Detergent-induced self-assembly and controllable photosensitizer activity of diester phenylene ethynylenes.

Photodynamic therapy, in which malignant tissue is killed by targeted light exposure following administration of a photosensitizer, can be a valuable treatment modality but currently relies on passive transport and local irradiation to avoid off-target oxidation. We present a system of excited-state control for truly local delivery of singlet oxygen. An anionic phenylene ethynylene oligomer is initially quenched by water, producing minimal fluorescence and no measurable singlet oxygen generation. When presented with a binding partner, in this case an oppositely charged surfactant, changes in solvent microenvironment result in fluorescence unquenching, restoration of intersystem crossing to the triplet state, and singlet oxygen generation, as assayed by transient absorption spectroscopy and chemical trapping. This solvation-controlled photosensitizer model has possible applications as a theranostic agent for, for example, amyloid diseases.

Efficient Long-Range, Directional Energy Transfer through DNA-Templated Dye Aggregates.

The benzothiazole cyanine dye K21 forms dye aggregates on double-stranded DNA (dsDNA) templates. These aggregates exhibit a red-shifted absorption band, enhanced fluorescence emission, and an increased fluorescence lifetime, all indicating strong excitonic coupling among the dye molecules. K21 aggregate formation on dsDNA is only weakly sequence dependent, providing a flexible approach that is adaptable to many different DNA nanostructures. Donor (D)-bridge (B)-acceptor (A) complexes consisting of Alexa Fluor 350 as the donor, a 30 bp (9.7 nm) DNA templated K21 aggregate as the bridge, and Alexa Fluor 555 as the acceptor show an overall donor to acceptor energy transfer efficiency of ∼60%, with the loss of excitation energy being almost exclusively at the donor-bridge junction (63%). There was almost no excitation energy loss due to transfer through the aggregate bridge, and the transfer efficiency from the aggregate to the acceptor was about 96%. By comparing the energy transfer in templated aggregates at several lengths up to 32 nm, the loss of energy per nanometer through the K21 aggregate bridge was determined to be <1%, suggesting that it should be possible to construct structures that use much longer energy transfer "wires" for light-harvesting applications in photonic systems.

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.

Antifungal Properties of Cationic Phenylene Ethynylenes and Their Impact on ß-Glucan Exposure.

Candida species are the cause of many bloodstream infections through contamination of indwelling medical devices. These infections account for a 40% mortality rate, posing a significant risk to immunocompromised patients. Traditional treatments against Candida infections include amphotericin B and various azole treatments. Unfortunately, these treatments are associated with high toxicity, and resistant strains have become more prevalent. As a new frontier, light-activated phenylene ethynylenes have shown promising biocidal activity against Gram-positive and -negative bacterial pathogens, as well as the environmental yeast Saccharomyces cerevisiae In this study, we monitored the viability of Candida species after treatment with a cationic conjugated polymer [poly(p-phenylene ethynylene); PPE] or oligomer ["end-only" oligo(p-phenylene ethynylene); EO-OPE] by flow cytometry in order to explore the antifungal properties of these compounds. The oligomer was found to disrupt Candida albicans yeast membrane integrity independent of light activation, while PPE is able to do so only in the presence of light, allowing for some control as to the manner in which cytotoxic effects are induced. The contrast in killing efficacy between the two compounds is likely related to their size difference and their intrinsic abilities to penetrate the fungal cell wall. Unlike EO-OPE-DABCO (where DABCO is quaternized diazabicyclo[2,2,2]octane), PPE-DABCO displayed a strong propensity to associate with soluble ß-glucan, which is expected to inhibit its ability to access and perturb the inner cell membrane of Candida yeast. Furthermore, treatment with PPE-DABCO unmasked Candida albicans ß-glucan and increased phagocytosis by Dectin-1-expressing HEK-293 cells. In summary, cationic phenylene ethynylenes show promising biocidal activity against pathogenic Candida yeast cells while also exhibiting immunostimulatory effects.

Oligomeric Conjugated Polyelectrolytes Display Site-Preferential Binding to an MS2 Viral Capsid.

Opportunistic bacteria and viruses are a worldwide health threat prompting the need to develop new targeting modalities. A class of novel synthetic poly(phenylene ethynylene) (PPE)-based oligomeric conjugated polyelectrolytes (OPEs) have demonstrated potent wide-spectrum biocidal activity. A subset of cationic OPEs display high antiviral activity against the MS2 bacteriophage. The oligomers have been found to inactivate the bacteriophage and perturb the morphology of the MS2 viral capsid. However, details of the initial binding and interactions between the OPEs and the viruses are not well understood. In this study, we use a multiscale computational approach, including random sampling, molecular dynamics, and electronic structure calculations, to gain an understanding of the molecular-level interactions of a series of OPEs that vary in length, charge, and functional groups with the MS2 capsid. Our results show that OPEs strongly bind to the MS2 capsid protein assembly with binding energies of up to -30 kcal/mol. Free-energy analysis shows that the binding is dominated by strong van der Waals interactions between the hydrophobic OPE backbone and the capsid surface and strong electrostatic free energy contributions between the OPE charged moieties and charged residues on the capsid surface. This knowledge provides molecular-level insight into how to tailor the OPEs to optimize viral capsid disruption and increase OPE efficacy to target amphiphilic protein coats of icosahedral-based viruses.

Assessing the Sporicidal Activity of Oligo-p-phenylene Ethynylenes and Their Role as Bacillus Germinants.

A wide range of oligo-p-phenylene ethynylenes has been shown to exhibit good biocidal activity against both Gram-negative and Gram-positive bacteria. While cell death may occur in the dark, these biocidal compounds are far more effective in the light as a result of their ability to sensitize the production of cell-damaging reactive oxygen species. In these studies, the interactions of a specific cationic oligo-p-phenylene ethynylene with spore-forming Bacillus atrophaeus and Bacillus anthracis Sterne have been investigated. Flow cytometry assays are used to rapidly monitor cell death as well as spore germination. This compound effectively killed Bacillus anthracis Sterne vegetative cells (over 4 log reduction), presumably by severe perturbations of the bacterial cell wall and cytoplasmic membrane, while also acting as an effective spore germinant in the dark. While 2 log reduction of B. anthracis Sterne spores was observed, it is hypothesized that further killing could be achieved through enhanced germination.

Activating the antimicrobial activity of an anionic singlet-oxygen sensitizer through surfactant complexation.

Cationic oligo-p-phenylene ethynylenes have shown much promise as broad-spectrum light-activated antimicrobial compounds against both Gram-positive and Gram-negative bacteria. The anionic varieties, however, have weak biocidal activity. In this study, a complex is formed between a weakly biocidal anionic oligomer and a cationic surfactant, and the effects on their biocidal activity against Gram-negative E. coli and Gram-positive S. aureus are explored. The enhancement in biocidal activity that is observed when the complex is irradiated suggests that interfacial surfactant gives the complex a net-positive charge, allowing it to associate strongly with the bacterial membrane. The results of this study demonstrate a method for the enhancement of biocidal activity of singlet-oxygen sensitizers and corroborate the use of surfactants as trans-membrane drug-delivery agents.

AFM images of the dark biocidal action of cationic conjugated polyelectrolytes and oligomers on Escherichia coli.

Polymers and oligomers with conjugated phenylene ethynylene or thiophene ethynylene backbones have been shown to be potent antimicrobials. The mechanisms by which they act have been unclear, though AFM imaging of Escherichia coli cells before and after exposure to two such biocides, PPE-Th polymer and EO-OPE-1(C3), shows their effects on cell surface structure. Dried, unexposed E. coli cells could be imaged at resolution high enough to discern the physical structure of the cell surfaces, including individual porin proteins and their distribution on the cell. Exposure to 30 µg/mL PPE-Th polymer caused major cell surface disruption due to either emulsification of the outer membrane or the formation of polymer aggregates or both. In contrast, exposure to 30 µg/mL EO-OPE-1(C3) oligomer did not cause large-scale membrane disruption but did cause apparent reorganization of the surface proteins into linear arrays or protein-lipid-OPE complexes that dominate on a small scale. E. coli cells were also successfully imaged underwater, allowing a real-time AFM image series as cells were exposed to 30 µg/mL EO-OPE-1(C3). Solution exposure caused the cell surfaces to noticeably increase their roughness over time. These results agree with proposed mechanisms for cell killing by PPE-Th and EO-OPE-1(C3) put forth by Wang et al.1 in which PPE-Th kills by large-scale disruption of the outer membrane and EO-OPE-1(C3) kills by membrane reorganization with possible pore formation.

Cationic oligo-p-phenylene ethynylenes form complexes with surfactants for long-term light-activated biocidal applications.

Cationic oligo-p-phenylene ethynylenes are highly effective light-activated biocides that deal broad-spectrum damage to a variety of pathogens, including bacteria. A potential problem arising in the long-term usage of these compounds is photochemical breakdown, which nullifies their biocidal activity. Recent work has shown that these molecules complex with oppositely-charged surfactants, and that the resulting complexes are protected from photodegradation. In this manuscript, we determine the biocidal activity of an oligomer and a complex formed between it and sodium dodecyl sulfate. The complexes are able to withstand prolonged periods of irradiation, continuing to effectively kill both Gram-negative and Gram-positive bacteria, while the oligomer by itself loses its biocidal effectiveness quickly in the presence of light. In addition, damage and stress responses induced by these biocides in both E. coli and S. aureus are discussed. This work shows that complexation with surfactants is a viable method for long-term light-activated biocidal applications.

Photochemistry of "end-only" oligo-p-phenylene ethynylenes: complexation with sodium dodecyl sulfate reduces solvent accessibility.

Cationic oligo-p-phenylene ethynylenes are very effective light-activated biocides and biosensors but degrade upon exposure to light. In this study, we explore the photochemistry of a class of "end-only" compounds from this series, which have cationic moieties on the ends of the backbone. Product characterization by mass spectrometry reveals that the photoreactivity of these molecules is higher than that of a previously studied oligomer and that the primary products of photolysis result from the addition of water or oxygen across the triple bond. In addition, a product suggesting the addition of peroxide or other reactive oxygen species across the triple bond was observed. To explore avenues by which the photodegradation of these compounds can be mitigated, the effects of complexation with sodium dodecyl sulfate micelles on their photochemistry was explored. Classical molecular dynamics simulations revealed that compounds that were protected from photolysis by SDS buried their phenylene ethynylene backbones into the interior of the micelle, protecting it from contact with water. This work has revealed a molecular basis for the protection of a novel class of light-activated biocides from irradiation that is consistent with the proposed photochemistry of these compounds. This information can be useful for developing photodegradation-resistant biocidal materials and applications for current compounds and leads to new molecular design.

Structural basis for aggregation mode of oligo-p-phenylene ethynylenes with ionic surfactants.

In this letter, the aggregation modes of two classes of ionic p-phenylene ethynylene oligomers with oppositely charged surfactants are studied. The location of the ionic side chains was found to influence the type of aggregate formed when an equivalent number of surfactant molecules are added to solution. When the charged groups were located at the terminal ends of the molecule, strong H-aggregates were observed to form. Alternatively, when the ionic groups were both located on opposite sides of the central phenyl ring, the formation of J-aggregates was observed. Interestingly, as the surfactant concentration approaches the critical micelle concentration, the weakly bound aggregates are dissociated and the absorbance spectrum returns to what is observed in water. This study reveals the structural basis for aggregation effects between molecules based on the p-phenylene ethynylene backbone, and gives an understanding of how to influence the aggregation mode of similar compounds.

When worlds collide: interactions at the interface between biological systems and synthetic cationic conjugated polyelectrolytes and oligomers.

This Feature Article focuses on recent progress made in elucidating the intermolecular interactions between a novel class of synthetic phenylene ethynylene (PPE)-based conjugated polyelectrolyte polymers (CPEs) and oligomers (OPEs) and multiscale cellular targets that give rise to their remarkable broad spectrum biocidal activity. We first review the interactions and self-assembly behaviors of the CPEs and OPEs with a set of vital biomolecules, including lipids, proteins, and nucleic acids, that reveal the potential pathways by which synthetic biocidal agents could exert toxicity. An overview of the antimicrobial effects and mechanisms of the CPEs and OPEs on multiple clinically relevant pathogens is then presented, with an emphasis on the morphological damage induced by the biocidal compounds toward the pathogens. Finally, we discuss the cytotoxicity of these materials against mammalian cells and human tissues to explore the potential applications of the CPEs and OPEs as antiseptics. We also pose some unanswered questions about their antimicrobial mechanisms, which provide direction for a future study.

Understanding the dark and light-enhanced bactericidal action of cationic conjugated polyelectrolytes and oligomers.

A multiscale investigation was carried out to study the dark and light-enhanced bactericidal mechanisms of poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPEs) and oligo-phenylene ethynylenes (OPEs). On the morphological scale, Gram-negative E. coli cells exposed to CPE and OPE compounds in the dark show damage to the cell envelope, plasma membrane, and in some cases the cytoplasm, while with UV-irradiation, E. coli sustained catastrophic damages to both the cell envelope and cytoplasm. In contrast, the Gram-positive S. epi bacteria appeared intact when exposed to CPE and OPE compounds in the dark but showed damages to the cell envelope with UV-irradiation. To better understand the molecular basis of CPE- and OPE-induced morphological changes and damages to bacteria, we investigated the effect of these compounds on model bacterial plasma membrane and bacterial proteins and plasmid DNA. Measurements of dark membrane perturbation activity of the CPEs and OPEs using model lipid membranes support a carpet or detergent-like mechanism by which the antimicrobial compounds induce membrane collapse and phase transitions. Under UV-irradiation, E. coli bacteria exposed to CPEs and OPEs showed covalent modifications and damages to both cellular protein and plasmid DNA, likely through oxidative pathways mediated by singlet oxygen and subsequent reactive oxygen species sensitized by the CPE and OPE compounds. Our finding thus show that the antimicrobial polymers and oligomers exert toxicity toward Gram-negative bacteria by disrupting the morphology and structures of cell envelope and cytoplasm, including cellular components such as proteins and DNA, while exert toxicity toward Gram-positive bacteria by binding to and disrupting just the cell wall.

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.

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.

Molecular dynamics simulation study of the interaction of cationic biocides with lipid bilayers: aggregation effects and bilayer damage.

A novel class of phenylene ethynylene polyelectrolyte oligomers (OPEs) has been found to be effective biocidal agents against a variety of pathogens. The mechanism of attack is not yet fully understood. Recent studies have shown that OPEs cause catastrophic damage to large unilamellar vesicles. This study uses classical molecular dynamics (MD) simulations to understand how OPEs interact with model lipid bilayers. All-atom molecular dynamics simulations show that aggregates of OPEs inserted into the membrane cause significant structural damage and create a channel, or pore, that allows significant leakage of water through the membrane on the 0.1 µs time scale.

Efficacy of end-only-functionalized oligo(arylene-ethynylene)s in killing bacterial biofilms.

Cationic end-only-functionalized oligo(arylene-ethynylene)s (EO-OPEs) have recently been found to be broad-spectrum and effective antimicrobial agents because of their unique structure and optical properties. In this study, we investigated their potential use for preventing and reducing Escherichia coli (E. coli) biofilms. The Calgary biofilm device (CBD) was used to form bacterial biofilms of E. coli; in these studies, the minimum inhibitory concentration (MIC) and the minimum biofilm eradication concentration (MBEC) were determined. E. coli biofilms uniformly grow on pegs of the CBD device lid. The MIC values determined for EO-OPEs are comparable to those found for standard antibiotics such as kanamycin (MIC = 11.2 µg/mL). About 10-30 times the concentration of EO-OPEs was required to eradicate E. coli biofilms and prevent regrowth in the dark. Near-UV irradiation of EO-OPEs enhanced their efficacy in killing biofilms.

Direct visualization of bactericidal action of cationic conjugated polyelectrolytes and oligomers.

The bactericidal mechanisms of poly(phenylene ethynylene) (PPE)-based cationic conjugated polyelectrolytes (CPE) and oligo-phenylene ethynylenes (OPE) were investigated using electron/optical microscopy and small-angle X-ray scattering (SAXS). The ultrastructural analysis shows that polymeric PPE-Th can significantly remodel the bacterial outer membrane and/or the peptidoglycan layer, followed by the possible collapse of the bacterial cytoplasm membrane. In contrast, oligomeric end-only OPE (EO-OPE) possesses potent bacteriolysis activity, which efficiently disintegrates the bacterial cytoplasm membrane and induces the release of bacterial cell content. Using single giant vesicles and SAXS, we demonstrated that the membrane perturbation mechanism of EO-OPE against model bacterial membranes results from a 3D membrane phase transition or perturbation.

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.