Toxicity of nanomixtures to human macrophages: Joint action of silver and polystyrene nanoparticles.

Increasing use of nano-enabled products provides many benefits in various industrial processes and medical applications, but it also raises concern about release of nanoparticles (NPs) into the environment and subsequent human exposure. While potential toxicity of individual NPs types has been well described in scientific literature, exposure and health-related effects of nanomixtures has been poorly described. This study aimed to evaluate the combined effect of silver (AgNP) and polystyrene NPs (PSNP) on the human macrophages. AgNP are one of the most commercialized NPs due to efficient antimicrobial activity, while PSNP are ubiquitous in terrestrial and aquatic environments due to plastic pollution and degradation of polystyrene-based products. Differentiated monocytic cell line THP-1 were used as an in vitro model of human macrophages. Multiple aspects of cellular response to AgNP-PSNP nanomixture were analyzed including cell death, induction of apoptosis, oxidative stress response, expression of pro- and anti-inflammatory cytokines, and nanomechanical properties of cells. NPs uptake was visualized by confocal microscopy and quantified using flow cytometry. Results show that nanomixture increased apoptosis and cell death, expression of IL-6, IL-8 and TNFa, oxidative stress and mitochondrial dysfunction in cells compared to AgNP and PSNP applied as single treatments, indicating mixture additive action. Anti-inflammatory cytokines IL1b, IL-4 and IL-10 were not affected by combined exposure compared to single NPs. Visualization of NPs uptake and internalization showed that AgNP and PSNP were localized mostly in cytoplasm, with small fraction of AgNP translocated into cell nuclei, which explain increased number of double-stranded DNA breaks following exposure of cells to AgNPs alone or in the mixture. Study outcomes represent clear warnings on the human co-exposure to AgNP and PSNP that needs to be implemented in risk assessment approaches towards toxic-free environment.

Cytotoxicity of nanomixture: Combined action of silver and plastic nanoparticles on immortalized human lymphocytes.

BACKGROUND: Silver nanoparticles (AgNP) are one of the most commercialized types of nanomaterials, with a wide range of applications owing to their antimicrobial activity. They are particularly important in hospitals and other healthcare settings, where they are used to maintain sterility of surfaces, textiles, catheters, medical implants, and more. However, AgNP can not only harm bacteria, but also damage mammalian cells and tissue. While the potential toxicity of AgNP is an understood risk, there is a lack of data on their toxicity in combination with polymeric materials, especially plastic nanoparticles such as polystyrene nanoparticles (PSNP) that can be released from surfaces of polystyrene devices during their medical use. AIM: This study aimed to investigate combined effect of AgNP and nanoplastics on human immune response. METHODS: Cells were treated with a range of PSNP and AgNP concentrations, either applied alone or in combination. Cytotoxicity, induction of apoptosis, generation of oxidative stress, uptake efficiency, intracellular localization and nanomechanical cell properties were selected as exposure biomarkers. RESULTS: Collected experimental data showed that nanomixture induced oxidative stress, apoptosis and mortality of Jurkat cells stronger than its individual components. Cell treatment with AgNP/PSNP mixture also significantly changed cell mechanical properties, evidenced by reduction of cells' Young Modulus. CONCLUSION: AgNP and PSNP showed additive toxic effects on immortalized human lymphocytes, evidenced by increase in cellular oxidative stress, induction of apoptosis, and reduction of cell stiffness. These results have important implications for using AgNP and PSNP in medical contexts, particularly for long-term medical implants.

Anisaxins, helical antimicrobial peptides from marine parasites, kill resistant bacteria by lipid extraction and membrane disruption.

An infecting and propagating parasite relies on its innate defense system to evade the host's immune response and to survive challenges from commensal bacteria. More so for the nematode Anisakis, a marine parasite that during its life cycle encounters both vertebrate and invertebrate hosts and their highly diverse microbiotas. Although much is still unknown about how the nematode mitigates the effects of these microbiota, its antimicrobial peptides likely play an important role in its survival. We identified anisaxins, the first cecropin-like helical antimicrobial peptides originating from a marine parasite, by mining available genomic and transcriptomic data for Anisakis spp. These peptides are potent bactericidal agents in vitro, selectively active against Gram-negative bacteria, including multi-drug resistant strains, at sub-micromolar concentrations. Their interaction with bacterial membranes was confirmed by solid state NMR (ssNMR) and is highly dependent on the peptide concentration as well as peptide to lipid ratio, as evidenced by molecular dynamics (MD) simulations. MD results indicated that an initial step in the membranolytic mode of action involves membrane bulging and lipid extraction; a novel mechanism which may underline the peptides' potency. Subsequent steps include membrane permeabilization leading to leakage of molecules and eventually cell death, but without visible macroscopic damage, as shown by atomic force microscopy and flow cytometry. This membranolytic antibacterial activity does not translate to cytotoxicity towards human peripheral blood mononuclear cells (HPBMCs), which was minimal at well above bactericidal concentrations, making anisaxins promising candidates for further drug development. STATEMENT OF SIGNIFICANCE: Witnessing the rapid spread of antibiotic resistance resulting in millions of infected and dozens of thousands dying worldwide every year, we identified anisaxins, antimicrobial peptides (AMPs) from marine parasites, Anisakis spp., with potent bactericidal activity and selectivity towards multi-drug resistant Gram-negative bacteria. Anisaxins are membrane-active peptides, whose activity, very sensitive to local peptide concentrations, involves membrane bulging and lipid extraction, leading to membrane permeabilization and bacterial cell death. At the same time, their toxicity towards host cells is negligible, which is often not the case for membrane-active AMPs, therefore making them suitable drug candidates. Membrane bulging and lipid extraction are novel concepts that broaden our understanding of peptide interactions with bacterial functional structures, essential for future design of such biomaterials.

The proline-rich myticalins from Mytilus galloprovincialis display a membrane-permeabilizing antimicrobial mode of action.

Bivalve mollusks are continuously exposed to potentially pathogenic microorganisms living in the marine environment. Not surprisingly, these filter-feeders developed a robust innate immunity to protect themselves, which includes a broad panel of antimicrobial peptides. Among these, myticalins represent a recently discovered family of linear cationic peptides expressed in the gills of Mytilus galloprovincialis. Even though myticalins and insect and mammalian proline-rich antimicrobial peptides (PrAMPs) share a similar amino acid composition, we here show that none of the tested mussel peptides use a non-lytic mode of action relying on the bacterial transporter SbmA. On the other hand, all the tested myticalins perturbed and permeabilized the membranes of E. coli BW25113, as shown by flow-cytometry and atomic force microscopy. Circular dichroism spectra revealed that most myticalins did not adopt recognizable secondary structures in the presence of amphipathic environments, such as biological membranes. To explore possible uses of myticalins for biotech, we assessed their biocompatibility with a human cell line. Non-negligible cytotoxic effects displayed by myticalins indicate that their optimization would be required before their further use as lead compounds in the development of new antibiotics.

The mode of antibacterial action of quaternary N-benzylimidazole salts against emerging opportunistic pathogens.

Quaternary ammonium compounds (QACs) are antimicrobial agents displaying a broad spectrum of activity due to their mechanism of action targeting the bacterial membrane. The emergence of bacterial resistance to QACs, especially in times of pandemics, requires the continuous search for new and potent QACs structures. Here we report the synthesis and biological evaluation of QACs based on imidazole derivative, N-benzylimidazole. The antimicrobial activity was tested against a range of pathogenic bacteria and fungi, both ATCC and clinical isolates, showing varying activities ranging in minimal inhibitory concentrations (MICs) from as low as 7 ng/mL. The most promising compound, N-tetradecyl derivative (BnI-14), proved to be very potent against bacterial biofilms, even at sub-MIC doses, suggesting interference with the bacterial growth and/or division process. The BnI-14 treatment induces bacterial membrane disruption, as observed by fluorescence spectroscopy and atomic force microscopy and it also binds to DNA indicating that bacterial membrane might not be the only cellular target of QACs. Most importantly, BnI-14 exhibits low toxicity to healthy human cell lines, suggesting that N-benzylimidazolium-based QACs may be promising new antimicrobial agents.

Probing the Mode of Antibacterial Action of Silver Nanoparticles Synthesized by Laser Ablation in Water: What Fluorescence and AFM Data Tell Us.

We aim to elucidate the mode of antibacterial action of the laser-synthesized silver colloid against Escherichia coli. Membrane integrity was studied by flow cytometry, while the strain viability of the treated culture was determined by plating. The spectrofluorometry was used to obtain the time development of the reactive oxygen species (ROS) inside the nanoparticle-treated bacterial cells. An integrated atomic force and bright-field/fluorescence microscopy system enabled the study of the cell morphology, Young modulus, viability, and integrity before and during the treatment. Upon lethal treatment, not all bacterial cells were shown to be permeabilized and have mostly kept their morphology with an indication of cell lysis. Young modulus of untreated cells was shown to be distinctly bimodal, with randomly distributed softer parts, while treated cells exhibited exponential softening of the stiffer parts in time. Silver nanoparticles and bacteria have shown a masking effect on the raw fluorescence signal through absorbance and scattering. The contribution of cellular ROS in the total fluorescence signal was resolved and it was proven that the ROS level inside the lethally treated cells is not significant. It was found that the laser-synthesized silver nanoparticles mode of antibacterial action includes reduction of the cell's Young modulus in time and subsequently the cell leakage.

Testing scalar diffraction theory: Gaussian beam on a slit.

We recorded diffraction patterns using a commercially available slit and sensor over a wide range of experimental circumstances, including near- and far-field regimes and oblique incidence at large angles. We then compared the measured patterns with theoretical intensity curves calculated via the numerical integration of formulas derived within the framework of scalar diffraction theory. Experiment and theory show particularly good agreement when the first Rayleigh-Sommerfeld (R-S) formula is used. The Kirchhoff formula, though problematic in the context of mathematical consistency, agrees with the first R-S formula, even for large incidence angles, whereas the second R-S formula differs visibly. To obtain such a good agreement, we replaced the assumption of an incident plane wave with that of a Gaussian beam and implemented geometric corrections to account for slit imperfections. These results reveal how the scope of scalar diffraction theory can be extended with a small set of auxiliary assumptions.

Bacteria Exposed to Silver Nanoparticles Synthesized by Laser Ablation in Water: Modelling E. coli Growth and Inactivation.

This study is aimed to better understand the bactericidal mode of action of silver nanoparticles. Here we present the production and characterization of laser-synthesized silver nanoparticles along with growth curves of bacteria treated at sub-minimal and minimal inhibitory concentrations, obtained by optical density measurements. The main effect of the treatment is the increase of the bacterial apparent lag time, which is very well described by the novel growth model as well as the entire growth curves for different concentrations. The main assumption of the model is that the treated bacteria uptake the nanoparticles and inactivate, which results in the decrease of both the nanoparticles and the bacteria concentrations. The lag assumes infinitive value for the minimal inhibitory concentration treatment. This apparent lag phase is not postponed bacterial growth. It is a dynamic state in which the bacterial growth and death rates are close in value. Our results strongly suggest that the predominant mode of antibacterial action of silver nanoparticles is the penetration inside the membrane.

A simple interaction-based E. coli growth model.

We present a simple growth model which was developed to explain Escherichia coli growth in batch culture. Optical density measurements are used to obtain E. coli growth curves for different inoculum sizes and nutrients concentrations. The model is described by two nonlinear mutually dependent differential equations that describe time evolution of bacteria and nutrients concentration. Introduction of the negative bacterium-bacterium interaction term is specific for the model and leads to the population decay. The proposed model describes entire experimental growth curves. The growth rate, as a function of initial nutrients concentration, follows the Monod function, whilst during the growth it decreases proportionally with the concentration of nutrients. The parameters in our equations can be related to the parameters of the logistic model. The proposed model can be applied to different E. coli strains and, because of the universality of the equations, might be applied to other bacterial strains.

Selection and redesign for high selectivity of membrane-active antimicrobial peptides from a dedicated sequence/function database.

Antimicrobial peptides (AMPs) are plausible candidates for the development of novel classes of antibiotics with a low tendency to elicit resistance. They often form lesions in the bacterial membrane making it hard for bacteria to develop permanent resistance. However, a potent antibacterial activity is often accompanied by excessive cytotoxicity towards host cells. Modifying known natural sequences, based on desirable biophysical properties, is expensive and time-consuming and often with limited success. 'Mutator' is a freely available web-based computational tool for suggesting residue variations that potentially increase a peptide's selectivity, based on the use of quantitative structure activity relationship (QSAR) criteria. Although proven to be successful, it has never been used to analyze multiple sequences simultaneously. Modifying the Mutator algorithm allowed screening of many sequences in the dedicated Database of Anuran Defense Peptides (DADP) and by implementing limited amino acid substitutions on appropriate candidates, propose 8 potentially selective AMPs called Dadapins. Two were chosen for testing, confirming the prediction and validating this approach. They were shown to efficiently inactivate bacteria by disrupting their membranes but to be non-toxic for host cells, as determined by flow cytometry and confirmed by atomic force microscopy (AFM).

Membrane-active antimicrobial peptide identified in Rana arvalis by targeted DNA sequencing.

Antimicrobial peptides (AMPs) are naturally produced, gene encoded molecules with a direct antimicrobial activity against pathogens, often also showing other immune-related properties. Anuran skin secretions are rich in bioactive peptides, including AMPs, and we have reported a novel targeted sequencing approach to identify novel AMPs simultaneously in different frog species, from small quantities of skin tissue. Over a hundred full-length peptides were identified from specimens belonging to five different Ranidae frog species, out of which 29 were novel sequences. Six of these were selected for synthesis and testing against a panel of Gram-negative and Gram-positive bacteria. One peptide, identified in Rana arvalis, proved to be a potent and broad-spectrum antimicrobial, active against ATCC bacterial strains and a multi-drug resistant clinical isolate. CD spectroscopy suggests it has a helical conformation, while surface plasmon resonance (SPR) that it may self-aggregate/oligomerize at the membrane surface. It was found to disrupt the bacterial membrane at sub-MIC, MIC and above-MIC concentrations, as observed by flow cytometry and/or visualized by atomic force microscopy (AFM). Only a limited toxicity was observed towards peripheral blood mononuclear cells (PBMC) with a more pronounced effect observed against the MEC-1 cell line.

Discovery of novel quaternary ammonium compounds based on quinuclidine-3-ol as new potential antimicrobial candidates.

Quaternary ammonium compounds (QACs) are amphiphilic molecules displaying a broad-spectrum of antibacterial activity. QACs are commonly used antiseptics in industrial, home and hospital settings. Given the emergence of the QAC-resistant bacteria, there is an urgent need to design new QACs with good antimicrobial activity, able to escape the host resistance mechanism. Therefore, a series of QACs derived from quinuclidine-3-ol and an alkyl chain of variable length (QOH-C3 to -C14), was designed and synthesized. The antimicrobial potential of the new monoquaternary QACs was surveyed against seventeen strains of emerging food spoilage and pathogenic microorganisms, including clinical multidrug-resistant ESKAPE isolates. The QOH-C14 proved to have the strongest antimicrobial activity. It was highly active against all pathogens tested, particularly against the Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.06 to 3.9 µg/mL, and fungi exerting the MIC90 between 0.12 and 3.9 µg/mL. The potency of QOH-C14, confirmed that alkyl chains are an important part of the structure with their lengths playing a critical role in bioactivity of these compounds. The atomic force microscopy images show the disruption of a cell membrane upon the treatment with QOH-C14. These results were additionally confirmed by flow cytometry and fluorescence microscopy. A relatively low toxicity toward healthy human cells underline that QOH-C14 has a potential as new QAC antimicrobial candidate.