Symposium SB05—Antimicrobial Materials Against Coronaviruses and Other Nosocomial Pathogens

Some of the pathogenic challenges currently facing the global community are becoming increasingly life-threatening. For instance, infectious microbes such as SARS-CoV-2 and MRSA constitute major healthcare concerns as invisible predators primarily, but not restricted to, stalking the elderly and immune-compromised. In response, various materials-related methods involving nanoparticles or surface functionalization have been proposed to combat this menace, but many are either limited to specific microbes or promote environmental contamination. In this work, we introduce two promising antimicrobial strategies in which several polymer films and coatings serve as exemplars to demonstrate their potential. The first focuses on embedding photosensitive dye molecules capable of generating singlet oxygen, a strong oxidizing agent, into polymer matrices as films, as well as coatings on polymer fibers and other substrates. In the presence of molecular oxygen and incoherent visible light, this system inactivates at least 99.9999% of two Gram-positive bacterial strains, as well as at least 99.9% of three Gram-negative bacterial strains (belonging to the ESKAPE family of bacteria primarily responsible for nosocomial infections) and three virus strains, in ca. 1 h under the conditions employed. Moreover, these materials retain their antimicrobial efficacy even after a month of exposure to light. Alternatively, a vastly different method employs anionic polymer surfaces that are inherently capable of promoting a dramatic pH reduction, resulting in a hostile acidic environment that rapidly kills at least 99.99% of the pathogens tested (including 3 drug-resistant bacteria such as MRSA, as well as C. difficile and the SARS-CoV-2 and HCoV229E viral strains) in less than 5 min. These materials afford highly effective and largely unexplored pathways to rapid, scalable and broad-spectrum microbial inactivation.

Coronavirus has affected the entire global community owing to its transmission through respiratory droplets. This has led to the mandatory usage of surgical masks for protection against this lethal virus in many countries. However, the currently available disposable surgical masks have limitations in terms of their hydrophobicity and reusability. Here, we report a single-step spray-coating technique for the formation of a superhydrophobic layer of single-walled carbon nanotubes (SWCNTs) on melt-blown polypropylene (PP) surgical mask. The sprayed SWCNTs form nanospike-like architecture on the PP surface, increasing the static contact angle for water from 113.6° ± 3.0° to 156.2° ± 1.8° and showing superhydrophobicity for various body fluids such as urine, tear, blood, sweat, and saliva. The CNT-coated surgical masks also display an outstanding photothermal response with an increase in their surface temperature to more than 90 °C within 30 s of 1 sun solar illumination, confirming its self-sterilization ability. Owing to the cumulative effect of the superhydrophobicity and photothermal performance of the SWCNTs, the CNT-coated masks show 99.99% higher bactericidal performance towards Escherichia coli than pristine masks. Further, the virucidal ability of the SWCNT-coated mask, tested by using virus-like particles, was found to be almost 99% under solar illumination. As the spray-coating method is easily scalable, the nanotube-coated mask provides cost-effective personal protection against respiratory diseases.

In recent years, the rise in antibiotic-resistant bacteria and other nosocomial pathogens has highlighted the need for passive, self-cleaning surfaces and personal protective equipment. The motivation to develop such materials has been emphasized in the past year with the outbreak of the coronavirus pandemic. Numerous classes of materials have been explored for such applications, including metal oxides, organic photosensitizers, and other compounds. Our interdisciplinary team is currently working with titania (TiO₂), a common photocatalytic agent known for its production of reactive oxygen species (ROS), in an attempt to lower the band-gap and increase light absorption in the visible region (>400 nm) and test for antimicrobial ability.
We reduce the bandgap into the visible spectrum via the introduction of mid-gap states using various doping techniques. In particular, we pursue cationic and self-doping methods with readily available components and develop a facile synthesis method. The procedure rapidly reaches completion under mild reaction conditions (ambient temperature, 1 atm) to produce visible-light absorbing TiO₂. Multiple characterization techniques are utilized to investigate the properties of the formed particles and determine the effect of the doping on the band-gap and the subsequent pathogen inactivation. Crystallographic structure, UV-Vis absorption properties, particle morphology, the cationic valence state and other properties are investigated to assess the materials’ viability for self-cleaning surfaces.
Materials are exposed to a variety of pathogens: methicillin resistant Staphylococcus aureus (MRSA), multi-drug resistant Acinetobacter baumannii (MDRAB), and human coronavirus 229E (HCoV-229E) to determine their overall antimicrobial efficacy through antimicrobial photodynamic inactivation (aPDI). Various concentrations of particles (5 and 10 mg/mL), light intensities (65 +/- 5 and 85 +/- 5 mW/cm²), and illumination times (30, 60, and 90 minutes) are tested. The particles exhibit promising antimicrobial properties with inactivation up to the limit of detection (6 log units, 99.9999%). Further investigations utilizing ROS scavengers and probes are performed to determine the primary mechanism of inactivation. Long term viability and photobleaching studies are also explored, demonstrating the particles’ ability to be exposed to intense illumination for extended periods with no effect on overall antimicrobial properties. Inductively coupled plasma optical emission spectroscopy is utilized to confirm dopants were not leaching from particles and acting as cytotoxic agents.
Several coating methods (slot-die and spray coating) are employed to test particle compatibility with surface application and reuse. This is done by slot-die and spray coating a UV-curable polymer matrix, containing photoactive TiO₂ particles onto glass slides and woven materials. Slot-die coating is chosen because it gives a uniform film across a variety of length scales and translates well to industrial surface coating applications. Similarly, spray coating can aid in applying these particles to personal protective equipment such as N-95 masks as well as other woven materials like curtains and upholstery commonly found in hospitals and other public areas. Further aPDI tests are being performed on coated materials to ascertain how well antimicrobial behavior can be transferred and utilized in the public sector.

Copper and it’s alloys have long been known for their antiviral and antibacterial properties, and in the course of the global pandemic, research on ways to accelerate their effectiveness have come to the forefront. Recent studies have reported that when the crystal size of bulk copper reduces towards the nanoscale, the anti-viral and anti-microbial efficiency increases non-linearly. The mechanisms for this are diverse, and include the increase in volume fraction of ionic pathways to the surface, oxidation-reduction, topological features and others. In this study, we investigate the antibacterial characteristics of nanocrystalline pure copper produced by severe plastic deformation (SPD), and based on a literature review and our own emerging data, attempt to unravel the mechanisms that promote bacteria or cell destruction as a function of grain size. The versatility of SPD methods allows for the fabrication of bulk components that, unlike thin-film coating approaches, may enable scalability of medical applications.

Currently, over a million people die each year due to some form of antibiotic-resistant infection. At the same time, the fast mutation of other pathogenic organisms – fungi, viruses and parasites – are also developing resistance to the drugs that are used to tackle them almost as quickly as new ones can be made. A devastating example is the recent outbreak of SARS-CoV-2 virus that began in December 2019 in China, and by March 15, 2021, the global case rate reached 120 million and 2.65 million people died around the world. This pandemic can be explained by the fact that today’s world is a global village, with many shared areas and surfaces (e.g., door handles, airplanes, and hospitals) that can serve as routes by which infectious diseases can easily spread around the world within hours. For instance, the SARS-CoV-2 virus has been shown to remain active for up to three days on plastic and stainless-steel door handles and up to 24 hours on cardboard. Some bacteria – including E. Coli and MRSA – can survive for several months on inanimate surfaces. Consequently, special attention has been devoted to the development of new material and surfaces with antimicrobial and antiviral properties that are less susceptible to resistance and can minimize the spread of pathogens at bay. Among different materials, copper (Cu) and silver (Ag) have been recognized as most widely used and well-documented materials that can effectively inactivate different microbes upon direct contact. However, these metals require a few hours of direct contact with the bacteria or virus that lands on its surface to completely inactivate them. Furthermore, it is impractical to make all high-risk surfaces in public places (e.g., buttons and door handles) and medical instruments out of Ag or Cu due to several limiting factors such as cost, weight and mechanical properties.
In this presentation, we will discuss two novel use of laser surface nano texturing and immobilization technologies to enhance the bacterial rate of killing of such metal surfaces (Cu and Ag) as well as effectively immobilize them onto other biologically inert metallic surface. This presentation is divided into two main sections, each showcasing one of these technologies and their applications. In the first section, we will describe a one-step laser nanotexturing process to enhance the bactericidal properties of Cu surfaces via concurrent selective modification of surface topography and chemistry of laser nanotextured copper. This process provides a unique and facile approach for creating robust Hierarchical Micro/Mesoporous structures directly onto metal surfaces in ambient conditions, eliminating complexities of chemical etching as well as metal nanoparticles synthesis and coating processes. In this section we will look at the surface chemistry and morphology that changes with the laser processing conditions and how these surface properties enhance the overall antibacterial properties of the Cu surface against a range of gram positive and negative antibiotic resistant bacteria.
In the second part of this talk, we will showcase the use of a combination of laser nanotexturing and laser immobilization to selectively reduce Ag ionic compounds directly onto the surface of biologically inert titanium metal implants. We will discuss the systematic experimental studies (e.g., elemental and surface morphology analysis) that were conducted with different laser processing condition and silver coating compositions which resulted in both improved cellular integration and antibacterial properties while having minimal change in the bulk mechanical properties of the titanium substrate. We will conclude this presentation by briefly commenting on our efforts to translate some of these technologies into clinical practice and the future directions.

Aiming to develop scalable, potent, color variable, and comfortable antimicrobial textiles that can help reducing infection transmission in healthcare facilities, we explored photodynamic antimicrobial polyethylene terephthalate (PET) /cotton blended fabric (TC) comprising of photosensitizer-conjugated cotton fibers and disperse dye dyed PET fibers. A small library of TC blended fabrics was constructed wherein the PET fibers dyed with different traditional disperse dyes dominated the color of the fabrics and thereby enables variable color expression, while cotton fibers were covalently coupled with thionine acetate as photosensitizer generating photobiocidal under illumination. Physical (SEM, CLSM, TGA and mechanical strength), colorimetric (K/S and CIELab values) characterizations were employed to investigate the resultant fabrics and the photooxidation studies with DPBF demonstrated the ability of these materials to generate reactive oxygen species (i.e., singlet oxygen) upon visible light illumination. The best results of these materials demonstrated the 99.985% (~3.82 log unit reduction, P = 0.00208) photodynamic inactivation of Gram-positive S. Aureus, and achieved detection limit 99.99% inactivation (4 log unit reduction, P ≤ 0.0001) against Gram-negative E. coli upon illumination with visible light (60 min; ~300 mW/cm²; λ ≥ 420 nm). Different disperse dyes on the fabric were observed no photophysical competition relation, leading to lower aPDI efficiency, with photosensitizer and furthermore, playing a ‘protection’ role for photosensitizer during the photobleaching process which ends up improving the photostability of these materials. Taken together, these results suggested the feasibility of low cost, scalable and color variable thionine conjugated TC blended fabrics as potent self-disinfecting textiles.

Bacterial biofilms are communities of microbes encased within a self-produced slimy extracellular polymeric substance (EPS) attached to a surface. Biofilms can enable bacteria to survive in hostile environments and release fresh bacteria into the environment. Compared to their planktonic counterparts, biofilm bacteria embedded in layers of EPS are highly resistant to antibiotics and conventional disinfection strategies as compared to their planktonic counterparts.. Particularly, medical implant-associated biofilms accounts for significant increase in chronic diseases and mortality worldwide.
To eliminate biofilm colonization on medical implants, various strategies including antibiotics, disinfectants, cationic materials, and anti-biofilm coatings have been attempted so far with some success. Surfaces coated with gold nanorods (GNRs) have been demonstrated to be promising as they are capable of eradicating bacterial biofilm upon exposure to near-infrared (NIR) radiation. When exposed to NIR, GNRs can generate plasmonic photothermal heat that can inhibit microbial biofilms and thereby, reduce implant failures and the need for revision surgeries. However, the performances of these surfaces may differ as the fabrication methods vary. In addition, the biocompatibility and anti-biofilm performances of such functionalized surfaces prepared by various methods are not yet fully investigated.
This study addresses the regulatory knowledge gap in evaluating safety and effectiveness of GNR-coated titanium surfaces with antibiofilm activity. Herein, we have used medical grade titanium as a model material and coated GNRs by different techniques via thiol- or amine-terminated silane. Successful deposition of GNRs was confirmed and quantified by scanning electron microscope and X-Ray spectroscopies. The results showed that the more polar amine-terminated silane achieved a higher amount of deposited nanorods on titanium. Biocompatibility testing was conducted in vitro on human osteoblast, macrophages, and fibroblast cells by following ISO-10993 Part 5: Tests for in vitro cytotoxicity. Titanium coated with GNRs solutions up to 200 µg/mL did not show significant cytotoxicity and were favorable to cell attachment and proliferation. In addition, toxicological assessments, such as cytotoxicity and measurement of inflammatory cytokine levels, are conducted on fabricated surfaces exposed to NIR. Anti-biofilm performance of the GNR-coated surfaces in the presence or absence of NIR will be tested against Staphylococcus aureus and Nontuberculous mycobacterial biofilms. The results of this research will help the FDA assess medical device submissions that use similar nanomaterial-based anti-biofilm technology.

Hydrophobic coatings rely on chemical apolarity and hierarchical roughness to achieve high contact angles and low roll-off angles that lead to self-cleaning and antibacterial properties. Current hydrophobic coatings tend to be delicate and lose their properties easily when subjected to water spray. Many of these coatings become impaled (pinned) with water droplets immediately after incremental hydrodynamic wear from water spray. This hydrodynamic wear differs from seemingly more aggressive mechanical wear including scratching with sandpaper - a common approach used to claim both self-similarity of a material and extreme robustness against wear. However, the hydrodynamic wear of these self-similar materials can quickly erode the hydrophobic constituents, exposing more hydrophilic materials that lead to quick wetting out despite the self-similarity of the greater material. In this work, we systematically study several approaches that have claimed robustness against wear and find that most approaches fall short of application requirements for withstanding a simple onslaught of water spray. Through quantifying hydrodynamic wear, we have also been able to demonstrate an advancement on the leading systems through developing a novel superhydrophobic material. We compare a popular off-the-shelf industry solution to these recent academic works, as well as offer an analytical model that nicely predicts the hydrodynamic wear of these systems.

Silver nanoparticles (Ag NPs) have well-studied antimicrobial properties effective against many types of bacteria and fungi. Fine Ag NPs have also demonstrated antiviral activity against H1N1 (influenza), SARS-CoV-2, and other viruses.^[1,2] The antimicrobial properties of Ag NPs are due to the release of silver ions that inhibit respiratory enzymes of bacteria and fungi. In its main antiviral mechanism, Ag NPs bind to and inactivate membrane proteins, thereby preventing the virus from entering the host cell.^[1] Applications of Ag NPs in textiles have gained worldwide attention due to the importance of antimicrobial textiles for wound dressings, medical equipment and staff uniforms, bedsheets, and others. Because textiles serve as a medium for microbe growth and cross-contamination, there is a growing demand for antimicrobial textiles. To reduce waste, especially since the textile industry produces over 10 million tons of waste cotton cloth globally each year, this study seeks to synthesize Ag NPs on discarded cotton muslin tailoring cloth.^[3] Cotton fabric can be deweaved into cellulose fibers, which act as a stabilizing agent on which silver nanoparticles can be synthesized through the reduction of silver nitrate with trace amounts of sodium borohydride.

Fabric was deweaved by stirring in a 3:1 by volume ratio solution of 0.5 M citric acid and 0.5 M sodium nitrate at 50 °C and 600 rpm for 15 minutes. After drying, the fibers were used for the synthesis of silver nanoparticles in two ways. Both samples were treated with a solution of 20 mL silver nitrate (0.01 M) and 2-5 drops of sodium borohydride (0.01 M) until a color change was observed. The first sample was placed for 20 minutes at room temperature, while the second sample was ultrasonicated for 20 minutes at 40 °C. Both samples were then air-dried. The solution used in the synthesis process, which contained silver nitrate, sodium borohydride, and silver nanoparticles, was then analyzed using UV-Vis spectrometry. Results showed a peak at 325 nm, which is characteristic of silver nanoparticles.^[4] The samples were then observed with SEM to visualize the size and features of the nanoparticles, and EDX was simultaneously performed to verify the existence of silver. The fibers heated in the ultrasonicator showed a higher silver signal in EDX than the non-heated samples, and the silver coating was present on more parts of the fibers in the ultrasonicated sample. The size of the nanoparticles was below the resolution of the SEM. X-ray photoelectron spectroscopy showed that the silver in the nanoparticles existed in both elemental and oxidized forms, both of which are antimicrobial.^[5]

We then performed an antibacterial test using S. aureus and E. coli. Deweaved fibers without Ag NPs did not exhibit any antibacterial properties, while both species of bacteria showed zones of exclusion when the Ag NP-containing fibers were used. The zone of exclusion was larger in the culture of S. aureus, suggesting that silver nanoparticles may be more effective against Gram-positive bacteria. Experiments are underway in order to determine the antiviral properties of the silver coating against H1N1 influenza. Hence, this technique has the potential to produce antiviral and antibacterial fibers that can be incorporated into textiles.

[1] Mori, Y., et. al, Nanoscale Res. Lett., 2013, 8, 93
[2] Jeremiah, S., et. al, Biochem. Biophys. Res. Commun., 2020, 533, 195
[3] Mohamed S., et. al, Polymers, 2021, 13, 626
[4] Santos, K. de O., et. al, J. Phys. Chem. C., 2012, 116, 4594
[5] Dharmaraj, D., et. al, J. Drug Deliv. Sci. Technol., 2021, 61, 102111

Efforts to control hospital acquired infections (HAIs) have been hampered by the emergence of drug-resistant pathogens, necessitating the pursuit of advanced functional materials that are capable of the self-disinfection of such microbes in hospital environments. To that end, we have explored the feasibility of antimicrobial photodynamic inactivation (aPDI) of bacteria and viruses using photodynamic materials, as well as pathogen inactivation using "pH-drop" based materials. In vitro aPDI studies were performed against bacteria and viruses employing photosensitizer-embedded or conjugated nanofibrillated cellulose, polyacrylonitrile or nylon nanofibers, dual-dyed wool/acrylic blended fibers, olefinic block copolymers, and spray coatings. Pathogens were cultured, deposited onto the materials, and subsequently illuminated with visible light (400–700 nm, 65-80 mW/cm², 5-60 min), and their survivability was determined via colony counting or plaque assay methods. For natural polymer scaffolds, cellulose-porphyrin conjugates (either as nanocrystals, nanofibers, or paper sheets) were found to be highly effective against a broad spectrum of pathogens: our best results demonstrated that S. aureus, A. baumannii, P. aeruginosa and K. pneumoniae all exhibited photodynamic inactivation by 99.99+%, as well as inactivation of dengue-1 virus (>99.995%), influenza A (~99.5%), and human adenovirus-5 (~99%). As an alternative strategy, non-covalent approaches to photodynamic materials using artificial polymers were also explored: i) using electrospinning, cationic porphyrin and BODIPY photosensitizers were embedded into polyacrylonitrile and nylon nanofibers, and the resultant nonwoven materials possessed both antibacterial and antiviral activities; ii) using melt-pressing, we developed a photosensitizer-embedded olefinic block copolymer that exhibited excellent antimicrobial properties against a range of microbes, including Gram-positive and Gram-negative drug-resistant bacteria, as well as against enveloped and non-enveloped viruses. More recently, we have explored photodynamic coatings on polymer microfibers for pathogen inactivation, and have demonstrated population reductions of >99.9999 and 99.6% for S. aureus and antibiotic-resistant E. coli, respectively, after exposure to visible light for 1 h. In response to the current COVID-19 pandemic, we also confirmed that these coated fibers can inactivate a human common cold coronavirus serving as a surrogate for the SARS-CoV-2 virus. In a non-photodynamic approach, excellent antimicrobial properties were acheived through the use of charged multiblock polymers wherein the midblock was selectively sulfonated: the resultant hydrophilic and water-swellable polymers led to self-sterilizing surfaces that rapidly act (killing more than 99.9999% in just 5 min) against a wide range of Gram-positive and -negative bacteria, as well as enveloped and non-enveloped viruses. Together, these results demonstrate that such materials may have widespread applicability for non-specific pathogen disinfection, and further research may lead to their application in hospitals and healthcare-related industries where novel materials with the capability of reducing the rates of transmission of a wide range of bacteria, viruses, and fungi, particularly of antibiotic resistant strains, are desired.

Biofilms and microbial colonies are ubiquitous on nearly any substrate exposed to water and can easily spread microorganisms causing severe conditions in water filtration systems. To defeat the formation of biofilm, deposition of silver nanoparticles (AgNPs) on water treatment membranes has been widely exploited through various techniques including physical vapor deposition and chemical vapor deposition, however, the deposited AgNPs are not effectively anchored to the surface. In this study, we functionalized the surface of a carbon membrane via a CO₂ laser in order to generate oxygen containing species to aid perfect attachment of AgNPs on the membrane surface while providing a superhydrophilic surface. Various characterizations including SEM, XRD, and EDX displayed the nanotextures, enhanced surface roughness, and significant alteration of surface chemistry caused by laser processing. To validate the strong AgNPs attachment to the surface, vigorous mechanical agitation as well as probe sonication were performed. Finally, direct contact killing along with antibiofouling properties of laser functionalized carbon membrane with immobilized AgNPs was performed and compared to the pristine membrane. The results demonstrated an exceptional antibacterial of the laser functionalized membrane to fully eradicate the pathogenic bacterial strains of Escherichia coli and Pseudomonas aeruginosa. It is believed that the developed technology opens new opportunities towards a rapid, scalable, and cost-effective preparation technique for fabrication of carbon membranes with outstanding antimicrobial activities suitable for water purification applications.

The ability of bacteria to form complex structures called biofilms create numerous human health and industrial problems. These 3D architectures are defined by the extracellular polymer substance (EPS) that consists of a protective assembly of biomolecules surrounding encased planktonic bacterial cells. The current strategies for targeting biofilms weaken the EPS layer but are typically organic in nature and are subject to enzymatic degradation. Nanoparticles are a promising new class of anti-biofilm agents for their highly tunable physical and chemical properties, stability, and ability to interact with biological molecules, namely proteins. Here, we report the anti-biofilm activity of l-cysteine or d-cysteine derived chiral graphene quantum dots (L/D-GQDs) against amyloid rich staphylococcus aureus (S. aureus) biofilms. Both enantiomers are capable of biofilm disruption. Notably, D-GQDs displayed higher affinity for amyloid fibers causing increased biofilm dispersal compared to L-GQDs. Chiral GQDs mimic peptide binding proteins, interfering with the self-assembly phenol soluble modulins(PSMs) , the monomeric form of amyloid fibers responsible for biofilm structuring and multiple other virulence factors. The effect of chiral GQDs on key PSM functions including cytolysis, neutrophil production, and anti-biofilm properties were also explored. The chiral interactions between GQDs and biofilm components give rise to a highly tunable antibiofilm NP platform.

The world is facing a growing challenge of antibiotic resistance, making diseases assumed benign with antibiotic treatment once again a looming threat to all members of society. Surfaces play an important role in the transmission of bacterial pathogens. Not only can bacteria survive on surfaces, but in aqueous surroundings, surfaces allow the formation of biofilms, stable bacterial ecosystems that make it extremely difficult to remove bacterial contamination. Preventing bacterial transmission on surfaces is relevant in a variety of settings such as public transport, sanitary facilities, electronic devices, food production and in particular, health care. Hospitals are fertile breeding grounds for antibiotic resistance, as patients gather there, bringing many different types of pathogens. Compromised immune systems combined with the widespread use of antibiotic agents promote the development of antibiotic resistance.

Therefore, there is a growing push to develop antibacterial and bactericidal surfaces to reduce the transmission of bacterial pathogens. Common approaches towards designing antibacterial surface employ the release of biocidal agents. Though effective in some applications, biocide releasing systems can contribute to antibiotic resistance, as release rates decrease with time. Furthermore, biocide release can result in undesired pollution of its environment. Especially nanoparticles, commonly used as biocide agents, have toxic effects on other organisms even at low concentration.

To kill bacteria while avoiding the use of biocidal agents, nanotopographies can be designed to mechanically kill adhered bacteria by rupturing the cell membrane during the adhesion process. This form of bactericidal action was initially discovered on cicada and dragonfly wings, which inspired the production of nanostructures on black silicon. Further studies have been conducted on graphene, titanium and gold, as well as stiff polymers such as PMMA and polystyrene. However, the mechanism leading to bacteria death is not yet fully understood. Recent literature points to differences in bactericidal effects of extremely sharp nanostructures such as vertical graphene and carbon nanotubes that could cut the bacterial membrane versus wider nanopillars or nanocones. It is speculated that the latter functions by exerting lateral force on the membrane that leads to membrane rupture.

In order to make use of bactericidal properties of nanotopographies in a wide range of applications, it is necessary to fabricate such surfaces on a large variety of materials. Polymers are widely used in food, sanitation and health settings and often times soft or flexible surfaces are required. It is therefore necessary to understand the interplay between pillar geometry and elastic modulus on the bactericidal mechanism. Furthermore, fabrication methods need to permit low-cost upscaling to large surface areas of varying geometries.

We have developed a nanoimprint lithography process for rapid, simple and scalable fabrication of nanopillar arrays using UV-curable polymeric resins, therefore permitting the use of a wide range of polymers with varying material properties. In addition, surface material properties can differ from the bulk material by combining polymer foil as a carrier with UV-curable resin for the surface topography.

Using this process, we have fabricated nanopillar arrays of different pillar diameters, spacings and materials in order to investigate the effect of pillar geometry and elastic modulus on bactericidal efficacy. After incubation of samples in bacteria-PBS suspension, the number of live and dead cells on each array are quantified via fluorescent live/dead staining. High-resolution SEM imaging shows the change in bacterial morphology and degree of pillar-bending.

Our results help to shed light on bactericidal mechanisms of nanopillars and determine pillar diameter, periodicity and stiffness in order to optimize bactericidal action.

Nosocomial bacterial infections are a major health crisis, often occurring due to bacterial contamination of medical devices or implants. Antimicrobial coatings are promising materials for preventing bacterial contamination of medical devices. Protein-based materials offer a sustainable and biocompatible strategy for combatting bacterial infections, either by harnessing antimicrobial properties of native proteins or through the fabrication of antimicrobial-loaded protein scaffolds. However, the high aqueous solubility of proteins often results in materials with poor aqueous stability and mechanical properties. Therefore, current strategies rely on physical or chemical treatments to enhance the stability of protein-based materials and prevent rapid degradation in biological milieus. Thermal treatment is an additive-free approach for stabilizing protein films, but proteins are prone to denaturation at high temperatures leading to loss of function.

We employed a thermal treatment approach in fluorous media (hereafter referred to as fluorous-cured or FC) to enhance aqueous stability of protein films while minimizing protein denaturation. We hypothesized that the fluorous environment prevents rearrangement of proteins at the interface during heat treatment thereby minimizing denaturation. Through this strategy, we developed anti-fouling protein coatings for medical implants, by harnessing the native properties of BSA (Bovine serum albumin). FC-BSA films retained most of the secondary structure of the native protein but remained stable in aqueous media. FC-BSA films were hydrophilic and had an overall negative charge, similar to native BSA. The negative charge of the film prevents bacterial contamination through electrostatic repulsion. Conformal BSA coatings were generated on dental implants (screws) by dip-coating in BSA solution followed fluorous-curing. FC-BSA coated dental implants successfully resisted contamination from E. coli.

Anti-fouling FC-BSA films provided passive protection against bacterial contamination by preventing bacterial adhesion on the surface. However, they cannot actively eliminate bacteria as BSA is not an inherently biocidal protein. We hypothesized that electrostatic interactions may be utilized to load antibiotics into FC-BSA films to impart antibacterial activity. FC-BSA films were loaded with a cationic antibiotic colistin sulfate by incubated the films in antibiotic solution overnight. Release of colistin was modulated by ionic strength of supernatant – high ionic strength results in slow release of colistin while low ionic strength results in a rapid burst-release. The colistin-loaded FC- BSA not only retained their anti-fouling behavior while also eliminated bacteria in the vicinity of the dental implant.

Colistin-loaded FC-BSA films are biocompatible antibacterial coatings for medical implants, offering passive protection in the form of anti-fouling behavior as well as active antimicrobial activity through release of antibacterial agents. Thermal treatment in fluorous media offers an additive-free, sustainable, and biodegradable approach for fabricating protein-based materials while retaining the native properties of protein precursors.

The field of nanotechnology has grown immensely in the last couple of decades owing to the unique properties exhibited by nanomaterials. One such class of nanomaterials are the metal oxide nanoparticles or nanostructures ranging in size from 1 to 100 nm and available in several different shapes. These metal oxide nanostructures find applications in many fields including catalysis, drug delivery, sensing, semiconductors etc. Along with these aforementioned applications, metal oxide nanostructures are favorable candidates as antimicrobial agents.
In the present seminar, the synthesis, characterization and testing of metal oxide nanostructures as antimicrobial agents will be discussed. The influence of nanostructure size and shape on their antimicrobial performance will be evaluated. Additionally, the influence of underlying substrates of the nanostructures on antimicrobial performance will also be addressed. Finally, a tentative guideline will be provided to obtain the best performing antimicrobial nanostructures through the above-mentioned investigation.

Carbon Nanomaterials such as carbon nanotubes as well as graphene and related materials, present many exceptional properties, including antimicrobial ones. The transfer of the latter through their incorporation into materials has generated a growing interest especially the last ten years [1]. However, due to their intrinsic toxicity not only for microbes but also potentially for humans and the environment, their potential release in use conditions is a central issue in terms of practical applications.
Our work stands at the interface between materials science / chemistry and biology, thanks to the fruitful collaboration between our two laboratories. It focuses on the comparison of different carbon nanomaterials against 2 kinds of pathogens: bacteria and viruses. After deposition of carbon nanomaterials onto a surface, we measure the antibacterial activity against both gram + (S. aureus) and gram - (P. aeruginosa) bacteria. In the current context of the COVID-19 pandemic, we also investigate the antiviral activity of the same carbon nanomaterials against SARS-CoV-2. This work is especially important in the current context of the worldwide withdrawal from the market of face masks containing "graphene" for safety reasons, while the literature about the actual antiviral activity of such nanomaterials is clearly deficient.
Our most recent results will be presented and discussed considering the many technical challenges associated to such research.
Reference:
L. Giraud, A. Tourrette, E. Flahaut
Carbon, 182, (2021), 463-483
"Carbon nanomaterials-based polymer-matrix nanocomposites for antimicrobial applications: a review"

Biofilms are a major issue for industries from global shipping to health underpinning significant markets for antifouling paints and coatings, and antimicrobial surfaces. To combat biofilm growth porous/textured solid surfaces with a lubricant locked-in to the structure by capillary forces have been used to create a stable hemi-solid/hemi-liquid surface or a continuous liquid lubricant coating – a so-called Slippery Liquid Infused Porous Surface (SLIPS). However, performance under flow conditions where there is shear stress at the surface, such as in catheters, remains a concern due to flow-induced depletion of the lubricant. Here, we report an anti-biofilm surface strategy using liquid-like solid surfaces where the risk of lubricant loss is completely removed. Our coating is a Slippery Omniphobic Covalently Attached Liquid-like (SOCAL) surface, developed as an ultra-slippery non-pinning surface for sessile droplets. This surface displays similar wetting properties to SLIPS through its grafted PDMS coating that behaves as a liquid phase approximately 150^oC above its glass transition temperature. However, since it does not flow and is optically transparent, mechanically durable and thermally stable, it offers significant potential for stable anti-biofilm coatings. In this study, SOCAL surface reduced biofilm formation of Staphylococcus epidermidis and Pseudomonas aeruginosa, two key biofilm forming bacterial pathogens, by 3 to 4 orders of magnitude compared to polydimethylsiloxane (PDMS) in both static and dynamic culture conditions. This liquid-like solid surface approach offers a new strategy for biofilm resistant surfaces under flow conditions and may find application in medical devices, biosensors and many other areas where biofilms are problematic.

Biofouling is a global problem that stemmed from the gradual accumulation of biomolecules and microorganisms, such as barnacles and algae, on a submerged surface. It causes pitting and crevice corrosion of vessels' metal surfaces and increases the drag on vessels while sailing. An affordable solution in this regard requires developing a non-toxic, eco-friendly, and sustainable-in-all-marine-environments anti-biofouling coating. Trimethylamine-N-oxide (TMAO), a naturally occurring zwitterionic biopolymer found in the deepwater sea fish, is a promising candidate to this end. Its two oppositely charged moieties are directly connected, possessing zero zwitterionic separation. Experimentally, the antibiofouling property of zwitterions increases as the zwitterionic separation decreases. Employing ab initio molecular dynamics, we thus investigate hydration of TMAO and another zwitterion with a larger separation, carboxybetaine (CBAA1). Our simulations show an inhomogeneous water distribution for both TMAO and CBAA1. Due to the strong hydrogen bonding, a condensed layer of water surrounds the negative moieties. In contrast, the positive moieties have less hydration due to the shielding of three methyl groups.
While comparing the hydrogen bond strength between the zwitterionic oxygens and water molecules, TMAO has far stronger bonding than CBAA1, as validated and quantified by the geometric and quantum theory of atoms in molecules analyses, respectively. Moreover, we utilize the symmetry-adapted perturbation theory (SAPT) to investigate the zwitterion-water interactions. Our SAPT analysis demonstrates that the electrostatic interaction is the dominant attractive term in zwitterions' interactions with hydrogen-bonded-water molecules. The charge transfer between zwitterionic oxygen and water hydrogen plays a critical role in hydrogen bonding, although it is not the dominant term. In the case of TMAO, it is larger, indicating the more rigidity of hydrogen bonding in TMAO than in CBAA1. Our analysis elucidates the origin of the solid antibiofouling behavior of TMAO.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or Coronavirus Disease 2019 (COVID-19), characterized as a pandemic at 2020 march, is affecting 216 countries and territories around the world. The issues of hygiene to defend against germs and viruses have risen significantly. For antiviral and antibacterial, it is important to prevent anti-microbial and anti-fouling on the surface because of transmission of pathogens droplets and surface contact.
We thus developed multi-functionalized polyurethane coating materials and techniques to satisfy both strategies. PU has been widely used commercially and can be controlled with a variety of mechanical properties, such as hardness, toughness, elasticity and so on due to chemical bonding and physical interaction.
Firstly, we synthesized perfluoro tert butanol-hexamethylene diisocyanate (PFtB-HDI) containing of an umbrella-like structure for anti-fouling of airborne infectious particles (< 5 µm in diameter). The fluorine-containing PU coatings have highly hydrophobic properties, scratch resistance and durability after laundry with commercial detergent, which means the coating has a potential of reusability and decreasing waste plastic. The PFtB-HDI-PU-coated fabric had an improved aerosol protection performance without changing its air and moisture permeability.
Secondly, we prepared a quaternary ammonium alkyl chain-isocyanate (QAC-NCO) for coating with curing and bacterio cidal effect. The coatings were prepared with reacting polyol (pHEMA or PEG) and QAC-NCO. This coating contains QCA as a killer of bacteria and urethane bond for durability on the outside and inside. After QAC-NCO coating, percent (%) of bacteria killed for Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) were decreased up to 99.8% and 98.4%, respectively. Our coating systems is based on a polyurethane coating. Therefore, it has a high potential of realizing good performance by applying it to many fields where urethane coating is used.

Photosensitizers immobilized onto various matrixes as self-disinfecting materials has been widely reported in the last few decades. However, the antimicrobial photodynamic inactivation (aPDI) efficiency of those materials was restricted by different factors including the photosensitizer itself, immobilization strategy, light availability, illumination conditions, etc. In view of this, we have focused on the potentiation of photoactive materials in which different photosensitizers have been embedded to either electrospun nanofibers (Mater. Sci. Eng. C, 2021, 111502), bacterial cellulose (BC) (Cellulose 2020, 27, 991-1007), or traditional textile fabrics (ACS Appl. Mater. Interfaces 2019, 11, 29557−29568). The photodynamic antimicrobial membrane comprised of electrospun cellulose acetate (CA) microfibers into which the photosensitizer protoporphyrin IX (PpIX) was in situ embedded (abbr. PPIX/CA) achieved a 99.8% reduction in Gram-positive S. aureus after illumination (Xe lamp, 500 W, λ ≥ 420 nm; 30 min), with a lower level of reduction (86.6%) for Gram-negative E. coli. Potentiation with potassium iodide was found to be an effective way to further enhance the antimicrobial efficacy of the PpIX/CA microfibrous membrane, achieving 99.9999% (6 log units) inactivation of both S. aureus and E. coli in the presence of 25 and 100 mM KI, respectively. As an alternative strategy, we have embedded another promising photosensitizer molybdenum disulfide (MoS₂) into BC membrane by a facile in-situ growth followed by a dip-coating process to coat a chitosan layer on its surface (abbr. BC/MoS₂-CS). When compared with BC/MoS₂ membrane, which showed a 95% reduction of S. aureus and 99.9% inactivation of E. coli, the addition of chitosan led to a satisfying synergistic effect between chitosan and MoS₂, showing a 99.998% reduction of S. aureus and 99.988% inactivation of E. coli. More recently, we have developed photosensitizer-luminous powder dual-coated fabrics using silk-screen printing, and demonstrated that this strategy is able to produce antimicrobial materials working not only in light-available area but also in the dark. Our conclusion is that those photosensitizer-embedded materials, that are not so desirable in anti-microbial efficacy, may still have widespread applicability when appropriate potentiation method was employed.
– References –
1. T. Wang, H. Ke, S. Chen, J. Wang, W. Yang, X. Cao, J. Liu, Q. Wei, R. A. Ghiladi and Q. Wang “Porous Protoporphyrin IX-Embedded Cellulose Diacetate Electrospun Microfibers in Antimicrobial Photodynamic Inactivation“ Mater. Sci. Eng. C, 2021, 111502.
2. T. Wang, L. Xu, H. Shen, X. Cao, Q. Wei, R. A. Ghiladi, and Q. Wang “Photoinactivation of Bacteria by Hypocrellin-Grafted Bacterial Cellulose” Cellulose 2020, 27, 991-1007
3. W. Chen, J. Chen, L. Li, X. Wang, Q. Wei, R. A. Ghiladi, and Q. Wang “Wool/Acrylic Blended Fabrics as Next Generation Photodynamic Antimicrobial Materials” ACS Appl. Mater. Interfaces 2019, 11, 29557−29568.

Infectious diseases caused by pathogenic bacteria and viruses represent a huge global challenge in public health and have prompted the in-demand search for novel and effective alternative antimicrobial agents. We have recently developed carbon dots (CDots) as a new class of effective and efficient visible/natural light-activated antimicrobial agents, with demonstrated ability against a wide range of bacteria and viruses. CDots are surface-functionalized small carbon nanoparticles (CNPs), represent the nanoscale carbon allotrope at zero-dimension. Attributed to the π-plasmon-associated electronic transitions, CDots have remarkable broad optical absorptions in the entire visible spectrum, extending into both near-UV and near-IR, making them excellent visible light-excitable agents. Mechanistically, upon photoexcitation on CDots, there are rapid charge transfers and separation to form electrons and holes redox pairs, followed up the radiative recombinations of the separated redox pairs resulting in emissive excited states, which are responsible for photodynamic production of classical reactive oxygen species (ROS). The combined action of the initially separated redox pairs and the generated classical ROS are responsible for the observed uniquely effective photoinduced antimicrobial function of CDots.
We have demonstrated the highly effective photoactive antimicrobial activities of CDots against various bacteria and viruses, ranging from laboratory model bacteria (E. coli, Bacillus Subtilis), pathogenic foodborne bacteria (Listeria, Salmonella), to multi-drug resistant (MDR) nosocomial pathogens (Enterococcus), as well as various viruses (model MS2 virus, vesicular stomatitis virus (VSV), and marine norovirus (MNV)). For the bacterial species tested, the treatments with 0.1 mg/mL CDots under 1 h household light illumination achieved up to more than 7 log viable cell reductions, demonstrating the high efficiency of photoinactivation by CDots. For viruses tested, the treatment with 5 mg/mL to viral samples containing 10⁶ PFU/mL under 1 h household light illumination inactivated all the viruses, diminishing viral infectivity to their host cells. Bacterial damages by CDots treatments, including elevated levels of lipid peroxidation, increased membrane permeability, and cytoplasmic structural disruptions, are evident. Degradation in viral proteins and genomic RNAs are also observed.
Along with these demonstrated highly effective antimicrobial activities, our studies on property-function correlations have revealed that the optical properties, the surface functional groups and charges, and the passivation layers of CDots are highly correlated with their antimicrobial activities, which make CDots tunable and expandable material platforms for further improvement. With their tunable structures and uniquely advantageous properties together with their non-toxic nature, CDots present new opportunities for effective control and prevention of infectious diseases.
Acknowledgement: The research was supported by NSF grants #1855905, #2102021 , #2102056, and USDA grant #2019-67018-29689.

Medical textiles have a need for repellency to body fluids such as blood, urine, or sweat that may contain infectious vectors that contaminate surfaces and spread to other individuals. Similarly, viral repellency has yet to be demonstrated and long-term mechanical durability is a major challenge. In this work, we demonstrate a simple, durable, and scalable coating on nonwoven polypropylene textile that is both superhemophobic and antivirofouling. The treatment consists of polytetrafluoroethylene (PTFE) nanoparticles in a solvent thermally sintered to polypropylene (PP) microfibers, which creates a robust, low-surface-energy, multilayer, and multilength scale rough surface. The treated textiles demonstrate a static contact angle of 158.3 ± 2.6° and hysteresis of 4.7 ± 1.7° for fetal bovine serum and reduce serum protein adhesion by 89.7 ± 7.3% (0.99 log). The coated textiles reduce the attachment of adenovirus type 4 and 7a virions by 99.2 ± 0.2% and 97.6 ± 0.1% (2.10 and 1.62 log), respectively, compared to noncoated controls. The treated textiles provide these repellencies by maintaining a Cassie–Baxter state of wetting where the surface area in contact with liquids is reduced by an estimated 350 times (2.54 log) compared to control textiles. Moreover, the treated textiles exhibit unprecedented mechanical durability, maintaining their liquid, protein, and viral repellency after extensive and harsh abrasion and washing. The multilayer, multilength scale roughness provides for mechanical durability through self-similarity, and the samples have high-pressure stability with a breakthrough pressure of about 255 kPa. These properties highlight the potential of durable, repellent coatings for medical gowning, scrubs, or other hygiene textile applications.

Polyethylenimine in the presence of organic reducing agent allow the formation of functionalized Silver nanoparticles of controlled particle
size distribution. The nanoparticles exhibited potent antimicrobial activity against drug resistant bacterial and also to viruses. as a function of size of silver nnoprticles. Fluorescence imaging of Ag-NPs revealed selective transfusion of Ag-NPs across the cell membrane as a function of the polymeric MW; differential interaction of the cytoplasmic proteins during antimicrobial activity was observed and will be discussed during presentation.

The COVID-19 pandemic has made the general public more aware of the threat of viruses. With that comes interest in how long pathogens are able to survive on surfaces, to help minimize potential exposures. Hypochlorous Acid, or HOCL is an endogenous substance found in mammals, and effective against a wide range of microorganisms1. This research will determine the efficacy of Hypochlorous Acid for eliminating the H1N1 Virus, which is responsible for a highly contagious respiratory disease2, and identify the survival rate on various surfaces.
To identify the reduction rate of H1N1, a plaque assay was executed to calculate the PFU of H1N1 plated on PLA (Polylactic Acid), Polystyrene (280K), and Aluminum at different timeframes. The samples were allocated into vials with 0.2% BSA in MEM (minimum essential media) and vortexed to promote uniformity. Subsequently, the new virus solution underwent a ten-fold serial dilution to perform a plaque assay. The virus was then replaced by the gum medium (0.2% BSA, 0.5% TrypLE Select, 1% PenStrep and 25% Tragacanth gum). Lastly, the plates were placed in the incubator for 3 days and later stained with 1% crystal violet, with 4% formaldehyde and 50% methanol dye. The PFU of each well was counted and documented.
In relation to time (in hours), drying in a room temperature environment was significantly more susceptible to viruses surviving than at 4°C and -20°C, despite the variance in surfaces. In a 4°C refrigerator, the penny, an impure copper surface, had the lowest H1N1 survival surface rate (blank) in coin currencies. For the 24 hour series, PLA—at room temperature—achieved the most resistance to the H1N1 virus with a 2-log reduction. Most notably, an Aluminum surface drying for 48 hours produced a 3-log reduction, which resulted in a 99.9% H1N1 elimination.
At room temperature, all tested surfaces reached antiviral capabilities (at least 4-log reduction or 99.99% elimination) between 24 and 50 hours; however, this span significantly increases the likelihood of pathogens to spread via surfaces. Therefore, the addition of Hypochlorous Acid, a common disinfectant, was deemed necessary to increase the log reduction of H1N1 in a shorter time frame.
To observe the disinfecting effect of Hypochlorous Acid, 350 and 510 ppm (low and high) concentrations were used. Firstly, the H1N1 virus was deposited on three different surfaces: Polystyrene (280K), Aluminum, and PLA. The plated virus samples were then fogged with HOCI at 2.5 minutes, 5 minutes, 7.5 minutes, and 10 minutes with a 5 minute interval of rest for the different concentrations. For H1N1 deposited on all surfaces, there was an increasing correlation between log reduction and time (in minutes) for both chlorine concentrations, denoting that longer fogging cycles eliminates more virus particles. Specifically, Aluminum and Polystyrene infected surfaces exposed to high chloride concentration were considered antiviral between 5 and 7.5 minutes. For low chloride concentration, surfaces plated with H1N1 produced a 4-log reduction between 7.5 and 10 minutes. Therefore, these results provide promising evidence that makes hypochlorous acid fogging, a cheap and readily available substance, a potential method of disinfecting with high efficacy.
1Block, Michael S, and Brian G Rowan. “Hypochlorous Acid: A Review.” Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons vol. 78,9 (2020): 1461-1466. doi:10.1016/j.joms.2020.06.029 2Lee, Spike W., et al. “Risk Overgeneralization in Times of a Contagious Disease Threat.” Frontiers in Psychology, vol. 11, 2020, doi:10.3389/fpsyg.2020.01392.

Candida species are the most common fungi isolated from nosocomial infections (e.g., hospital-acquired infections), and biofilms formed by these organisms are associated with drastically enhanced resistance against most antimicrobial agents. Nowadays, also rare fungal species of low pathogenic potential, or even species never described before as a cause of disease, are being more commonly detected in health-care settings. Thus, an intensive search and development of the novel anti-fungal agents is of current interest. This study reports the impact of Ca₂Fe₂O₅ nanoporous powder on yeast Candida utilis – as a fungal model - at different phases of growth, i.e., early exponential, mid-log and stationary phases. After 120 min incubation, the fungicidal activity of nanoporous powder was observed, i.e., log reduction of 2.81 and 2.58 for mid-log and stationary culture, respectively, reaching the maximum of 4 log reduction after 7 days. The early exponential culture of C. utilis showed an enhanced resistance to Ca₂Fe₂O₅ with a log reduction £0.4 during the 7 days exposure. Our results not only showed that Ca₂Fe₂O₅ have the potential to effectively decrease the C. utilis cell growth, but also indicated the importance of the physiological state of the test culture. Obtained results may contribute to better understanding of the difference in fungicidal efficacy of antimicrobial materials.

Acknowledgments: This work has been supported by the European Regional Development Fund within the Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment” (No. 1.1.1.2/VIAA/2/18/331).

Materials and methods of collecting bioaerosol for performing analysis on potential biological threats is at an all-time high after over a year of battling COVID-19. Currently, filter-based air samplers capture the highest yield and ratio, while liquid impingers offer the best chance to maintain pathogen viability. In this body of work, we present a novel approach to filter-based filtration by fabricating liquid nets. Infusing polytetrafluoroethylene filters with a perfluoropolyether oil resulted in improved rate of release of captured Escherichia coli aerosols onto culture plates. Furthermore, wetting meltblown polypropylene filters with the same perfluoropolyether oil improved the rate of release, as well as the overall colonies transferred and cultured. The efficiency of bioaerosol transfer demonstrated by the liquid-infused filters could provide a better representation of the airborne pathogens within an environment, enabling informed actions by infectious disease experts and medical professionals.