Probing Diffusive Dynamics of Natural Tubule Nanoclays with Machine Learning.

Reproducibility of the experimental results and object of study itself is one of the basic principles in science. But what if the object characterized by technologically important properties is natural and cannot be artificially reproduced one-to-one in the laboratory? The situation becomes even more complicated when we are interested in exploring stochastic properties of a natural system and only a limited set of noisy experimental data is available. In this paper we address these problems by exploring diffusive motion of some natural clays, halloysite and sepiolite, in a liquid environment. By using a combination of dark-field microscopy and machine learning algorithms, a quantitative theoretical characterization of the nanotubes' rotational diffusive dynamics is performed. Scanning the experimental video with the gradient boosting tree method, we can trace time dependence of the diffusion coefficient and probe different regimes of nonequilibrium rotational dynamics that are due to contacts with surfaces and other experimental imperfections. The method we propose is of general nature and can be applied to explore diffusive dynamics of various biological systems in real time.

Nanomechanical Atomic Force Microscopy to Probe Cellular Microplastics Uptake and Distribution.

The concerns regarding microplastics and nanoplastics pollution stimulate studies on the uptake and biodistribution of these emerging pollutants in vitro. Atomic force microscopy in nanomechanical PeakForce Tapping mode was used here to visualise the uptake and distribution of polystyrene spherical microplastics in human skin fibroblast. Particles down to 500 nm were imaged in whole fixed cells, the nanomechanical characterization allowed for differentiation between internalized and surface attached plastics. This study opens new avenues in microplastics toxicity research.

Label-free identification of microplastics in human cells: dark-field microscopy and deep learning study.

The development of an automatic method of identifying microplastic particles within live cells and organisms is crucial for high-throughput analysis of their biodistribution in toxicity studies. State-of-the-art technique in the data analysis tasks is the application of deep learning algorithms. Here, we propose the approach of polystyrene microparticle classification differing only in pigmentation using enhanced dark-field microscopy and a residual neural network (ResNet). The dataset consisting of 11,528 particle images has been collected to train and evaluate the neural network model. Human skin fibroblasts treated with microplastics were used as a model to study the ability of ResNet for classifying particles in a realistic biological experiment. As a result, the accuracy of the obtained classification algorithm achieved up to 93% in cell samples, indicating that the technique proposed will be a potent alternative to time-consuming spectral-based methods in microplastic toxicity research.

Biodistribution of Quantum Dots-Labelled Halloysite Nanotubes: A Caenorhabditis elegans In Vivo Study.

Halloysite is a promising building block in nanoarchitectonics of functional materials, especially in the development of novel biomaterials and smart coatings. Understanding the behavior of materials produced using halloysite nanotubes within living organisms is essential for their safe applications. In this study, quantum dots of different compositions were synthesized on the surface of modified clay nanotubes, and the biodistribution of this hybrid material was monitored within Caenorhabditis elegans nematodes. The influence of the modification agent as well as the particles' composition on physicochemical properties of hybrid nanomaterials was investigated. Several microscopy techniques, such as fluorescence and dark-field microscopy, were compared in monitoring the distribution of nanomaterials in nematodes' organisms. The effects of QDs-halloysite composites on the nematodes' life cycle were investigated in vivo. Our fluorescent hybrid probes induced no acute toxic effects in model organisms. The stable fluorescence and low toxicity towards the organisms suggest that the proposed synthesis procedure yields safe nanoarchitectonic materials that will be helpful in monitoring the behavior of nanomaterials inside living cells and organisms.

Clay Nanotube Immobilization on Animal Hair for Sustained Anti-Lice Protection.

Topical administration of drugs is required for the treatment of parasitic diseases and insect infestations; therefore, fabrication of nanoscale drug carriers for effective insecticide topical delivery is needed. Here we report the enhanced immobilization of halloysite tubule nanoclay onto semiaquatic capybaras which have hydrophobic hair surfaces as compared to their close relatives, land-dwelling guinea pigs, and other agricultural livestock. The hair surface of mammals varies in hydrophobicity having a cortex surrounded by cuticles. Spontaneous 1-2 µm thick halloysite hair coverages on the semi-aquatic rodent capybara, non-aquatic rodent guinea pig, and farm goats were compared. The best coating was found for capybara due to the elevated 5 wt% wax content. As a result, we suggest hair pretreatment with diluted wax for enhanced nanoclay adsorption. The formation of a stable goat hair coverage with a 2-3 µm halloysite layer loaded with permethrin insecticide allowed for long-lasting anti-parasitic protection, enduring multiple rain wettings and washings. We expect that our technology will find applications in animal parasitosis protection and may be extended to prolonged human anti-lice treatment.

Dark-field/hyperspectral microscopy for detecting nanoscale particles in environmental nanotoxicology research.

Nanoscale contaminants (including engineered nanoparticles and nanoplastics) pose a significant threat to organisms and environment. Rapid and non-destructive detection and identification of nanosized materials in cells, tissues and organisms is still challenging, although a number of conventional methods exist. These approaches for nanoparticles imaging and characterisation both inside the cytoplasm and on the cell or tissue outer surfaces, such as electron or scanning probe microscopies, are unquestionably potent tools, having excellent resolution and supplemented with chemical analysis capabilities. However, imaging and detection of nanomaterials in situ, in wet unfixed and even live samples, such as living isolated cells, microorganisms, protozoans and miniature invertebrates using electron microscopy is practically impossible, because of the elaborate sample preparation requiring chemical fixation, contrast staining, matrix embedding and exposure into vacuum. Atomic force microscopy, in several cases, can be used for imaging and mechanical analysis of live cells and organisms under ambient conditions, however this technique allows for investigation of surfaces. Therefore, a different approach allowing for imaging and differentiation of nanoscale particles in wet samples is required. Dark-field microscopy as an optical microscopy technique has been popular among researchers, mostly for imaging relatively large specimens. In recent years, the so-called "enhanced dark field" microscopy based on using higher numerical aperture light condensers and variable numerical aperture objectives has emegred, which allows for imaging of nanoscale particles (starting from 5 nm nanospheres) using almost conventional optical microscopy methodology. Hyperspectral imaging can turn a dark-field optical microscope into a powerful chemical characterisation tool. As a result, this technique is becoming popular in environmental nanotoxicology studies. In this Review Article we introduce the reader into the methodology of enhanced dark-field and dark-field-based hyperspectral microscopy, covering the most important advances in this rapidly-expanding area of environmental nanotoxicology.

Recent advances in the design of inorganic and nano-clay particles for the treatment of brain disorders.

Inorganic materials, in particular nanoclays and silica nanoparticles, have attracted enormous attention due to their versatile and tuneable properties, making them ideal candidates for a wide range of biomedical applications, such as drug delivery. This review aims at overviewing recent developments of inorganic nanoparticles (like porous or mesoporous silica particles) and different nano-clay materials (like montmorillonite, laponites or halloysite nanotubes) employed for overcoming the blood brain barrier (BBB) in the treatment and therapy of major brain diseases such as Alzheimer's, Parkinson's, glioma or amyotrophic lateral sclerosis. Recent strategies of crossing the BBB through invasive and not invasive administration routes by using different types of nanoparticles compared to nano-clays and inorganic particles are overviewed.

Worms eat oil: Alcanivorax borkumensis hydrocarbonoclastic bacteria colonise Caenorhabditis elegans nematodes intestines as a first step towards oil spills zooremediation.

The environmental hazards of oil spills cannot be underestimated. Bioremediation holds promise among various approaches to tackle oil spills in soils and sediments. In particular, using oil-degrading bacteria is an efficient and self-regulating way to remove oil spills. Using animals for oil spills remediation is in its infancy, mostly due to the lack of efficient oil-degrading capabilities in eukaryotes. Here we show that Caenorhabditis elegans nematodes survive for extended periods (up to 22 days) on pure crude oil diet. Moreover, we report for the first time the use of Alcanivorax borkumensis hydrocarbonoclastic bacteria for colonisation of C. elegans intestines, which allows for effective digestion of crude oil by the nematodes. The worms fed and colonised by A. borkumensis demonstrated the similar or even better longevity, resistance against oxidative and thermal stress and reproductivity as those animals fed with Escherichia coli bacteria (normal food). Importantly, A. borkumensis-carrying nematodes were able to accumulate oil droplet from oil-contaminated soils. Artificial colonisation of soil invertebrates with oil-degrading bacteria will be an efficient way to distribute microorganisms in polluted soil, thus opening new avenues for oil spills zooremediation.

Halloysite/Keratin Nanocomposite for Human Hair Photoprotection Coating.

We propose a novel keratin treatment of human hair by its aqueous mixtures with natural halloysite clay nanotubes. The loaded clay nanotubes together with free keratin produce micrometer-thick protective coating on hair. First, colloidal and structural properties of halloysite/keratin dispersions and the nanotube loaded with this protein were investigated. Above the keratin isoelectric point (pH = 4), the protein adsorption into the positive halloysite lumen is favored because of the electrostatic attractions. The ζ-potential magnitude of these core-shell particles increased from -35 (in pristine form) to -43 mV allowing for an enhanced colloidal stability (15 h at pH = 6). This keratin-clay tubule nanocomposite was used for the immersion treatment of hair. Three-dimensional-measuring laser scanning microscopy demonstrated that 50-60% of the hair surface coverage can be achieved with 1 wt % suspension application. Hair samples have been exposed to UV irradiation for times up to 72 h to explore the protection capacity of this coating by monitoring the cysteine oxidation products. The nanocomposites of halloysite and keratin prevent the deterioration of human hair as evident by significant inhibition of cysteic acid. The successful hair structure protection was also visually confirmed by atomic force microscopy and dark-field hyperspectral microscopy. The proposed formulation represents a promising strategy for a sustainable medical coating on the hair, which remediates UV irradiation stress.

Selective Antimicrobial Effects of Curcumin@Halloysite Nanoformulation: A Caenorhabditis elegans Study.

Alterations in the normal gastrointestinal microbial community caused by unhealthy diet, environmental factors, and antibiotic overuse may severely affect human health and well-being. Novel antimicrobial drug formulations targeting pathogenic microflora while not affecting or even supporting symbiotic microflora are urgently needed. Here we report fabrication of a novel antimicrobial nanocontainer based on halloysite nanotubes loaded with curcumin and protected with a dextrin outer layer (HNTs+Curc/DX) and its effective use to suppress the overgrowth of pathogenic bacteria in Caenorhabditis elegans nematodes. Nanocontainers have been obtained using vacuum-facilitated loading of hydrophobic curcumin into halloysite lumens. We have applied UV-vis and infrared spectroscopy, thermogravimetry and microscopy to characterize the HNTs+Curc/DX nanocontainers. In experiments in vitro we found that HNTs+Curc/DX effectively suppressed the growth of Serratia marcescens cells, whereas Escherichia coli bacteria were not affected. We applied HNTs+Curc/DX nanocontainers to alleviate the S. marcescens infection in C. elegans nematodes in vivo. The nematodes ingest HNTs+Curc/DX at 4-6 ng per worm, which results in improvement of the nematodes' fertility and life expectancy. Remarkably, treatment of S. marcescens-infected nematodes with HNTs+Curc/DX nanocontainers completely restored the longevity, demonstrating the enhanced bioavailability of hydrophobic curcumin. We believe that our results reported here open new avenues for fabrication of effective antimicrobial nanoformulations based on hydrophobic drugs and clay nanotubes.

Antimicrobial Applications of Clay Nanotube-Based Composites.

Halloysite nanotubes with different outer surface/inner lumen chemistry (SiO2/Al2O3) are natural objects with a 50 nm diameter hollow cylindrical structure, which are able to carry functional compounds both inside and outside. They are promising for biological applications where their drug loading capacity combined with a low toxicity ensures the safe interaction of these nanomaterials with living cells. In this paper, the antimicrobial properties of the clay nanotube-based composites are reviewed, including applications in microbe-resistant biocidal textile, paints, filters, and medical formulations (wound dressings, drug delivery systems, antiseptic sprays, and tissue engineering scaffolds). Though halloysite-based antimicrobial materials have been widely investigated, their application in medicine needs clinical studies. This review suggests the scalable antimicrobial nano/micro composites based on natural tubule clays and outlines research and development perspectives in the field.

Self-assembly of clay nanotubes on hair surface for medical and cosmetic formulations.

While most hair care formulations are developed on the basis of surfactants or polymers, we introduce self-assembly coating of micro and nanoparticles as the underlying principle for hair modification, protection and enhancement. Halloysite clay nanotubes formed by rolled sheets of aluminosilicate kaolin assemble on the surface of hair forming a robust multilayer coverage. Prior to the application, clay nanotubes were loaded with selected dyes or drug allowing for hair coloring or medical treatment. This facile process is based on a 3-minute application of 1 wt% aqueous dispersion of color/drug loaded halloysite resulting in a ca. 3 µm thick uniform hair surface coating. This technique, which employs a very safe, biocompatible and inexpensive material, is ubiquitous with respect to the species of source of hair and additives in solvent, making it viable as an excipient for conventional medical and veterinarian formulations.

Atomic force microscopy for imaging and nanomechanical characterisation of live nematode epicuticle: A comparative Caenorhabditis elegans and Turbatrix aceti study.

Atomic force microscopy (AFM), a powerful tool in interdisciplinary biomedical research, has been applied here to investigate the surface of live nematodes epicuticle. We have used AFM in PeakForce Tapping non-resonant imaging and nanomechanical characterisation mode to investigate and compare the surface features of epicuticle of two free-living microscopic nematodes, Caenorhabditis elegans and Turbatrix aceti. We have successfully immobilised live anesthetized adult nematodes on glass supports using either layer-by-layer-deposited polyelectrolyte films or bioadhesive coatings, which allowed for imaging the living nematodes in native environment. We have obtained AFM images and corresponding nanomechanical maps of annular rings and furrows, demonstrating the differences in topography and structure between the species. Our results demonstrate that AFM in PeakForce Tapping mode can be used to image and characterise surfaces of relatively-large live immobilised multicellular organisms, which can be further applied to a number of invertebrates.

Nematode Epicuticle Visualisation by PeakForce Tapping Atomic Force Microscopy.

The free-living soil nematode Caenorhabditis elegans has become an iconic experimental model animal in biology. This transparent animal can be easily imaged using optical microscopy to visualise its organs, tissues, single cells and subcellular events. The epicuticle of C. elegans nematodes has been studied at nanoscale using transmission and scanning (SEM) electron microscopies. As a result, imaging artefacts can appear due to embedding the worms into resins or coating the worms with a conductive gold layer. In addition, fixation and contrasting may also damage the cuticle. Conventional tapping mode atomic force microscopy (AFM) can be applied to image the cuticle of the dried nematodes in air, however this approach also suffers from imaging defects. Ideally, the nematodes should be imaged under conditions resembling their natural environment. Recently, we reported the use of PeakForce Tapping AFM mode for the successful visualisation and numerical characterisation of C. elegans nematode cuticle both in air and in liquid ( Fakhrullina et al., 2017 ). We imaged the principal nematode surface structures and characterised mechanical properties of the cuticle. This protocol provides the detailed description of AFM imaging of liquid-immersed C. elegans nematodes using PeakForce Tapping atomic force microscopy.

Nanoscale imaging and characterization of Caenorhabditis elegans epicuticle using atomic force microscopy.

Here we introduce PeakForce Tapping non-resonance atomic force microscopy for imaging and nanomechanical mapping of Caenorhabditis elegans nematodes. The animals were imaged both in air and water at nanoscale resolution. Layer-by-layer glass surface modification was employed to secure the worms for imaging in water. Microtopography of head region, annuli, furrows, lateral alae and tail region was visualized. Analysis of nanoscale surface features obtained during AFM imaging of three larval and adult hermaphrodite nematodes in natural environment allowed for numerical evaluation of annuli periodicity, furrows depth and annuli roughness. Nanomechanical mapping of surface deformation, Young modulus and adhesion confirms that the mechanical properties of the nematode cuticle are non-uniform. Overall, PeakForce Tapping AFM is a robust and simple approach applicable for nanoscale three-dimensional imaging and characterization of C. elegans nematodes.

Nano-labelled cells-a functional tool in biomedical applications.

Nanotechnology offers an unprecedented number of opportunities for biomedical research, utilizing the unusual functionalities of nanosized materials. Here we describe the recent advances in fabrication and utilization of nanoparticle-labelled cells. We present a brief overview of the most promising techniques, namely layer-by-layer polyelectrolyte assembly on cells and intracellular and extracellular labelling with magnetic nanoparticles. Several important practical application of nanofucntionalized cells, including tissue engineering and tumour therapy, are reviewed.