promising class of antiviral drugs for combating these viruses, as they can prevent infection at the site of viral entry, i.e., the respiratory tract. Here we used a broad-spectrum entry inhibitor, composed of a ß-cyclodextrin backbone, functionalized with 11-mercapto-1-undecanesulfonate (CD-MUS) that is capable of neutralizing a variety of viruses that employ heparan sulfate proteoglycans (HSPG) to infect host cells. CD-MUS inactivates viral particles irreversibly by binding to viral attachment proteins through a multivalent binding mechanism. In the present study, we show that CD-MUS is well tolerated when administered to the respiratory tract of mice. Based on this, we developed an inhalable spray-dried powder formulation that fits the size requirements for lung deposition and disperses well upon use with the Cyclops dry powder inhaler (DPI). Using an in vitro dose-response assay, we show that the compound retained its activity against RSV after the spray drying process. Our study sets the stage for further in vivo studies, exploring the efficacy of pulmonary administered CD-MUS in animal models of RSV infection.

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).

Antiviral Mechanism of Virucidal Sialic Acid Modified Cyclodextrin.

We have reported that CD-6'SLN [6-sialyllactosamine (6'SLN)-modified ß-cyclodextrin (CD)] can be a potential anti-influenza drug because it irreversibly deactivates virions. Indeed, in vivo, CD-6'SLN improved mice survival in an H1N1 infection model even when administered 24 h post-infection. Although CD-6'SLN was designed to target the viral envelope protein hemagglutinin (HA), a natural receptor of 6'SLN, it remains unclear whether other targets exist. In this study, we confirm that CD-6'SLN inhibits the influenza virus through an extracellular mechanism by interacting with HA, but not with neuraminidase (NA), despite the latter also having a binding pocket for the sialyl group. We find that CD-6'SLN interacts with the viral envelope as it elicits the release of a fluorophore embedded in the membrane. Two similar compounds were designed to test separately the effect of 6'SLN and of the undecyl moiety that links the CD to 6'SLN. Neither showed any interaction with the membrane nor the irreversible viral inhibition (virucidal), confirming that both components are essential to membrane interaction and virucidal action. Unlike similar antiviral cyclodextrins developed against other viruses, CD-6'SLN was not able to decapsulate viral RNA. Our findings support that combining viral protein-specific epitopes with hydrophobic linkers provides a strategy for developing antiviral drugs with a virucidal mechanism.

Photodynamic treatment of multidrug-resistant bacterial infection using indium phosphide quantum dots.

Infections caused by multidrug-resistant (MDR) bacteria pose an impending threat to humanity, as the evolution of MDR bacteria outpaces the development of effective antibiotics. In this work, we use indium phosphide (InP) quantum dots (QDs) to treat infections caused by MDR bacteria via photodynamic therapy (PDT), which shows superior bactericidal efficiency over common antibiotics. PDT in the presence of InP QDs results in high-efficiency bactericidal activity towards various bacterial species, including Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. Upon light absorption, InP QDs generate superoxide (O2˙-), which leads to efficient and selective killing of MDR bacteria while mammalian cells remain intact. The cytotoxicity evaluation reveals that InP QDs are bio- and blood-compatible in a wide therapeutic window. For the in vivo study, we drop a solution of InP QDs at a concentration within the therapeutic window onto MDR S. aureus-infected skin wounds of mice and perform PDT for 15 min. InP QDs show excellent therapeutic and prophylactic efficacy in treating MDR bacterial infection. These findings show that InP QDs have great potential to serve as antibacterial agents for MDR bacterial infection treatment, as an effective and complementary alternative to conventional antibiotics.

Monovalent ion-mediated charge-charge interactions drive aggregation of surface-functionalized gold nanoparticles.

Monolayer-protected metal nanoparticles (NPs) are not only promising materials with a wide range of potential industrial and biological applications, but they are also a powerful tool to investigate the behaviour of matter at nanoscopic scales, including the stability of dispersions and colloidal systems. This stability is dependent on a delicate balance between attractive and repulsive interactions that occur in the solution, and it is described in quantitative terms by the classic Derjaguin-Landau-Vewey-Overbeek (DLVO) theory, that posits that aggregation between NPs is driven by van der Waals interactions and opposed by electrostatic interactions. To investigate the limits of this theory at the nanoscale, where the continuum assumptions required by the DLVO theory break down, here we investigate NP dimerization by computing the Potential of Mean Force (PMF) of this process using fully atomistic MD simulations. Serendipitously, we find that electrostatic interactions can lead to the formation of metastable NP dimers at physiological ion concentrations. These dimers are stabilized by complexes formed by negatively charged ligands belonging to distinct NPs that are bridged by positively charged monovalent ions present in solution. We validate our findings by collecting tomographic EM images of NPs in solution and by quantifying their radial distribution function, that shows a marked peak at interparticle distance comparable with that of MD simulations. Taken together, our results suggest that not only van der Waals interactions, but also electrostatic interactions mediated by monovalent ions at physiological concentrations, contribute to attraction between nano-sized charged objects at very short length scales.

Peptide-Grafted Nontoxic Cyclodextrins and Nanoparticles against Bacteriophage Infections.

One of the biggest threats for bacteria-based bioreactors in the biotechnology industry is infections caused by bacterial viruses called bacteriophages. More than 70% of companies admitted to encountering this problem. Despite phage infections being such a dangerous and widespread risk, to date, there are no effective methods to avoid them. Here we present a peptide-grafted compounds that irreversibly deactivate bacteriophages and remain safe for bacteria and mammalian cells. The active compounds consist of a core (cyclodextrin or gold nanoparticle) coated with a hydrophobic chain terminated with a peptide selective for bacteriophages. Such peptides were selected via a phage display technique. This approach enables irreversible deactivation of the wide range of T-like phages (including the most dangerous in phage infections, phage T1) at 37 °C in 1 h. We show that our compounds can be used directly inside the environment of the bioreactor, but they are also a safe additive to stocks of antibiotics and expression inducers (such as isopropyl ß-d-1-thiogalactopyranoside, i.e., IPTG) that cannot be autoclaved and are a common source of phage infections.

An anti-influenza combined therapy assessed by single cell RNA-sequencing.

Influenza makes millions of people ill every year, placing a large burden on the healthcare system and the economy. To develop a treatment against influenza, we combined virucidal sialylated cyclodextrins with interferon lambda and demonstrated, in human airway epithelia, that the two compounds inhibit the replication of a clinical H1N1 strain more efficiently when administered together rather than alone. We investigated the mechanism of action of the combined treatment by single cell RNA-sequencing analysis and found that both the single and combined treatments impair viral replication to different extents across distinct epithelial cell types. We showed that each cell type comprises multiple sub-types, whose proportions are altered by H1N1 infection, and assessed the ability of the treatments to restore them. To the best of our knowledge this is the first study investigating the effectiveness of an antiviral therapy against influenza virus by single cell transcriptomic studies.

Lignin: A Sustainable Antiviral Coating Material.

Transmission of viruses through contact with contaminated surfaces is an important pathway for the spread of infections. Antiviral surface coatings are useful to minimize such risks. Current state-of-the-art approaches toward antiviral surface coatings either involve metal-based materials or complex synthetic polymers. These approaches, however, even if successful, will have to face great challenges when it comes to large-scale applications and their environmental sustainability. Here, an antiviral surface coating was prepared by spin-coating lignin, a natural biomass residue of the paper production industry. We show effective inactivation of herpes simplex virus type 2 (>99% after 30 min) on a surface coating that is low-cost and environmentally sustainable. The antiviral mechanism of the lignin surface was investigated and is attributed to reactive oxygen species generated upon oxidation of lignin phenols. This mechanism does not consume the surface coating (as opposed to the release of a specific antiviral agent) and does not require regeneration. The coating is stable in ambient conditions, as demonstrated in a 6 month aging study that did not reveal any decrease in antiviral activity. This research suggests that natural compounds may be used for the development of affordable and sustainable antiviral coatings.

Potent Virustatic Polymer-Lipid Nanomimics Block Viral Entry and Inhibit Malaria Parasites In Vivo.

Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanoparticles that mimic functional components of host cell membranes, yielding multivalent nanomimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer-lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition.