An Effective Approach to the Disinfection of Pathogens: Cationic Conjugated Polyelectrolytes and Oligomers.

The synthetic cationic conjugated polyelectrolytes and oligomers have demonstrated great effectiveness and versatility as antimicrobial materials. They have the ability to eliminate or render inactive various pathogens, including viruses like SARS-CoV-2, bacteria, and fungi. These pathogens can be rapidly eradicated when the polyelectrolytes and oligomers are applied as sprays, wipes, or coatings on solid surfaces. Inactivation of the pathogens occurs through two distinct processes: a non-light-activated process similar to Quats, and a more efficient and faster process that is triggered by light. These materials possess fluorescence and photosensitizing properties, enabling prolonged protection when coated on surfaces. The level of fluorescence exhibited by samples applied to nonfluorescent surfaces serves as an indicator of the coating's integrity and viability, making it easily detectable. Importantly, these materials demonstrate low toxicity towards mammalian cells and human skin, allowing for their safe use. While they can serve as durable coatings for pathogen protection, extended exposure to visible or ultraviolet light leads to their photochemical degradation. Our research also suggests that these materials act against pathogens through nonspecific mechanisms, minimizing the likelihood of pathogens developing resistance and rendering the materials ineffective.

Partial Destabilization of Amyloid-ß Protofibril by Methionine Photo-Oxidation: A Molecular Dynamic Simulation Study.

Selective photosensitized oxidation of amyloid protein aggregates is being investigated as a possible therapeutic strategy for treating Alzheimer's disease (AD). Photo-oxidation has been shown to degrade amyloid-ß (Aß) aggregates and ameliorate aggregate toxicity in vitro and reduce aggregate levels in the brains of AD animal models. To shed light on the mechanism by which photo-oxidation induces fibril destabilization, we carried out an all-atom molecular dynamics (MD) simulation to examine the effect of methionine (Met35) oxidation on the conformation and stability of a ß-sheet-rich Aß9-40 protofibril. Analyses of up to 1 µs simulations showed that the oxidation of the Met35 residues, which resulted in the addition of hydrophilic oxygens in the fibril core, reduced the overall conformational stability of the protofibril. Specifically, Met35 disrupted the hydrophobic interface that stabilizes the stacking of the two hexamers that comprise the protofibril. The oxidized protofibril is more solvent exposed and exhibits more backbone flexibility. However, the protofibril retained the underlying U-shaped architecture of each peptide upon oxidation, and although some loss of ß-sheets occurred, a significant portion remained. Our simulation results are thus consistent with our experimental observation that photo-oxidation of Aß40 fibril resulted in the dis-agglomeration and fragmentation of Aß fibrils but did not cause complete disruption of the fibrillar morphology or ß-sheet structures. The partial destabilization of Aß aggregates supports the further development of photosensitized platforms for the targeting and clearing of Aß aggregates as a therapeutic strategy for treating AD.

Selective In Vitro and Ex Vivo Staining of Brain Neurofibrillary Tangles and Amyloid Plaques by Novel Ethylene Ethynylene-Based Optical Sensors.

The identification of protein aggregates as biomarkers for neurodegeneration is an area of interest for disease diagnosis and treatment development. In this work, we present novel super luminescent conjugated polyelectrolyte molecules as ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-ß (Aß) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE12- and cationic OPE24+, as stains for fibrillar protein pathology in brain sections of transgenic mouse (rTg4510) and rat (TgF344-AD) models of Alzheimer's disease (AD) tauopathy, and post-mortem brain sections from human frontotemporal dementia (FTD). OPE12- displayed selectivity for PHFs in fluorimetry assays and strong staining of neurofibrillary tangles (NFTs) in mouse and human brain tissue sections, while OPE24+ stained both NFTs and Aß plaques. Both OPEs stained the brain sections with limited background or non-specific staining. This novel family of sensors outperformed the gold-standard dye Thioflavin T in sensing capacities and co-stained with conventional phosphorylated tau (AT180) and Aß (4G8) antibodies. As the OPEs readily bind protein amyloids in vitro and ex vivo, they are selective and rapid tools for identifying proteopathic inclusions relevant to AD. Such OPEs can be useful in understanding pathogenesis and in creating in vivo diagnostically relevant detection tools for neurodegenerative diseases.

Controlled and Selective Photo-oxidation of Amyloid-ß Fibrils by Oligomeric p-Phenylene Ethynylenes.

Photodynamic therapy (PDT) has been explored as a therapeutic strategy to clear toxic amyloid aggregates involved in neurodegenerative disorders such as Alzheimer's disease. A major limitation of PDT is off-target oxidation, which can be lethal for the surrounding cells. We have shown that a novel class of oligo-p-phenylene ethynylenes (OPEs) exhibit selective binding and fluorescence turn-on in the presence of prefibrillar and fibrillar aggregates of disease-relevant proteins such as amyloid-ß (Aß) and α-synuclein. Concomitant with fluorescence turn-on, OPE also photosensitizes singlet oxygen under illumination through the generation of a triplet state, pointing to the potential application of OPEs as photosensitizers in PDT. Herein, we investigated the photosensitizing activity of an anionic OPE for the photo-oxidation of Aß fibrils and compared its efficacy to the well-known but nonselective photosensitizer methylene blue (MB). Our results show that, while MB photo-oxidized both monomeric and fibrillar conformers of Aß40, OPE oxidized only Aß40 fibrils, targeting two histidine residues on the fibril surface and a methionine residue located in the fibril core. Oxidized fibrils were shorter and more dispersed but retained the characteristic ß-sheet rich fibrillar structure and the ability to seed further fibril growth. Importantly, the oxidized fibrils displayed low toxicity. We have thus discovered a class of novel theranostics for the simultaneous detection and oxidization of amyloid aggregates. Importantly, the selectivity of OPE's photosensitizing activity overcomes the limitation of off-target oxidation of traditional photosensitizers and represents an advancement of PDT as a viable strategy to treat neurodegenerative disorders.

Rapid and Effective Inactivation of SARS-CoV-2 with a Cationic Conjugated Oligomer with Visible Light: Studies of Antiviral Activity in Solutions and on Supports.

This paper presents results of a study of a new cationic oligomer that contains end groups and a chromophore affording inactivation of SARS-CoV-2 by visible light irradiation in solution or as a solid coating on paper wipes and glass fiber filtration substrates. A key finding of this study is that the cationic oligomer with a central thiophene ring and imidazolium charged groups gives outstanding performance in both the killing of E. coli bacterial cells and inactivation of the virus at very short times. Our introduction of cationic N-methyl imidazolium groups enhances the light activation process for both E. coli and SARS-CoV-2 but dampens the killing of the bacteria and eliminates the inactivation of the virus in the dark. For the studies with this oligomer in solution at a concentration of 1 µg/mL and E. coli, we obtain 3 log killing of the bacteria with 10 min of irradiation with LuzChem cool white lights (mimicking indoor illumination). With the oligomer in solution at a concentration of 10 µg/mL, we observe 4 log inactivation (99.99%) in 5 min of irradiation and total inactivation after 10 min. The oligomer is quite active against E. coli on oligomer-coated paper wipes and glass fiber filter supports. The SARS-CoV-2 is also inactivated by oligomer-coated glass fiber filter papers. This study indicates that these oligomer-coated materials may be very useful as wipes and filtration materials.

Understanding the Photochemical Properties of Polythiophene Polyelectrolyte Soft Aggregates with Sodium Dodecyl Sulfate for Antimicrobial Activity.

The threat of antibiotic-resistant bacteria is an ever-increasing problem in public health. In this report, we examine the photochemical properties with a proof-of-principle biocidal assay for a novel series of regio-regular imidazolium derivative poly-(3-hexylthiophene)/sodium dodecyl sulfate (P3HT-Im/SDS) materials from ultrafast sub-ps dynamics to µs generation of reactive oxygen species (ROS) and 30 min biocidal reactivity with Escherichia coli (E. coli). This broad series encompassing pure P3HT-Im to cationic, neutral, and anionic P3HT-Im/SDS materials are all interrogated by a variety of techniques to characterize the physical material structure, electronic structure, and antimicrobial activity. Our results show that SDS complexation with P3HT-Im results in aggregate materials with reduced ROS generation and light-induced anti-microbial activity. However, our characterization reveals that the presence of non-aggregated or lightly SDS-covered polymer segments is still capable of ROS generation. Full encapsulation of the P3HT-Im polymer completely deactivates the light killing pathway. High SDS concentrations, near and above critical micelle concentration, further deactivate all anti-microbial activity (light and dark) even though the P3HT-Im regains its electronic properties to generate ROS.

Tau and Membranes: Interactions That Promote Folding and Condensation.

Tau misfolding and assembly is linked to a number of neurodegenerative diseases collectively described as tauopathies, including Alzheimer's disease (AD) and Parkinson's disease. Anionic cellular membranes, such as the cytosolic leaflet of the plasma membrane, are sites that concentrate and neutralize tau, primarily due to electrostatic interactions with tau's microtubule binding repeat domain (RD). In addition to electrostatic interactions with lipids, tau also has interactions with membrane proteins, which are important for tau's cellular functions. Tau also interacts with lipid tails to facilitate direct translocation across the membrane and can form stable protein-lipid complexes involved in cell-to-cell transport. Concentrated tau monomers at the membrane surface can form reversible condensates, change secondary structures, and induce oligomers, which may eventually undergo irreversible crosslinking and fibril formation. These ß-sheet rich tau structures are capable of disrupting membrane organization and are toxic in cell-based assays. Given the evidence for relevant membrane-based tau assembly, we review the emerging hypothesis that polyanionic membranes may serve as a site for phase-separated tau condensation. Membrane-mediated phase separation may have important implications for regulating tau folding/misfolding, and may be a powerful mechanism to spatially direct tau for native membrane-mediated functions.

Highly Effective Inactivation of SARS-CoV-2 by Conjugated Polymers and Oligomers.

In the present study, we examined the inactivation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by synthetic conjugated polymers and oligomers developed in our laboratories as antimicrobials for bacteria, fungi, and nonenveloped viruses. The results show highly effective light-induced inactivation with several of these oligomers and polymers including irradiation with near-UV and visible light. In the best case, one oligomer induced a 5-log reduction in pfu/mL within 10 min. In general, the oligomers are more active than the polymers; however, the polymers are active with longer wavelength visible irradiation. Although not studied quantitatively, the results show that in the presence of the agents at concentrations similar to those used in the light studies, there is essentially no dark inactivation of the virus. Because three of the five materials/compounds examined are quaternary ammonium derivatives, this study indicates that conventional quaternary ammonium antimicrobials may not be active against SARS-CoV-2. Our results suggest several applications involving the incorporation of these materials in wipes, sprays, masks, and clothing and other personal protection equipment that can be useful in preventing infections and the spreading of this deadly virus and future outbreaks from similar viruses.

Computational Investigation of the Binding Dynamics of Oligo p-Phenylene Ethynylene Fluorescence Sensors and Aß Oligomers.

Amyloid protein aggregates are pathological hallmarks of neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's (PD) diseases and are believed to be formed well before the onset of neurodegeneration and cognitive impairment. Monitoring the course of protein aggregation is thus vital to understanding and combating these diseases. We have recently demonstrated that a novel class of fluorescence sensors, oligomeric p-phenylene ethynylene (PE)-based electrolytes (OPEs) selectively bind to and detect prefibrillar and fibrillar aggregates of AD-related amyloid-ß (Aß) peptides over monomeric Aß. In this study, we investigated the binding between two OPEs, anionic OPE12- and cationic OPE24+, and to two different ß-sheet rich Aß oligomers using classical all-atom molecular dynamics simulations. Our simulations have revealed a number of OPE binding sites on Aß oligomer surfaces, and these sites feature hydrophobic amino acids as well as oppositely charged amino acids. Binding energy calculations show energetically favorable interactions between both anionic and cationic OPEs with Aß oligomers. Moreover, OPEs bind as complexes as well as single molecules. Compared to free OPEs, Aß protofibril bound OPEs show backbone planarization with restricted rotations and reduced hydration of the ethyl ester end groups. These characteristics, along with OPE complexation, align with known mechanisms of binding induced OPE fluorescence turn-on and spectral shifts from a quenched, unbound state in aqueous solutions. This study thus sheds light on the molecular-level details of OPE-Aß protofibril interactions and provides a structural basis for fluorescence turn-on sensing modes of OPEs.

Highly Effective Inactivation of SARS-CoV-2 by Conjugated Polymers and Oligomers.

The current Covid-19 Pandemic caused by the highly contagious SARS-CoV-2 virus has proven extremely difficult to prevent or control. Currently there are few treatment options and very few long-lasting disinfectants available to prevent the spread. While masks and protective clothing and social distancing may offer some protection, their use has not always halted or slowed the spread. Several vaccines are currently undergoing testing; however there is still a critical need to provide new methods for inactivating the virus before it can spread and infect humans. In the present study we examined the inactivation of SARS-CoV-2 by synthetic conjugated polymers and oligomers developed in our laboratories as antimicrobials for bacteria, fungi and non-enveloped viruses. Our results show that we can obtain highly effective light induced inactivation with several of these oligomers and polymers including irradiation with near-UV and visible light. With both the oligomers and polymers, we can reach several logs of inactivation with relatively short irradiation times. Our results suggest several applications involving the incorporation of these materials in wipes, sprays, masks and clothing and other Personal Protection Equipment (PPE) that can be useful in preventing infections and the spreading of this deadly virus and future outbreaks from similar viruses.

Lipid membrane templated misfolding and self-assembly of intrinsically disordered tau protein.

The aggregation of the intrinsically disordered tau protein into highly ordered ß-sheet-rich fibrils is implicated in the pathogenesis of a range of neurodegenerative disorders. The mechanism of tau fibrillogenesis remains unresolved, particularly early events that trigger the misfolding and assembly of the otherwise soluble and stable tau. We investigated the role the lipid membrane plays in modulating the aggregation of three tau variants, the largest isoform hTau40, the truncated construct K18, and a hyperphosphorylation-mimicking mutant hTau40/3Epi. Despite being charged and soluble, the tau proteins were also highly surface active and favorably interacted with anionic lipid monolayers at the air/water interface. Membrane binding of tau also led to the formation of a macroscopic, gelatinous layer at the air/water interface, possibly related to tau phase separation. At the molecular level, tau assembled into oligomers composed of ~ 40 proteins misfolded in a ß-sheet conformation at the membrane surface, as detected by in situ synchrotron grazing-incidence X-ray diffraction. Concomitantly, membrane morphology and lipid packing became disrupted. Our findings support a general tau aggregation mechanism wherein tau's inherent surface activity and favorable interactions with anionic lipids drive tau-membrane association, inducing misfolding and self-assembly of the disordered tau into ß-sheet-rich oligomers that subsequently seed fibrillation and deposition into diseased tissues.

Quantitative Determination of Dark and Light-Activated Antimicrobial Activity of Poly(Phenylene Ethynylene), Polythiophene, and Oligo(Phenylene Ethynylene) Electrolytes.

Much recent effort has been directed toward the development of novel antimicrobial materials able to defeat new and antibiotic resistant pathogens. In this report, we study the efficacy of cationic poly(phenylene ethynylene), polythiophene, and oligo(phenylene ethynylene) electrolytes against laboratory strains of Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. The focus of the study is to quantitatively evaluate the speed and extent of dark and light-activated antimicrobial activity. Using cell plating with serial dilutions, we determined that planktonic bacteria suspensions exposed to the antimicrobials (at 10 µg/mL) result in several log kills at 10 min both in the dark and under UV irradiation (360 nm) for all eight synthetic antimicrobials. However, there are significant differences in the ease of killing the different pathogens. In most trials, there is significantly greater killing under light-irradiation, indicating these materials may be used as versatile disinfectants.

Passive Immunotherapies Targeting Amyloid Beta and Tau Oligomers in Alzheimer's Disease.

Alzheimer's disease (AD) is historically difficult to treat, in part because of the inaccessible nature of brain pathology. Amyloid beta and tau proteins drive pathology by forming toxic oligomers that eventually deposit as insoluble amyloid plaques and neurofibrillary tangles. Recent clinical studies suggest that effective drugs must specifically target oligomers, not native monomers or insoluble fibrils. Passive immunotherapy is a promising pharmaceutical strategy used to specifically target these oligomers in situ. Using the specificity of antibodies coupled with the natural power of the body's immune response, this treatment provides an opportunity for safe clearance of pathogenic protein species from the brain. Passive immunotherapies against amyloid beta and tau oligomers have progressed to clinical trials, with many currently in progress. Biochemical studies of antibody-oligomer complexes have helped identify previously unknown toxic epitopes, thus providing knowledge to the AD field as a whole. This mini-review focuses on the efforts to develop passive immunotherapy treatments for AD and discusses the knowledge gained from recent failures and clinical trials in progress.

Fibrillar and Nonfibrillar Amyloid Beta Structures Drive Two Modes of Membrane-Mediated Toxicity.

In Alzheimer's disease, the amyloid-beta peptide (Aß) is implicated in neuronal toxicity via interactions with the cell membrane. Monomeric Aß (Aßm) is intrinsically disordered, but it can adopt a range of aggregated conformations with varying toxicities from short fibrillar oligomers (FO), to globular nonfibrillar oligomers (NFO), and full-length amyloid fibrils. NFO is considered to be the most toxic, followed by fibrils, and finally Aßm. To elucidate molecular-level membrane interactions that contribute to their different toxicities, we used liquid surface X-ray scattering and Langmuir trough insertion assays to compare Aßm, FO, and NFO surface activities and interactions with anionic DMPG lipid monolayers at the air/water interface. All Aß species were highly surface active and rapidly adopted ß-sheet rich structures upon adsorption to the air/water interface. Likewise, all Aß species had affinity for the anionic membrane. Aßm rapidly converted to ß-sheet rich assemblies upon binding the membrane, and these aggregated structures of Aßm and FO disrupted hexagonally packed lipid domains and resulted in membrane thinning and instability. In contrast, NFO perturbed membrane structure by extracting lipids from the air/water interface and causing macroscale membrane deformations. Altogether, our results support two models for membrane-mediated Aß toxicity: fibril-induced reorganization of lipid packing and NFO-induced membrane destabilization and lipid extraction. This work provides a structural understanding of Aß neurotoxicity via membrane interactions and aids the effort in understanding early events in Alzheimer's disease and other neurodegenerative diseases.

Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature.

Biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb3. This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb3 carbohydrate and ceramide moieties modulated the toxin's capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.

Membrane-mediated fibrillation and toxicity of the tau hexapeptide PHF6.

The aggregation of the tau protein into neurofibrillary tangles is believed to correlate with cognitive decline in several neurodegenerative disorders, including Alzheimer's disease. Recent studies suggest that tau's interactions with the cell membrane could serve as a toxicity pathway and also enhance fibrillation into paired helical filaments (PHFs). Conformational changes associated with tau-membrane interactions are poorly understood, and their characterization could improve our understanding of tau pathogenicity. In this study, we investigated the molecular level structural changes associated with the interaction of the tau hexapeptide PHF6 with model lipid membranes and characterized the effects of these interactions on membrane stability and peptide fibrillation. We used two PHF6 forms, the aggregation-prone PHF6 with N-terminal acetylation (Ac-PHF6) and the non-aggregation prone PHF6 with a standard N terminus (NH3+-PHF6). We found that both PHF6 peptides are neurotoxic and exhibit similar membrane-mediated changes, consisting of: 1) favorable interactions with anionic membranes, 2) membrane destabilization through lipid extraction, and 3) membrane-mediated fibrillation. The rate at which these changes occurred was the main difference between the two peptides. NH3+-PHF6 displayed slow membrane-mediated fibrillation after 6 days of incubation, whereas Ac-PHF6 adopted a ß-sheet conformation at the surface of the membrane within hours. Ac-PHF6 interactions with the membrane were also accompanied by membrane invagination and rapid membrane destabilization. Overall, our results reveal that membrane interactions could play a critical role in tau toxicity and fibrillation, and highlight that unraveling these interactions is important for significantly advancing the development of therapeutic strategies to manage tau-associated neurodegenerative diseases.

High Selectivity and Sensitivity of Oligomeric p-Phenylene Ethynylenes for Detecting Fibrillar and Prefibrillar Amyloid Protein Aggregates.

Misfolding and aggregation of amyloid proteins into fibrillar aggregates is a central pathogenic event in neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's diseases (PD). Currently, there is a lack of reliable sensors for detecting the range of protein aggregates involved in disease etiology, particularly the prefibrillar aggregate conformations that are more neurotoxic. In this study, the fluorescent sensing of two novel oligomeric p-phenylene ethynylenes (OPEs), anionic OPE1- and cationic OPE2+, for detecting prefibrillar and fibrillar aggregates of AD-associated amyloid-ß (Aß40 and Aß42) and PD-associated α-synuclein proteins (wildtype, and single mutants A30P, E35K, and A53T) over their monomeric counterparts, were tested. Furthermore, the performance of OPEs was evaluated and compared to thioflavin T (ThT), the most widely used fibril dye. Our results show that OPE1- and OPE2+ exhibited aggregate-specific binding inducing large fluorescence turn-on and spectral shifts based on a combination of backbone planarization, hydrophobic unquenching, and superluminescent OPE complex formation sensing modes. OPEs exhibited higher selectivity, higher binding affinity, and comparable limits of detection for Aß40 fibrils compared to ThT. OPE2+ exhibited the largest fluorescence turn-on and highest sensitivity. Significantly, OPEs detected prefibrillar aggregates of Aß42 and α-synuclein that ThT failed to detect. The superior sensing performance, the nonprotein specific detection, and the ability to selectively detect fibrillar and prefibrillar amyloid protein aggregates point to the potential of OPEs to overcome the limitations of existing probes and promise significant advancement in the detection of the myriad of protein aggregates involved in the early stages of AD and PD.

A Retrospective: 10 Years of Oligo(phenylene-ethynylene) Electrolytes: Demystifying Nanomaterials.

In this retrospective, we first reviewed the synthesis of the oligo(phenylene-ethynylene) electrolytes (OPEs) we created in the past 10 years. Since the general antimicrobial activity of these OPEs had been reported in our previous account in Langmuir, we are focusing only on the unusual spectroscopic and photophysical properties of these OPEs and their complexes with anionic scaffolds and detergents in this Feature Article. We applied classical all-atom MD simulations to study the hydrogen bonding environment in the water surrounding the OPEs with and without detergents present. Our finding is that OPEs could form a unit cluster or unit aggregate with a few oppositely charged detergent molecules, indicating that the photostability and photoreactivity of these OPEs might be considerably altered with important consequences to their activity as antimicrobials and fluorescence-based sensors. Thus, in the following sections, we showed that OPE complexes with detergents exhibit enhanced light-activated biocidal activity compared to either OPE or detergent individually. We also found that similar complexes between certain OPEs and biolipids could be used to construct sensors for the enzyme activity. Finally, the OPEs could covalently bind to microsphere surfaces to make a bactericidal surface, which is simpler and more ordered than the surface grafted from microspheres with polyelectrolytes. In the Conclusions and Prospects section, we briefly summarize the properties of OPEs developed so far and future areas for investigation.