The influence of size and surface chemistry on the bioavailability, tissue distribution and toxicity of gold nanoparticles in zebrafish (Danio rerio).

Gold nanoparticles (AuNPs) are widely used in biomedicine and their specific properties including, size, geometrics, and surface coating, will affect their fate and behaviour in biological systems. These properties are well studied for their intended biological targets, but there is a lack of understanding on the mechanisms by which AuNPs interact in non-target organisms when they enter the environment. We investigated the effects of size and surface chemistry of AuNPs on their bioavailability, tissue distribution and potential toxicity using zebrafish (Danio rerio) as an experimental model. Larval zebrafish were exposed to fluorescently tagged AuNPs of different sizes (10-100 nm) and surface modifications (TNFα, NHS/PAMAM and PEG), and uptake, tissue distribution and depuration rates were measured using selective-plane illumination microscopy (SPIM). The gut and pronephric tubules were found to contain detectable levels of AuNPs, and the concentration-dependent accumulation was related to the particle size. Surface addition of PEG and TNFα appeared to enhance particle accumulation in the pronephric tubules compared to uncoated particles. Depuration studies showed a gradual removal of particles from the gut and pronephric tubules, although fluorescence indicating the presence of the AuNPs remained in the pronephros 96 h after exposure. Toxicity assessment using two transgenic zebrafish reporter lines, however, revealed no AuNP-related renal injury or cellular oxidative stress. Collectively, our data show that AuNPs used in medical applications across the size range 40-80 nm, are bioavailable to larval zebrafish and some may persist in renal tissue, although their presence did not result in measurable toxicity with respect to pronephric organ function or cellular oxidative stress for short term exposures.

Exploration of utility of combined optical photothermal infrared and Raman imaging for investigating the chemical composition of microcalcifications in breast cancer.

Microcalcifications play an important role in cancer detection. They are evaluated by their radiological and histological characteristics but it is challenging to find a link between their morphology, their composition and the nature of a specific type of breast lesion. Whilst there are some mammographic features that are either typically benign or typically malignant often the appearances are indeterminate. Here, we explore a large range of vibrational spectroscopic and multiphoton imaging techniques in order to gain more information about the composition of the microcalcifications. For the first time, we validated the presence of carbonate ions in the microcalcifications by O-PTIR and Raman spectroscopy at the same time, the same location and the same high resolution (0.5 µm). Furthermore, the use of multiphoton imaging allowed us to create stimulated Raman histology (SRH) images which mimic histological images with all chemical information. In conclusion, we established a protocol for efficiently analysing the microcalcifications by iteratively refining the area of interest.

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

Combined effects of exposure to engineered silver nanoparticles and the water-soluble fraction of crude oil in the marine copepod Calanus finmarchicus.

While it is likely that ENPs may occur together with other contaminants in nature, the combined effects of exposure to both ENPs and environmental contaminants are not studied sufficiently. In this study, we investigated the acute and sublethal toxicity of PVP coated silver nanoparticles (AgNP) and ionic silver (Ag+; administered as AgNO3) to the marine copepod Calanus finmarchicus. We further studied effects of single exposures to AgNPs (nominal concentrations: low 15 µg L-1 NPL, high 150 µg L-1 NPH) or Ag+ (60 µg L-1), and effects of co-exposure to AgNPs, Ag+ and the water-soluble fraction (WSF; 100 µg L-1) of a crude oil (AgNP + WSF; Ag++WSF). The gene expression and the activity of antioxidant defense enzymes SOD, CAT and GST, as well as the gene expression of HSP90 and CYP330A1 were determined as sublethal endpoints. Results show that Ag+ was more acutely toxic compared to AgNPs, with 96 h LC50 concentrations of 403 µg L-1 for AgNPs, and 147 µg L-1 for Ag+. Organismal uptake of Ag following exposure was similar for AgNP and Ag+, and was not significantly different when co-exposed to WSF. Exposure to AgNPs alone caused increases in gene expressions of GST and SOD, whereas WSF exposure caused an induction in SOD. Responses in enzyme activities were generally low, with significant effects observed only on SOD activity in NPL and WSF exposures and on GST activity in NPL and NPH exposures. Combined AgNP and WSF exposures caused slightly altered responses in expression of SOD, GST and CYP330A1 genes compared to the single exposures of either AgNPs or WSF. However, there was no clear pattern of cumulative effects caused by co-exposures of AgNPs and WSF. The present study indicates that the exposure to AgNPs, Ag+, and to a lesser degree WSF cause an oxidative stress response in C. finmarchicus, which was slightly, but mostly not significantly altered in combined exposures. This indicated that the combined effects between Ag and WSF are relatively limited, at least with regard to oxidative stress.

Ultra-low timing jitter, Ti:Al2O3 synchronization for stimulated Raman scattering and pump-probe microscopy.

SIGNIFICANCE: Stimulated Raman scattering (SRS) and pump-probe microscopy are implementations of multiphoton microscopy that acquire high-resolution, label-free images of live samples encoded with molecular contrast. Most commercial multiphoton microscopes cannot access these techniques since they require sample illumination by two temporally synchronized ultrafast pulse trains. We present a compact and robust way of synchronizing an additional Ti:sapphire laser with a conventional single-beam multiphoton microscope to realize an instrument that can acquire images with enhanced molecular specificity. AIM: A passive optical synchronization scheme for a pair of commercially available, unmodified modelocked Ti:sapphire lasers was developed. The suitability of this synchronization scheme for advanced biomedical microscopy was investigated. APPROACH: A pair of modelocked Ti:sapphire lasers were aligned in master-slave configuration. Five percent of the master laser output was used to seed the modelocking in the slave laser cavity. The timing jitter of the master and slave pulse trains was characterized using an optical autocorrelator. The synchronized output of both lasers was coupled into a laser scanning microscope and used to acquire spectral focusing SRS and pump-probe microscopy images from biological and nonbiological samples. RESULTS: A timing jitter between the modelocked pulse trains of 0.74 fs was recorded. Spectral focusing SRS allowed spectral discrimination of polystyrene and polymethyl methacrylate beads. Pump-probe microscopy was used to record excited state lifetime curves from hemoglobin in intact red blood cells. CONCLUSION: Our work demonstrates a simple and robust method of upgrading single-beam multiphoton microscopes with an additional ultrafast laser. The resulting dual-beam instrument can be used to acquire label-free images of sample structure and composition with high biochemical specificity.

Microstructure and antibacterial efficacy of graphene oxide nanocomposite fibres.

Antibacterial polymer nanocomposite fibre meshes containing graphene oxide (GO) nanosheets were successfully prepared by pressurised gyration. The morphological and chemical composition of the resulting fibre meshes were determined using Scanning Electron Microscopy (SEM), Raman spectroscopy, Raman mapping and Fourier-Transform Infrared Spectroscopy (FT-IR). SEM showed the fibres to have an average diameter increasing from ~1-4 µm as the GO loading increased. FT-IR and Raman spectroscopy confirmed the inclusion of GO nanosheets on the fibre surface. The antibacterial potential of GO nanocomposite fibres were investigated using Escherichia coli K12. Average bacterial reduction ranged from 46 to 85 % with results favouring the strongest bioactivities of the nanocomposite containing 8 wt% of GO. Finally, bacterial toxicity of the nanocomposites was evaluated by reactive oxygen species (ROS) formation. A mechanism for the antibacterial behaviour of the nanocomposite fibres is presented. Stimulated Raman scattering imaging and spectra of the fibres post antibacterial studies showed flakes of GO distributed across the surface of the poly(methyl 2-methylpropenoate) (PMMA) fibres, which contribute to the high killing efficacy of the composites towards E. coli. GO nanosheets embedded in a polymer matrix have demonstrated the ability to retain their antibacterial properties, thus offering themselves as a promising antibacterial agent.

Author Correction: 4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

In situ chemically specific mapping of agrochemical seed coatings using stimulated Raman scattering microscopy.

Providing sufficient, healthy food for the increasing global population is putting a great deal of pressure on the agrochemical industry to maximize crop yields without sustaining environmental damage. The growth and yield of every plant with sexual reproduction, depends on germination and emergence of sown seeds, which is affected greatly by seed disease. This can be most effectively controlled by treating seeds with pesticides before they are sown. An effective seed coating treatment requires a high surface coverage and adhesion of active ingredients onto the seed surface and the addition of adhesive agents in coating formulations plays a key role in achieving this. Although adhesive agents are known to enhance seed germination, little is understood about how they affect surface distribution of actives and how formulations can be manipulated to rationally engineer seed coating preparations with optimized coverage and efficacy. We show, for the first time, that stimulated Raman scattering microscopy can be used to map the seed surface with microscopic spatial resolution and with chemical specificity to identify formulation components distributed on the seed surface. This represents a major advance in our capability to rationally engineer seed coating formulations with enhanced efficacy.

Clinical applications of infrared and Raman spectroscopy: state of play and future challenges.

Vibrational spectroscopies, based on infrared absorption and/or Raman scattering provide a detailed fingerprint of a material, based on the chemical content. Diagnostic and prognostic tools based on these technologies have the potential to revolutionise our clinical systems leading to improved patient outcome, more efficient public services and significant economic savings. However, despite these strong drivers, there are many fundamental scientific and technological challenges which have limited the implementation of this technology in the clinical arena, although recent years have seen significant progress in addressing these challenges. This review examines (i) the state of the art of clinical applications of infrared absorption and Raman spectroscopy, and (ii) the outstanding challenges, and progress towards translation, highlighting specific examples in the areas of in vivo, ex vivo and in vitro applications. In addition, the requirements of instrumentation suitable for use in the clinic, strategies for pre-processing and statistical analysis in clinical spectroscopy and data sharing protocols, will be discussed. Emerging consensus recommendations are presented, and the future perspectives of the field are assessed, particularly in the context of national and international collaborative research initiatives, such as the UK EPSRC Clinical Infrared and Raman Spectroscopy Network, the EU COST Action Raman4Clinics, and the International Society for Clinical Spectroscopy.

Correction: Clinical applications of infrared and Raman spectroscopy: state of play and future challenges.

Correction for 'Clinical applications of infrared and Raman spectroscopy: state of play and future challenges' by Matthew J. Baker, et al., Analyst, 2018, DOI: 10.1039/c7an01871a.

Nanoparticulate peptide delivery exclusively to the brain produces tolerance free analgesia.

The delivery of peptide drugs to the brain is challenging, principally due to the blood brain barrier and the low metabolic stability of peptides. Exclusive delivery to the brain with no peripheral exposure has hitherto not been demonstrated with brain quantification data. Here we show that polymer nanoparticles encapsulating leucine5-enkephalin hydrochloride (LENK) are able to transport LENK exclusively to the brain via the intranasal route, with no peripheral exposure and nanoparticle localisation is observed within the brain parenchyma. Animals dosed with LENK nanoparticles (NM0127) showed a strong anti-nociceptive response in multiple assays of evoked and on going pain whereas animals dosed intranasally with LENK alone were unresponsive. Animals did not develop tolerance to the anti-hyperalgesic activity of NM0127 and NM0127 was active in morphine tolerant animals. A microparticulate formulation of clustered nanoparticles was prepared to satisfy regulatory requirements for nasal dosage forms and the polymer nanoparticles alone were found to be biocompatible, via the nasal route, on chronic dosing.

4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.

Functional neuroimaging, using genetically-encoded Ca2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.

Imaging microscopic distribution of antifungal agents in dandruff treatments with stimulated Raman scattering microscopy.

Treatment of dandruff condition usually involves use of antidandruff shampoos containing antifungal agents. Different antifungal agents show variable clinical efficacy based on their cutaneous distribution and bioavailability. Using stimulated Raman scattering (SRS), we mapped the distribution of unlabeled low-molecular weight antifungal compounds zinc pyrithione (ZnPT) and climbazole (CBZ) on the surface of intact porcine skin with cellular precision. SRS has sufficient chemical selectivity and sensitivity to detect the agents on the skin surface based on their unique chemical motifs that do not occur naturally in biological tissues. Moreover, SRS is able to correlate the distribution of the agents with the morphological features of the skin using the CH 2 stretch mode, which is abundant in skin lipids. This is a significant strength of the technique since it allows the microscopic accumulation of the agents to be correlated with physiological features and their chemical environment without the use of counter stains. Our findings show that due to its lower solubility, ZnPT coats the surface of the skin with a sparse layer of crystals in the size range of 1 to 4 ?? ? m . This is consistent with the current understanding of the mode of action of ZnPT. In contrast, CBZ being more soluble and hydrophobic resulted in diffuse homogeneous distribution. It predominantly resided in microscopic lipid-rich crevasses and penetrated up to 60 ?? ? m into the infundibular spaces surrounding the hair shaft. The ability of the SRS to selectively map the distribution of agents on the skin's surface has the potential to provide insight into the mechanisms underpinning the topical application of antifungal or skin-active agents that could lead to the rational engineering of enhanced formulations.

Limiting the level of tertiary amines on polyamines leads to biocompatible nucleic acid vectors.

We have designed an efficient, synthetic nucleic acid vector, which is relatively non-toxic. [N-(2-ethylamino)-6-O-glycolchitosan - EAGC] polymers were 10-50 fold less toxic than Lipofectamine 2000, able to complex DNA, mRNA and siRNA into positively charged (zeta potential=+40 - 50mV), 50-450nm nanoparticles. The level of tertiary amine N-2-ethylamino substitution (DStert) was inversely proportional to the IC50 of the EAGC polymers in the A431 cell line: IC50=6.18DStert-0.9, r2=0.9991. EAGC polyplexes were stable against a heparin challenge, able to protect the nucleic acids from nuclease degradation and achieve levels of transfection comparable to Lipofectamine 2000 formulations. The relative biocompatibility of the vector allowed 10 fold higher doses of DNA (1µg compared to 0.1µg per well with Lipofectamine 2000) and siRNA (10.7µg per well vs 1.3µg with Lipofectamine 2000) to be applied to cells, when compared to Lipofectamine 2000. Finally intranasal application of EAGC - siRNA complexes resulted in siRNA transfer to the neurons of the olfactory bulb.

Making microscopy count: quantitative light microscopy of dynamic processes in living plants.

Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking.

Effect of Microplastic on the Gills of the Shore Crab Carcinus maenas.

Microscopic plastic debris (microplastics, <5 mm in diameter) is ubiquitous in the marine environment. Previous work has shown that microplastics may be ingested and inhaled by the shore crab Carcinus maenas, although the biological consequences are unknown. Here, we show that acute aqueous exposure to polystyrene microspheres (8 µm) with different surface coatings had significant but transient effects on branchial function. Microspheres inhaled into the gill chamber had a small but significant dose-dependent effect on oxygen consumption after 1 h of exposure, returning to normal levels after 16 h. Ion exchange was also affected, with a small but significant decrease in hemolymph sodium ions and an increase in calcium ions after 24 h post-exposure. To further asses the effects on osmoregulation, we challenged crabs with reduced salinity after microplastic exposure. Neither microspheres nor natural sediments altered the crab's response to osmotic stress regardless of plastic concentration added. Carboxylated (COOH) and aminated (NH2) polystyrene microspheres were distributed differently across the gill surface, although neither had a significant adverse impact on gill function. These results illustrate the extent of the physiological effects of microplastics compared to the physiological resilience of shore crabs in maintaining osmoregulatory and respiratory function after acute exposure to both anthropogenic plastics and natural particles.

Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels - A Strategy for Brain Cancer Treatments.

PURPOSE: The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression. METHODS: Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg(-1)) or ethanolic lomustine (6.5 mg kg(-1)) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg(-1)) or ethanolic lomustine (daily 1.2 mg kg(-1) - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated. RESULTS: The MET formulation resulted in modest brain targeting (brain/ bone AUC0-4h ratios for MET and ethanolic lomustine = 0.90 and 0.53 respectively and brain/ liver AUC0-4h ratios for MET and ethanolic lomustine = 0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes. CONCLUSIONS: Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.

Drug delivery into microneedle-porated nails from nanoparticle reservoirs.

This study demonstrates the potential of polymeric nanoparticles as drug reservoirs for sustained topical drug delivery into microneedle-treated human nail. Laser scanning confocal microscopy was used to image the delivery of a fluorescent model compound from nanoparticles into the nail. A label-free imaging technique, stimulated Raman scattering microscopy, was applied, in conjunction with two-photon fluorescence imaging, to probe the disposition of nanoparticles and an associated lipophilic 'active' in a microneedle-porated nail. The results provide clear evidence that the nanoparticles function as immobile reservoirs, sequestered on the nail surface and in the microneedle-generated pores, from which the active payload can be released and diffuse laterally into the nail over an extended period of time.

Molecular diffusion in the human nail measured by stimulated Raman scattering microscopy.

The effective treatment of diseases of the nail remains an important unmet medical need, primarily because of poor drug delivery. To address this challenge, the diffusion, in real time, of topically applied chemicals into the human nail has been visualized and characterized using stimulated Raman scattering (SRS) microscopy. Deuterated water (D2O), propylene glycol (PG-d8), and dimethyl sulphoxide (DMSO-d6) were separately applied to the dorsal surface of human nail samples. SRS microscopy was used to image D2O, PG-d8/DMSO-d6, and the nail through the O-D, -CD2, and -CH2 bond stretching Raman signals, respectively. Signal intensities obtained were measured as functions of time and of depth into the nail. It was observed that the diffusion of D2O was more than an order of magnitude faster than that of PG-d8 and DMSO-d6. Normalization of the Raman signals, to correct in part for scattering and absorption, permitted semiquantitative analysis of the permeation profiles and strongly suggested that solvent diffusion diverged from classical behavior and that derived diffusivities may be concentration dependent. It appeared that the uptake of solvent progressively undermined the integrity of the nail. This previously unreported application of SRS has permitted, therefore, direct visualization and semiquantitation of solvent penetration into the human nail. The kinetics of uptake of the three chemicals studied demonstrated that each altered its own diffusion in the nail in an apparently concentration-dependent fashion. The scale of the unexpected behavior observed may prove beneficial in the design and optimization of drug formulations to treat recalcitrant nail disease.

Tracing engineered nanomaterials in biological tissues using coherent anti-Stokes Raman scattering (CARS) microscopy - A critical review.

Nanomaterials (NMs) are used in an extremely diverse range of products and are increasingly entering the environment, driving a need to better understand their potential health effects in both humans and wildlife. A major challenge in nanoparticle (eco)toxicology is the ability to localise NMs post exposure, to enable more targeted biological effects analyses. A range of imaging techniques have been applied to do so, but they are limited, requiring either extensive processing of the material, staining or use of high intensity illumination that can lead to photo damage and/or have limited tissue penetration. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free imaging technique, providing contrast based on the intrinsic molecular vibrations of a specimen, circumventing the need for chemical perturbation by exogenous labels. CARS uses near infra-red excitation wavelengths which allow microscopy at depths of several hundred microns in intact tissues and minimises photo-damage to live and delicate samples. Here we provide an overview of the CARS process and present a series of illustrative examples demonstrating its application for detecting NMs within biological tissues, ranging from isolated cells to whole organisms and including materials spanning metals to polymers. We highlight the advantages of this technique which include chemically selective live imaging and substantial depth penetration, but we also discuss its limitations when applied to nanotoxicology, which most notably include the lack of resolution for studies on single nanoparticles.