Microplastics in biosolids: A review of ecological implications and methods for identification, enumeration, and characterization.

Biosolids, or treated sludge, are by-products of the wastewater treatment processes and are commonly used in agricultural applications to enrich soil nutrients. However, it contains microplastics, plastic particles with a diameter below 1 mm. Microplastics exist and accumulate in the environment, which can have major impacts on the ecosystem. Despite their abundance in the environment, there are to date no standardized methods for their enumeration and characterization. A literature review was conducted focusing on the occurrence of microplastics at wastewater treatment plants, particularly in the solid waste stream, and their influence on the soil ecosystem where biosolids is applied. We found a conflicting evidence to which extent microplastics negatively impact the ecosystem. Some reported either a direct negative impact of microplastics or because of microplastic interaction with other soil contaminants. Meanwhile, other studies showed no effect or at certain amount of microplastics on the ecosystem. We also found that microplastics size, shape, type, concentration, and exposure time play a critical role in their ecological impacts. However, currently, there is no unified approach for microplastics identification and characterization in solid waste resulting in a various and incomparable data. Therefore, utilizing standardized methods for microplastics analysis must be considered as the initial step to better understand the impact of microplastics onto the environment. We suggest a method's scaling comparison as a practical approach to select and develop techniques based on cost, time, data obtained, accuracy, and sensitivity criteria. Further research into the ecotoxicity of microplastics and continuous monitoring of biosolid applications are also necessary.

Model architectures for bacterial membranes.

The complex composition of bacterial membranes has a significant impact on the understanding of pathogen function and their development towards antibiotic resistance. In addition to the inherent complexity and biosafety risks of studying biological pathogen membranes, the continual rise of antibiotic resistance and its significant economical and clinical consequences has motivated the development of numerous in vitro model membrane systems with tuneable compositions, geometries, and sizes. Approaches discussed in this review include liposomes, solid-supported bilayers, and computational simulations which have been used to explore various processes including drug-membrane interactions, lipid-protein interactions, host-pathogen interactions, and structure-induced bacterial pathogenesis. The advantages, limitations, and applicable analytical tools of all architectures are summarised with a perspective for future research efforts in architectural improvement and elucidation of resistance development strategies and membrane-targeting antibiotic mechanisms. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-021-00913-7.

Increasing Antibiotic Susceptibility: The Use of Cationic Gold Nanoparticles in Gram-Negative Bacterial Membrane Models.

Antibiotic resistance will be one of the most prominent challenges to health-care systems in the coming decades, with the OECD predicting that up to 2.4 million deaths will be caused between 2015 and 2050 by drug-resistant bacterial infections in first-world countries alone, with infections costing health-care systems billions of dollars each year. Developing new methods to increase bacterial susceptibility toward drugs is an important step in treating resistant infections. Here, the synergistic effects of gold nanoparticles and the antibiotic drug colistin sulfate have been examined. A tethered lipid bilayer membrane was used to mimic a Gram-negative bacterial cell membrane. Exposing the membrane to gold nanoparticles prior to adding the antibiotic significantly increased the effect of the antibiotic on the membrane. Cationic gold nanoparticles could thus be used to enhance bacterial susceptibility to antibiotics, leading to a more potent treatment.

Cellular interactions with polystyrene nanoplastics-The role of particle size and protein corona.

Plastic waste is ubiquitously spread across the world and its smaller analogs-microplastics and nanoplastics-raise particular health concerns. While biological impacts of microplastics and nanoplastics have been actively studied, the chemical and biological bases for the adverse effects are sought after. This work explores contributory factors by combining results from in vitro and model mammalian membrane experimentation to assess the outcome of cell/nanoplastic interactions in molecular detail, inspecting the individual contribution of nanoplastics and different types of protein coronae. The in vitro study showed mild cytotoxicity and cellular uptake of polystyrene (PS) nanoplastics, with no clear trend based on nanoplastic size (20 and 200 nm) or surface charge. In contrast, a nanoplastic size-dependency on bilayer disruption was observed in the model system. This suggests that membrane disruption resulting from direct interaction with PS nanoplastics has little correlation with cytotoxicity. Furthermore, the level of bilayer disruption was found to be limited to the hydrophilic headgroup, indicating that transmembrane diffusion was an unlikely pathway for cellular uptake-endocytosis is the viable mechanism. In rare cases, small PS nanoplastics (20 nm) were found in the vicinity of chromosomes without a nuclear membrane surrounding them; however, this was not observed for larger PS nanoplastics (200 nm). We hypothesize that the nanoplastics can interact with chromosomes prior to nuclear membrane formation. Overall, precoating PS particles with protein coronae reduced the cytotoxicity, irrespective of the corona type. When comparing the two types, the extent of reduction was more apparent with soft than hard corona.

The Membrane Composition Defines the Spatial Organization and Function of a Major Acinetobacter baumannii Drug Efflux System.

Acinetobacter baumannii is one of the world's most problematic nosocomial pathogens. The combination of its intrinsic resistance and ability to acquire resistance markers allow this organism to adjust to antibiotic treatment. Despite being the primary barrier against antibiotic stress, our understanding of the A. baumannii membrane composition and its impact on resistance remains limited. In this study, we explored how the incorporation of host-derived polyunsaturated fatty acids (PUFAs) is associated with increased antibiotic susceptibility. Functional analyses of primary A. baumannii efflux systems indicated that AdeB-mediated antibiotic resistance was impacted by PUFA treatment. Molecular dynamics simulations of AdeB identified a specific morphological disruption of AdeB when positioned in the PUFA-enriched membrane. Collectively, we have shown that PUFAs can impact antibiotic efficacy via a vital relationship with antibiotic efflux pumps. Furthermore, this work has revealed that A. baumannii's unconditional desire for fatty acids may present a possible weakness in its multidrug resistance capacity. IMPORTANCE Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options. Due to their relatively high abundance at the host-pathogen interface, PUFAs and other fatty acid species not commonly synthesized by A. baumannii may be actively acquired by A. baumannii during infection and change the biophysical properties of the membrane beyond that studied in standard laboratory culturing media. Our work illustrates how the membrane phospholipid composition impacts membrane protein function, which includes an important multidrug efflux system in extensively-drug-resistant A. baumannii. This work emphasizes the need to consider including host-derived fatty acids in in vitro analyses of A. baumannii. On a broader scope, this study presents new findings on the potential health benefits of PUFA in individuals at risk of contracting A. baumannii infections or those undergoing antibiotic treatment.

Reviewing nanoplastic toxicology: It's an interface problem.

Multiple international agencies have recently raised environmental and health concerns regarding plastics in nanoforms (nanoplastics), but there is insufficient knowledge of their properties to allow for an accurate risk assessment to be conducted and any risks managed. For this reason, research into the toxicity of nanoplastics has focused strongly on documenting their impacts on biological organisms. One scope of this review is to summarise the recent findings on the adverse effects on biological organisms and strategies which can be adopted to advance our understanding of nanoplastic properties and their toxicity. Specifically, a mechanistic approach has already been employed in nanotoxicology, which focuses on the cause-and-effect relationships to establish a tool that predicts the biological impacts based on nanoparticle characteristics. Identifying the chemical and biological bases behind the observed biological effects (such as in vitro cellular response) is a major challenge, due to the intricate nature of nanoparticle-biological molecule complexes and an unawareness of their interaction with other biological targets, particularly at interfacial level. An exemplary case includes protein corona formation and ecological molecule corona (eco-corona) for nanoplastics. Therefore, the second scope of this review is to discuss recent findings and importance of (for both non-plastic and plastic nanoparticles) coronae formation and structure. Finally, we discuss the opportunities provided by model system approaches (model protein corona and lipid bilayer) to deepen the understanding of the above-mentioned perspectives, and corroborate the findings from in vitro experiments.

Solid-supported lipid bilayers - A versatile tool for the structural and functional characterization of membrane proteins.

The cellular membrane is central to the development of single-and multicellular life, as it separates the delicate cellular interior from the hostile environment. It exerts tight control over entry and exit of substances, is responsible for signaling with other cells in multicellular organisms and prevents pathogens from entering the cell. In the case of bacteria and viruses, the cellular membrane also hosts the proteins enabling invasion of the host organism. In a very real sense therefore, the cellular membrane is central to all life. The study of the cell membrane and membrane proteins in particular has therefore attracted significant attention. Due to the enormous variety of tasks performed by the membrane, it is a highly complex and challenging structure to study. Ideally, membrane components would be studied in isolation from this environment, but unlike water soluble proteins, the amphiphilic environment provided by the cellular membrane is key to the structure and function of the cell membrane. Therefore, model membranes have been developed to provide an environment in which a membrane protein can be studied. This review presents a set of tools that enable the comprehensive characterization of membrane proteins: electrochemical tools, surface plasmon resonance, neutron scattering, the surface forces apparatus and atomic force microscopy are discussed, with a particular focus on experimental technique and data evaluation.

Structure of soft and hard protein corona around polystyrene nanoplastics-Particle size and protein types.

A major challenge in understanding nanoplastic toxicity (or nanoparticles in general) lies in establishing the causal relationships between its physical properties and biological impact. This difficulty can be attributed to surface alterations that follow the formation of a biological complex around the nanoplastic, as exemplified by protein coronae. The protein corona is known to be responsible for the biological response elicited, although its own structure and attributes remain unknown. We approach this knowledge gap by independently studying the structure of soft and hard coronae using neutron scattering techniques. We investigated the formation and the structure of corona proteins (human serum albumin and lysozyme) and the resulting protein corona complexes with polystyrene nanoplastics of different sizes (20 and 200 nm) and charges. Soft corona complexes (regardless of protein type) adopted a structure where the nanoplastics were surrounded by a loose protein layer (∼2-3 protein molecules thick). Hard corona complexes formed fractal-like aggregates, and the morphology of which is known to be harmful to cellular membranes. In most cases, hard-corona coated nanoplastics also formed fractal-like aggregates in solution. Nanoplastic size affected the structures of both the protein corona and the intrinsic protein: more significant conformational change was observed in the hard corona proteins around smaller nanoparticles compared to larger ones, as the self-association forces holding the nanoplastic/protein complex together were stronger. This also implies that protein-dependent biochemical processes are more likely to be disrupted by smaller polystyrene nanoplastics, rather than larger ones.

Investigating the Structure of Self-Assembled Monolayers Related to Biological Cell Membranes.

Tethered bilayer lipid membranes are solid supported lipid membranes, where the inner leaflet is covalently linked to the solid supported substrate through anchorlipids. These anchorlipids form a self-assembled monolayer, which serves as the basis of the membrane and also provides submembrane space. The molecular structure and composition of this monolayer has thus significant influence on the membrane structural and functional properties. The density of the self-assembled monolayer can be tailored by adding small molecules to the monolayer. Here, the structure of fully tethered and sparsely tethered monolayers, where the anchorlipid has been diluted with a small surface-active thiol, has been analyzed using neutral impact collision ion scattering spectroscopy, X-ray photoelectron spectroscopy, and metastable induced electron spectroscopy. Combination of these three techniques allowed description of the self-assembly process in detail. The monolayers have been characterized in terms of layer thickness and orientation of the lipids.

Biomedical applications of polyelectrolyte coated spherical gold nanoparticles.

Surface modified gold nanoparticles are becoming more and more popular for use in biomaterials due to the possibility for specific targeting and increased biocompatibility. This review provides a summary of the recent literature surrounding polyelectrolyte coatings on spherical gold nanoparticles and their potential biomedical applications. The synthesis and layer-by layer coating approach are briefly discussed together with common characterisation methods. The potential applications and recent developments in drug delivery, gene therapy, photothermal therapy and imaging are summarized as well as the effects on cellular uptake and toxicity. Finally, the future outlook for polyelectrolyte coated gold nanoparticles is explored, focusing on their use in biomedicine.

Soft and Hard Interactions between Polystyrene Nanoplastics and Human Serum Albumin Protein Corona.

Upon contact with biological fluids, the surface of nanoparticles is surrounded by many types of proteins, forming a so-called "protein corona". The physicochemical properties of the nanoparticle/corona complex depend predominantly on the nature of the protein corona. An understanding of the structure of the corona and the resulting complex provides insight into the structure-activity relationship. Here, we structurally evaluate the soft and hard components of the protein corona, formed from polystyrene (PS) nanoplastics and human serum albumin (HSA). Using circular dichroism spectroscopy to elucidate the structure of HSA within the complex, we establish the effect of nanoparticle size and pH on the nature of the protein corona formed- whether hard or soft. Despite the weak interaction between PS and the HSA corona, small angle neutron scattering revealed the formation of a complex structure that enhanced the intermolecular interactions between HSA proteins, PS particles, and the HS/PSA complexes. Fractal formation occurred under conditions where the interaction between PS and HSA was strong, and increasing HSA concentrations suppressed the degree of aggregation. The size of the nanoparticles directly influenced the nature of the protein corona, with larger particles favoring the formation of a soft corona, due to the decreased PS-HSA attraction.

Nanoparticles in an antibiotic-loaded nanomesh for drug delivery.

Antibiotic loaded nanomeshes were fabricated by electrospinning polycaprolactone, a biocompatible polymer, with 12.5% w/w Colistin, 1.4% w/w Vancomycin and either cationic or anionic gold nanoparticles in varying combinations. The resulting nanomeshes had different antibiotic release profiles, with citrate capped gold nanoparticles combined with Colistin having the highest sustained release over 14 days for a 4 mg, 1.5 cm2 nanomesh. The electrospinning parameters were optimised to ensure the spinning of a homogenous mesh and the addition of antibiotics was confirmed through 1H NMR and ATR-FTIR. This research, as a proof of concept, suggests an opportunity for fabricating nanomeshes which contain gold nanoparticles as a drug release mechanism for antibiotics.

A tethered bilayer lipid membrane that mimics microbial membranes.

A model membrane system has been developed, which mimics the outer membrane of Gram negative bacteria. The structure is based on a tethered monolayer which has been fused with vesicles containing lipopolysaccharide molecules. The effect of the composition of the monolayer and the lipids in the outer layer on the structural and electrical properties of the membrane has been investigated. By using electrochemical impedance spectroscopy as well as neutron scattering techniques, it could be shown that a relatively high tethering density and a small amount of diluting lipids in the outer membrane leaflet leads to the formation of a stable solid supported membrane. The influence of divalent ions on the membrane stability has been probed as well as the interaction of the bilayer with the antibiotic colistin. A number of different architectures were developed, suited to both the study of bacterial membrane proteins and the screening of antimicrobial activity of potential drug candidates.

Interaction of a synthetic antimicrobial peptide with a model bilayer platform mimicking bacterial membranes.

Tethered bimolecular lipid membranes are solid supported membrane systems, which provide a versatile model platform for the study of many membrane related processes. Here, such an architecture has been used to study the interaction of the small synthetic antimicrobial peptide, V4, with membranes of various mixed lipid compositions, including membranes containing bacterial lipids. By investigating the binding of the peptide using a range of surface analytical techniques such as surface plasmon resonance and surface plasmon field-enhanced fluorescence spectroscopy as well as electrochemical impedance spectroscopy, a clear preference of the peptide for negatively charged membranes over zwitterionic ones has been shown. Additionally, the interactions seemed to indicate a cooperative behavior for the peptide binding to a membrane.

Synthesis and Characterization of Novel Anchorlipids for Tethered Bilayer Lipid Membranes.

Tethered bilayer lipid membranes are versatile solid-supported model membrane systems. Core to these systems is an anchorlipid that covalently links a lipid bilayer to a support. The molecular structure of these lipids can have a significant impact on the properties of the resulting bilayer. Here, the synthesis of anchorlipids containing ester groups in the tethering part is described. The lipids are used to form bilayer membranes, and the resulting structures are compared with membranes formed using conventional anchorlipids or sparsely tethered membranes. All membranes showed good electrical sealing properties; the disulphide-terminated anchorlipids could be used in a sparsely tethered system without significantly reducing the sealing properties of the lipid bilayers. The sparsely tethered systems also allowed for higher ion transport across the membrane, which is in good correlation with higher hydration of the spacer region as seen by neutron scattering.

Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function.

Solid supported bilayer lipid membranes are model systems to mimic natural cell membranes in order to understand structural and functional properties of such systems. The use of a model system allows for the use of a wide variety of analytical tools including atomic force microscopy, impedance spectroscopy, neutron reflectometry, and surface plasmon resonance spectroscopy. Among the large number of different types of model membranes polymer-supported and tethered lipid bilayers have been shown to be versatile and useful systems. Both systems consist of a lipid bilayer, which is de-coupled from an underlying support by a spacer cushion. Both systems will be reviewed, with an emphasis on the effect that the spacer moiety has on the bilayer properties.

Cell-Free Synthesis of a Functional Membrane Transporter into a Tethered Bilayer Lipid Membrane.

Eukaryotic cell-free synthesis was used to incorporate the large and complex multispan plant membrane transporter Bot1 in a functional form into a tethered bilayer lipid membrane. The electrical properties of the protein-functionalized tethered bilayer were measured using electrochemical impedance spectroscopy and revealed a pH-dependent transport of borate ions through the protein. The efficacy of the protein synthesis has been evaluated using immunoblot analysis.

Membrane-drug interactions studied using model membrane systems.

The direct interaction of drugs with the cell membrane is often neglected when drug effects are studied. Systematic investigations are hindered by the complexity of the natural membrane and model membrane systems can offer a useful alternative. Here some examples are reviewed of how model membrane architectures including vesicles, Langmuir monolayers and solid supported membranes can be used to investigate the effects of drug molecules on the membrane structure, and how these interactions can translate into effects on embedded membrane proteins.

Oxidative Damage to Biomimetic Membrane Systems: In Situ Fe(II)/Ascorbate Initiated Oxidation and Incorporation of Synthetic Oxidized Phospholipids.

Damage to cellular membranes from oxidative stress has been implicated in aging related diseases. We report the effects of oxidative damage on the structure and properties of biomimetic phospholipid membrane systems. Two oxidation methods were used, in situ oxidation initiated using Fe(II) and ascorbate, and the incorporation of a synthetic "oxidized" phospholipid, PoxnoPC, into biomimetic membranes. The biomimetic systems employed included multibilayer stacks, tethered bilayers, and phospholipid monolayers studied using a combination of reflectometry, attenuated total reflection infrared spectroscopy, electrochemical impedance spectroscopy, and neutron diffraction. We show that oxidation with Fe(II) and ascorbate caused an increase in the order of the membrane, attributed to cross-linking of the phospholipids, and a change in the electrical permeability of the membrane, but no significant impact on the thickness or completeness of the membrane. Incorporation of PoxnoPC, on the other hand, had a larger impact on the structure of the membrane. Inversion of the aldehyde-terminated truncated sn-2 chain of PoxnoPC into the head group region was observed, along with a slight decrease in the thickness and order of the membrane.

Interaction of silver nanoparticles with tethered bilayer lipid membranes.

Silver nanoparticles are well-known for their antibacterial properties. However, the detailed mechanism describing the interaction between the nanoparticles and a cell membrane is not fully understood, which can impede the use of the particles in biomedical applications. Here, a tethered bilayer lipid membrane has been used as a model system to mimic a natural membrane and to study the effect of exposure to small silver nanoparticles with diameters of about 2 nm. The solid supported membrane architecture allowed for the application of surface analytical techniques such as electrochemical impedance spectroscopy and atomic force microscopy. Exposure of the membrane to solutions of the silver nanoparticles led to a small but completely reversible perturbation of the lipid bilayer.