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.

Yeast-encapsulated essential oils: a new perspective as an environmentally friendly larvicide.

BACKGROUND: Effective mosquito control approaches incorporate both adult and larval stages. For the latter, physical, biological, and chemical control have been used with varying results. Successful control of larvae has been demonstrated using larvicides including insect growth regulators, e.g. the organophosphate temephos, as well as various entomopathogenic microbial species. However, a variety of health and environmental issues are associated with some of these. Laboratory trials of essential oils (EO) have established the larvicidal activity of these substances, but there are currently no commercially available EO-based larvicides. Here we report on the development of a new approach to mosquito larval control using a novel, yeast-based delivery system for EO. METHODS: Food-grade orange oil (OO) was encapsulated into yeast cells following an established protocol. To prevent environmental contamination, a proprietary washing strategy was developed to remove excess EO that is adsorbed to the cell exterior during the encapsulation process. The OO-loaded yeast particles were then characterized for OO loading, and tested for efficacy against Aedes aegypti larvae. RESULTS: The composition of encapsulated OO extracted from the yeast microparticles was demonstrated not to differ from that of un-encapsulated EO when analyzed by high performance liquid chromatography. After lyophilization, the oil in the larvicide comprised 26-30 percentage weight (wt%), and is consistent with the 60-65% reduction in weight observed after the drying process. Quantitative bioassays carried with Liverpool and Rockefeller Ae. aegypti strains in three different laboratories presented LD50 of 5.1 (95% CI: 4.6-5.6) to 27.6 (95% CI: 26.4-28.8) mg/l, for L1 and L3/L4 mosquito larvae, respectively. LD90 ranged between 18.9 (95% CI: 16.4-21.7) mg/l (L1 larvae) to 76.7 (95% CI: 69.7-84.3) mg/l (L3/L4 larvae). CONCLUSIONS: The larvicide based on OO encapsulated in yeast was shown to be highly active (LD50 < 50 mg/l) against all larval stages of Ae. aegypti. These results demonstrate its potential for incorporation in an integrated approach to larval source management of Ae. aegypti. This novel approach can enable development of affordable control strategies that may have significant impact on global health.

Conjugated gold nanoparticles as a tool for probing the bacterial cell envelope: The case of Shewanella oneidensis MR-1.

The bacterial cell envelope forms the interface between the interior of the cell and the outer world and is, thus, the means of communication with the environment. In particular, the outer cell surface mediates the adhesion of bacteria to the surface, the first step in biofilm formation. While a number of ligand-based interactions are known for the attachment process in commensal organisms and, as a result, opportunistic pathogens, the process of nonspecific attachment is thought to be mediated by colloidal, physiochemical, interactions. It is becoming clear, however, that colloidal models ignore the heterogeneity of the bacterial surface, and that the so-called nonspecific attachment may be mediated by specific regions of the cell surface, whether or not the relevant interaction is ligand-mediate. The authors introduce surface functionalized gold nanoparticles to probe the surface chemistry of Shewanella oneidensis MR-1 as it relates to surface attachment to ω-substituted alkanethiolates self-assembled monolayers (SAMs). A linear relationship between the attachment of S. oneidensis to SAM modified planar substrates and the number of similarly modified nanoparticles attached to the bacterial surfaces was demonstrated. In addition, the authors demonstrate that carboxylic acid-terminated nanoparticles attach preferentially to the subpolar region of the S. oneidensis and obliteration of that binding preference corresponds in loss of attachment to carboxylic acid terminated SAMs. Moreover, this region corresponds to suspected functional regions of the S. oneidensis surface. Because this method can be employed over large numbers of cells, this method is expected to be generally applicable for understanding cell surface organization across populations.

Reusable nanoengineered surfaces for bacterial recruitment and decontamination.

Biofouling, or accumulation of unwanted biofilms, on surfaces is a major concern for public health and human industry. Materials either avoiding contamination (fouling resistant) and/or directly killing attached microbes (biocidal) have thus far failed to achieve the goal of eliminating biofouling; fouling resistant surfaces eventually foul and biocidal surfaces accumulate debris that eventually decrease their efficacy. Combined biocidal and fouling release materials offer the potential for both killing and removing debris and are promising candidates for reducing biofouling on manufactured materials. Interference lithography was used to create nanopatterns of initiators, which were then used to initiate atom transfer radical polymerization of the temperature-responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm) as a fouling release component. Biocidal activity was conferred by subsequent layer-by-layer deposition of cationic and anionic poly(phenylene ethynylenes) into the valleys between the PNIPAAm. For both Gram positive and Gram negative model bacteria, dark-regime biocidal activity was observed that did not increase upon exposure to light, suggesting that the mode of antimicrobial activity is due to ionic disruption of the cell wall. Subsequent to killing, bacteria and cellular debris were removed upon a temperature-induced phase transition of the PNIPAAm. These materials exhibited capture, killing, and release activity over multiple cycles of use.

Nanopatterned polymer brushes: conformation, fabrication and applications.

Surfaces with end-grafted, nanopatterned polymer brushes that exhibit well-defined feature dimensions and controlled chemical and physical properties provide versatile platforms not only for investigation of nanoscale phenomena at biointerfaces, but also for the development of advanced devices relevant to biotechnology and electronics applications. In this review, we first give a brief introduction of scaling behavior of nanopatterned polymer brushes and then summarize recent progress in fabrication and application of nanopatterned polymer brushes. Specifically, we highlight applications of nanopatterned stimuli-responsive polymer brushes in the areas of biomedicine and biotechnology.

Relationship between surface chemistry, biofilm structure, and electron transfer in Shewanella anodes.

A better understanding of how anode surface properties affect growth, development, and activity of electrogenic biofilms has great potential to improve the performance of bioelectrochemical systems such as microbial fuel cells. The aim of this paper was to determine how anodes with specific exposed functional groups (-N(CH3)3 (+), -COOH, -OH, and -CH3), created using ω-substituted alkanethiolates self-assembled monolayers attached to gold, affect the surface properties and functional performance of electrogenic Shewanella oneidensis MR-1 biofilms. A combination of spectroscopic, microscopic, and electrochemical techniques was used to evaluate how electrode surface chemistry influences morphological, chemical, and functional properties of S. oneidensis MR-1 biofilms, in an effort to develop improved electrode materials and structures. Positively charged, highly functionalized, hydrophilic surfaces were beneficial for growth of uniform biofilms with the smallest cluster sizes and intercluster diffusion distances, and yielding the most efficient electron transfer. The authors derived these parameters based on 3D morphological features of biofilms that were directly linked to functional properties of the biofilm during growth and that, during polarization, were directly connected to the efficiency of electron transfer to the anode. Our results indicate that substratum chemistry affects not only primary attachment, but subsequent biofilm development and bacterial physiology.

Nanopatterned antimicrobial enzymatic surfaces combining biocidal and fouling release properties.

Surfaces incorporating the antimicrobial enzyme, lysozyme, have been previously demonstrated to effectively disrupt bacterial cellular envelopes. As with any surface active antimicrobial, however, lysozyme-expressing surfaces become limited in their utility by the accumulation of dead bacteria and debris. Surfaces modified with environmentally responsive polymers, on the other hand, have been shown to reversibly attach and release both live and dead bacterial cells. In this work, we combine the antimicrobial activity of lysozyme with the fouling release capability of the thermally responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), which has a lower critical solution temperature (LCST) in water at ∼32 °C. Nanopatterned PNIPAAm brushes were fabricated using interferometric lithography followed by surface-initiated polymerization. Lysozyme was then adsorbed into the polymer-free regions of the substrate between the brushes to achieve a hybrid surface with switchable antimicrobial activity and fouling-release ability in response to the change of temperature. The temperature triggered hydration and conformational change of the nanopatterned PNIPAAm brushes provide the ability to temporally regulate the spatial concealment and exposure of adsorbed lysozyme. The biocidal efficacy and release properties of the hybrid surface were tested against Escherichia coli K12 and Staphylococcus epidermidis. The hybrid surfaces facilitated the attachment of bacteria at 37 °C for E. coli and 25 °C for S. epidermidis and when the temperature is above the LCST, collapsed and dehydrated PNIPAAm chains expose lysozyme to kill attached bacteria. Changing temperature across the LCST of PNIPAAm (e.g. from 37 °C to 25 °C for E. coli or from 25 °C to 37 °C for S. epidermidis) to induce a hydration transition of PNIPAAm promoted the release of dead bacteria and debris from the surfaces upon mild shearing. These results suggest that nano-engineered surfaces can provide an effective way for actively mitigating short term bacterial biofouling.

Nanopatterned smart polymer surfaces for controlled attachment, killing, and release of bacteria.

Model surfaces with switchable functionality based on nanopatterned, thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) brushes were fabricated using interferometric lithography combined with surface-initiated polymerization. The temperature-triggered hydration and conformational changes of nanopatterned PNIPAAm brushes reversibly modulate the spatial concealment and exposure of molecules that are immobilized in the intervals between nanopatterned brushes. A biocidal quaternary ammonium salt (QAS) was used to demonstrate the utility of nanopatterned PNIPAAm brushes to control biointerfacial interactions with bacteria. QAS was integrated into polymer-free regions of the substrate between nanopatterned PNIPAAm brushes. The biocidal efficacy and release properties of these surfaces were tested against Escherichia coli K12. Above the lower critical solution temperature (LCST) of PNIPAAm, desolvated, collapsed polymer chains facilitate the attachment of bacteria and expose QAS moieties that kill attached bacteria. Upon a reduction of the temperature below the LCST, swollen PNIPAAm chains promote the release of dead bacteria. These results demonstrate that nanopatterned PNIPAAm/QAS hybrid surfaces are model systems that exhibit an ability to undergo noncovalent, dynamic, and reversible changes in structure that can be used to control the attachment, killing, and release of bacteria in response to changes in temperature.

Thermodynamic analysis of marine bacterial attachment to oligo(ethylene glycol)-terminated self-assembled monolayers.

Colloidal models are frequently used to model the thermodynamics of bacterial attachment to surfaces. The most commonly used of such models is that proposed by van Oss, Chaudhury and Good, which includes both non-polar and polar (including hydrogen bonding) interactions between the attaching bacterium, the attachment substratum and the aqueous environment. We use this model to calculate the free energy of adhesion, ∆Gadh, for attachment of the marine bacterium Cobetia marina to well defined attachment substrata that systematically vary in their chemistry and their ability to attach bacteria, namely a series of oligo(ethylene glycol) (OEG) terminated self-assembled monolayers that vary in the number of OEG moieties. For this system, the values of ∆Gadh calculated using VCG do not correlate with observed attachment profiles. We examine the validity of a number of assumptions inherent in VCG and other colloidal models of adhesion, with special attention paid to those regarding bacterial surfaces.

Interfacial tension analysis of oligo(ethylene glycol)-terminated self-assembled monolayers and their resistance to bacterial attachment.

The fouling resistance of oligo(ethylene glycol) (OEG)-terminated self-assembled monolayers (SAMs) of alkanethiolates on gold has been well established. Although hydration of the OEG chains seems key to OEG-SAM resistance to macromolecular adsorption and cellular attachment, the details of how hydration prevents biofouling have been inferred largely through computational methods. Because OEG-SAMs of different lengths exhibit differing degrees of fouling resistance, the interactions between water and OEG-SAMs leading to fouling resistance can be deduced by comparing the properties of fouling and nonfouling OEG-SAMs. While all OEG-SAMs had similar water contact angles, contact angles taken with glycerol were able to individuate between different OEG-SAMs and between fouling and nonfouling OEG-SAMs. Subsequent estimation of surface and interfacial tension using a colloidal model showed that nonfouling surfaces are associated with an increased negative interfacial tension between those OEG-SAMs that resisted attachment and water. Further analysis of this interfacial tension experimentally confirmed current mathematical models that cite OEG-water hydrogen-bond formation as a driving force behind short-term fouling resistance. Finally, we found a correlation between solid-water interfacial tension and packing density and molecular density of ethylene glycol.

ARGET-ATRP synthesis and characterization of PNIPAAm brushes for quantitative cell detachment studies.

Stimuli responsive (or "smart") polymer brushes represent a non-toxic approach for achieving release of biofouling layers. Thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm) polymer brushes have been shown to modulate bacterial adhesion and release through transition between temperatures above and below the lower critical solution temperature (LCST ~32 °C) of PNIPAAm in water. In this article, we describe a convenient method to synthesize grafted PNIPAAm brushes over large areas for biological studies using a relatively simple and rapid method which allows atom transfer radical polymerization (ATRP) in presence of air using the activator regenerated electron transfer (ARGET) mechanism. PNIPAAm brushes were characterized using X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy, Fourier transform infrared spectroscopy, ellipsometry, and contact angle measurements. Our studies demonstrate that uniform, high purity PNIPAAm brushes with controlled and high molecular weight can be easily produced over large areas using ARGET-ATRP. We also report the use of a spinning disk apparatus to systematically and quantitatively study the detachment profiles of bacteria from PNIPAAm surfaces under a range (0-400 dyne/cm(2)) of shear stresses.

Conjugated-polyelectrolyte-grafted cotton fibers act as "micro flypaper" for the removal and destruction of bacteria.

We demonstrate herein a method for chemically modifying cotton fibers and cotton-containing fabric with a light-activated, cationic phenylene-ethynylene (PPE-DABCO) conjugated polyelectrolyte biocide. When challenged with Pseudomonas aeruginosa and Bacillus atropheaus vegetative cells from liquid suspension, light-activated PPE-DABCO effects 1.2 and 8 log, respectively, losses in viability of the exposed bacteria. These results suggest that conjugated polyelectrolytes retain their activity when grafted to fabrics, showing promise for use in settings where antimicrobial textiles are needed.

Experimental and theoretical examination of surface energy and adhesion of nitrifying and heterotrophic bacteria using self-assembled monolayers.

Biofilm-based systems, including integrated fixed-film activated sludge and moving bed bioreactors, are becoming increasingly popular for wastewater treatment, often with the goal of improving nitrification through the enrichment of ammonia and nitrite oxidizing bacteria. We have previously demonstrated the utility of self-assembled monolayers (SAMs) as tools for studying the initial attachment of bacteria to substrata systematically varying in physicochemical properties. In this work, we expanded these studies to bacteria of importance in wastewater treatment systems and we demonstrated attachment rates were better correlated with surface energy than with wettability (water contact angle). Toward the long-term goal of improving wastewater treatment performance through the strategic design of attachment substrata, the attachment rates of two autotrophic ammonia-oxidizing bacteria (Nitrosomonas europaea and Nitrosospira multiformis) and a heterotroph (Escherichia coli) were evaluated using SAMs with a range of wettabilities, surface energies, and functional properties (methyl, hydroxyl, carboxyl, trimethylamine, and amine terminated). Cell attachment rates were somewhat correlated with the water contact angles of the SAMs with polar terminal groups (hydroxyl, carboxyl, trimethylamine, and amine). Including all SAM surfaces, a better correlation was found for all bacteria between attachment rates and surface free energy, as determined using the Lewis Acid-Base approach. The ammonia-oxidizers had higher adhesion rates on the SAMs with higher surface energies than did the heterotroph. This work demonstrated the successful application of SAMs to determine the attachment surface preferences of bacteria important to wastewater treatment, and it provides guidance for a new area of research aimed at improving treatment performance through rational attachment surface design.

Engineered antifouling microtopographies: the role of Reynolds number in a model that predicts attachment of zoospores of Ulva and cells of Cobetia marina.

A correlation between the attachment density of cells from two phylogenetic groups (prokaryotic Bacteria and eukaryotic Plantae), with surface roughness is reported for the first time. The results represent a paradigm shift in the understanding of cell attachment, which is a critical step in the biofouling process. The model predicts that the attachment densities of zoospores of the green alga, Ulva, and cells of the marine bacterium, Cobetia marina, scale inversely with surface roughness. The size and motility of the bacterial cells and algal spores were incorporated into the attachment model by multiplying the engineered roughness index (ERI(II)), which is a representation of surface energy, by the Reynolds number (Re) of the cells. The results showed a negative linear correlation of normalized, transformed attachment density for both organisms with ERI(II) x Re (R(2) = 0.77). These studies demonstrate for the first time that organisms respond in a uniform manner to a model, which incorporates surface energy and the Reynolds number of the organism.

A low-cost, rapid deposition method for "smart" films: applications in mammalian cell release.

The "smart" polymer poly (N-isopropyl acrylamide), or pNIPAM, has been studied for bioengineering applications. The polymer's abrupt change in hydrophobicity near physiologic temperatures makes it ideal for use as a substrate in many applications, including protein separation and prevention of biofouling. To tether pNIPAM, many techniques such as plasma deposition, have been utilized, but most are expensive and require long equipment calibration or fabrication periods. Recently, a novel method for codepositing this smart polymer with a sol-gel, tetraethyl orthosilicate (TEOS), was developed. In this work, we adapt this technique for applications in mammalian cell attachment/detachment. In addition, we compare the effects of the pNIPAM/TEOS ratio to functionality using surface analysis techniques (XPS and contact angles). We found the optimal ratio to be 0.35 wt % pNIPAM/TEOS. Cell detachment from these substrates indicate that they would be ideal for applications that do not require intact cell sheets, such as biofouling prevention and protein separation, as this technique is a simple and affordable technique for pNIPAM deposition.

Attachment and detachment of bacteria on surfaces with tunable and switchable wettability.

Controlling accumulations of unwanted biofilms requires an understanding of the mechanisms that organisms use to interact with submerged substrata. While the substratum properties influencing biofilm formation are well studied, those that may lead to cellular or biofilm detachment are not. Surface-grafted stimuli-responsive polymers, such as poly (N-isopropylacrylamide) (PNIPAAm) release attached cells upon induction of environmentally-triggered phase changes. Altering the physicochemical characteristics of such polymeric systems for systematically studying release, however, can alter the phase transition. The physico-chemical changes of thin films of PNIPAAm grafted from initiator-modified self-assembled monolayers (SAMs) of omega-substituted alkanethiolates on gold can be altered by changing the composition of the underlying SAM, without affecting the overlying polymer. This work demonstrates that the ability to tune such changes in substratum physico-chemistry allows systematic study of attachment and release of bacteria over a large range of water contact angles. Such surfaces show great promise for studying a variety of interactions at the biointerface. Understanding of the source of this tunability will require further studies into the heterogeneity of such films and further investigation of interactions beyond those of water wettability.

CVD for the facile synthesis of hybrid nanobiomaterials integrating functional supramolecular assemblies.

In this letter, we present a simple one-step, versatile, scalable chemical vapor deposition (CVD)-based process for the encapsulation and stabilization of a host of single or multicomponent supramolecular assemblies (proteoliposomes, microbubbles, lipid bilayers, and photosynthetic antennae complexes and other biological materials) to form functional hybrid nanobiomaterials. In each case, it is possible (i) to form thin silica layers or gels controllably that enable the preservation of the supramolecular assembly over time and under adverse environmental conditions and (ii) to tune the structure of the silica gels so as to optimize solute accessibility while at the same time preserving functional dynamic properties of the encapsulated phospholipid assembly. The process allows precise temporal and spatial control of silica polymerization kinetics through the control of precursor delivery at room temperature and does not require or produce high concentrations of injurious chemicals that can compromise the function of biomolecular assemblies; it also does not require additives. This process differs from the conventional sol-gel process in that it does not involve the use of cosolvents (alcohols) and catalysts (acid or base).

Light and dark biocidal activity of cationic poly(arylene ethynylene) conjugated polyelectrolytes.

In this paper we report a study of cationic poly(arylene ethynylene) conjugated polyelectrolytes. The objective of the study was to compare the behavior of a polymer where a thiophene has replaced a phenyl ring in poly(phenylene ethynylene) polycations (PPE) previously investigated. Properties of solution phase and physisorbed suspensions of the polymer on microspheres were investigated. The photophysical properties of the polymer are evaluated and used to understand the striking differences in biocidal activity compared to the PPE polymers previously examined. The principal findings are that the thiophene polymer has remarkable dark biocidal activity against Pseudomonas aeruginosa strain PAO1 but very little light-activated activity. The low light-activated biocidal activity of the thiophene polymer is attributed to a highly aggregated state of the polymer in aqueous solutions and on microspheres as a physisorbed coating. This results in low triplet yields and a very poor sensitization of singlet oxygen and other reactive oxygen intermediates. The highly effective dark biocidal activity of the thiophene-containing polymers is attributed to its high lipophilicity and the presence of accessible quaternary ammonium groups. The difference in behavior among the polymers compared provides insights into the mechanism of the dark process and indicates that aggregation of polymer can reduce light activated biocidal activity by suppressing singlet oxygen generation.

Conjugated polyelectrolyte capsules: light-activated antimicrobial micro "Roach Motels".

Microcapsules consisting of alternating layers of oppositely charged poly(phenylene ethynylene)-type conjugated polyelectrolytes (CPEs) were prepared via layer-by-layer deposition onto MnCO3 template particles followed by dissolution of the template particles using an ethylenediaminetetraacetate solution. The resulting microcapsules exhibit bright-green fluorescence emission characteristics of the CPEs. Strong antimicrobial activity was observed upon mixing of polyelectrolyte capsules with Cobetia marina or Pseudomonas aeruginosa followed by white-light irradiation. It was demonstrated that the materials act as highly effective light-activated micro "Roach Motels" with greater than 95% kill after exposure to approximately 1 h of white light.

Light-induced biocidal action of conjugated polyelectrolytes supported on colloids.

A series of water soluble, cationic conjugated polyelectrolytes (CPEs) with backbones based on a poly(phenylene ethynylene) repeat unit structure and tetraakylammonium side groups exhibit a profound light-induced biocidal effect. The present study examines the biocidal activity of the CPEs, correlating this activity with the photophysical properties of the polymers. The photophysical properties of the CPEs are studied in solution, and the results demonstrate that direct excitation produces a triplet excited-state in moderate yield, and the triplet is shown to be effective at sensitizing the production of singlet oxygen. Using the polymers in a format where they are physisorbed or covalently grafted to the surface of colloidal silica particles (5 and 30 microm diameter), we demonstrate that they exhibit light-activated biocidal activity, effectively killing Cobetia marina and Pseudomonas aeruginosa. The light-induced biocidal activity is also correlated with a requirement for oxygen suggesting that interfacial generation of singlet oxygen is the crucial step in the light-induced biocidal activity.