Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins.

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Glycation changes molecular organization and charge distribution in type I collagen fibrils.

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils - potentially important in the microenvironment of actively dividing cells, such as cancer cells - disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

Poly(ADP-Ribose) Links the DNA Damage Response and Biomineralization.

Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.

In Vitro Targeting and Selective Killing of T47D Breast Cancer Cells by Purpurin and 5-Fluorouracil Anchored to Magnetic CNTs: Nitrene-Based Functionalization versus Uptake, Cytotoxicity, and Intracellular Fate.

Fe-encapsulated multiwall carbon nanotubes (Fe@MWCNTs) are candidates for magnetically targeted Drug Delivery Systems (mt-DDSs) against breast cancer. However, their full potential as versatile and biosafe vectors has yet to be developed. Key challenges that remain are relating surface functionalization to cytotoxicity and inducing selective cytotoxicity to cancer cells. We have studied quantitative uptake of pristine and functionalized Fe@MWCNTs (f-Fe@MWCNTs) in correlation to their in vitro cytotoxicity. Human monocyte macrophages (HMMs) and T47D breast cancer cells were selected as models to test selective cytotoxicity. [2+1]-Cycloaddition of nitrenes to Fe@MWCNTs yielded both effective functionalization and drug "tethering". Hydrophilization of Fe@MWCNTs was critical for efficient active cell uptake. f-Fe@MWCNTs were considerably more toxic to T47D cells than HMMs, in spite of longer exposure times of the latter. Eventually, Fe@MWCNTs loaded with 5-fluorouracil in a ß-cyclodextrin cage or with covalently linked purpurin emerged as the most cytotoxic and steerable in a magnetic field toward promising mt-DDSs.

Characterization and Visualization of Vesicles in the Endo-Lysosomal Pathway with Surface-Enhanced Raman Spectroscopy and Chemometrics.

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive vibrational fingerprinting technique widely used in analytical and biosensing applications. For intracellular sensing, typically gold nanoparticles (AuNPs) are employed as transducers to enhance the otherwise weak Raman spectroscopy signals. Thus, the signature patterns of the molecular nanoenvironment around intracellular unlabeled AuNPs can be monitored in a reporter-free manner by SERS. The challenge of selectively identifying molecular changes resulting from cellular processes in large and multidimensional data sets and the lack of simple tools for extracting this information has resulted in limited characterization of fundamental cellular processes by SERS. Here, this shortcoming in analysis of SERS data sets is tackled by developing a suitable methodology of reference-based PCA-LDA (principal component analysis-linear discriminant analysis). This method is validated and exemplarily used to extract spectral features characteristic of the endocytic compartment inside cells. The voluntary uptake through vesicular endocytosis is widely used for the internalization of AuNPs into cells, but the characterization of the individual stages of this pathway has not been carried out. Herein, we use reporter-free SERS to identify and visualize the stages of endocytosis of AuNPs in cells and map the molecular changes via the adaptation and advantageous use of chemometric methods in combination with tailored sample preparation. Thus, our study demonstrates the capabilities of reporter-free SERS for intracellular analysis and its ability to provide a way of characterizing intracellular composition. The developed analytical approach is generic and enables the application of reporter-free SERS to identify unknown components in different biological matrices and materials.

NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization.

Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15. The resulting spectra allowed us to refine an in vitro model of developing bone and to probe its detailed structure. The identification of an unexpected molecule, poly(adenosine diphosphate ribose), that may be implicated in calcification of the bone matrix, illustrates the analytical power of this approach.

The effect of particle agglomeration on the formation of a surface-connected compartment induced by hydroxyapatite nanoparticles in human monocyte-derived macrophages.

Agglomeration dramatically affects many aspects of nanoparticle-cell interactions. Here we show that hydroxyapatite nanoparticles formed large agglomerates in biological medium resulting in extensive particle uptake and dose-dependent cytotoxicity in human macrophages. Particle citration and/or the addition of the dispersant Darvan 7 dramatically reduced mean agglomerate sizes, the amount of particle uptake and concomitantly cytotoxicity. More surprisingly, agglomeration governed the mode of particle uptake. Agglomerates were sequestered within an extensive, interconnected membrane labyrinth open to the extracellular space. In spite of not being truly intracellular, imaging studies suggest particle degradation occurred within this surface-connected compartment (SCC). Agglomerate dispersion prevented the SCC from forming, but did not completely inhibit nanoparticle uptake by other mechanisms. The results of this study could be relevant to understanding particle-cell interactions during developmental mineral deposition, in ectopic calcification in disease, and during application of hydroxyapatite nanoparticle vectors in biomedicine.

The sequestration of hydroxyapatite nanoparticles by human monocyte-macrophages in a compartment that allows free diffusion with the extracellular environment.

Calcium phosphate and hydroxyapatite nanoparticles are extensively researched for medical applications, including bone implant materials, DNA and SiRNA delivery vectors and slow release vaccines. Elucidating the mechanisms by which cells internalize nanoparticles is fundamental for their long-term exploitation. In this study, we demonstrate that hydrophilic hydroxyapatite nanoparticles are sequestered within a specialized compartment called SCC (surface-connected compartment). This membrane-bound compartment is an elaborate labyrinth-like structure directly connected to the extracellular space. This continuity is demonstrated by in vivo 2-photon microscopy of ionic calcium using both cell-permeable and cell-impermeable dyes and by 3-D reconstructions from serial block-face SEM of fixed cells. Previously, this compartment was thought to be initiated specifically by exposure of macrophages to hydrophobic nanoparticles. However, we show that the SCC can be triggered by a much wider range of nanoparticles. Furthermore, we demonstrate its formation in A549 human lung epithelial cells, which are considerably less phagocytic than macrophages. EDX shows that extensive amounts of hydroxyapatite nanoparticles can be sequestered in this manner. We propose that SCC formation may be a means to remove large amounts of foreign material from the extracellular space, followed by slow degradation, may be to avoid excessive damage to surrounding cells or tissues.

Cellular uptake and cytotoxic impact of chemically functionalized and polymer-coated carbon nanotubes.

The impact of nanomaterials such as carbon nanotubes on biological matter is a topic of increasing interest and concern and requires a multifaceted approach to be resolved. A modified cytotoxic (lactate dehydrogenase (LDH)) assay is developed in an attempt to offer a valid and reliable methodology for screening carbon nanotube toxicity in vitro. Two of the most widely used types of surface-modified multiwalled carbon nanotubes (MWNTs) are tested: ammonium-functionalized MWNTs (MWNT-NH3+ ) and Pluronic F127 coated MWNTs (MWNT:F127). Chemically functionalized MWNTs show significantly greater cellular uptake into lung epithelial A549 cells compared to the non-covalently Pluronic F127-coated MWNTs. In spite of this, MWNT:F127 exhibit enhanced cytotoxicity according to the modified LDH assay. The validity of the modified LDH assay is further validated by direct comparison with other less reliable or accurate cytotoxicity assays. These findings indicate the reliability of the modified LDH assay as a screening tool to assess carbon nanotube cytotoxicity and illustrate that high levels of carbon nanotube cellular internalization do not necessarily lead to adverse responses.

Tunable chemistry and morphology of multi-wall carbon nanotubes as a route to non-toxic, theranostic systems.

Nanomedicine is one of the most promising areas of exploitation for multi-walled carbon nanotubes (MWNTs). These 'needle-like' nanovehicles are capable of carrying drug molecules via exo- and endohedral functionalisation and are steerable by an external magnetic field due to the presence of ferromagnetic nanoparticles in the nanotube core (up to 7.3wt.%). These properties make them promising candidates for drug targeting or MRI contrast agents. Particularly, oxidised and nitrogen-doped MWNTs exhibiting enhanced chemical reactivity compared to their unmodified precursors/analogues could be exploited in this field. Here, we assessed the toxicity and intracellular localisation of two different, chemically modified and unmodified nanotubes towards human macrophage cells using a range of toxicity and imaging techniques. Oxidised and N-doped MWNTs were not significantly toxic to HMMs in contrast to unmodified MWNTs. All types of MWNTs entered the cell via active phagocytosis/endocytosis, but also passively by 'self-injection' through the plasma membrane, and were ultimately found in the cytoplasm and possibly also the nucleus. The attained results carry hope to utilise functionalised nanotube vectors as non-cytotoxic controllable drug delivery systems.

Cellular uptake mechanisms of functionalised multi-walled carbon nanotubes by 3D electron tomography imaging.

Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH(3)(+)). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed that MWNT-NH(3)(+) were internalised in both phagocytic and non-phagocytic cells by any one of three mechanisms: (a) individually via membrane wrapping; (b) individually by direct membrane translocation; and (c) in clusters within vesicular compartments. At early time points following intracellular translocation, we noticed accumulation of nanotube material within various intracellular compartments, while a long-term (14-day) study using primary human macrophages revealed that MWNT-NH(3)(+) were able to escape vesicular (phagosome) entrapment by translocating directly into the cytoplasm.

In vivo carotid plaque MRI using quantitative T2* measurements with ultrasmall superparamagnetic iron oxide particles: a dose-response study to statin therapy.

This study investigates T(2)* quantification in carotid plaques before and after the administration of ultrasmall superparamagnetic iron oxide particles (USPIOs) in a cohort of patients receiving statin therapy. Phantom studies were performed using gels with varying concentrations of USPIOs. In the phantom study, 12 gels were prepared with a range of freely distributed concentrations of USPIO nanoparticles (0-0.05 mg/mL). Relative signal intensity measurements were obtained from a T(2)*-weighted sequence as well as quantitative T(2)* (qT(2)*) measurements. In the patient study, 40 patients with >40% carotid stenosis were randomised to low- and high-dose statin therapy (10 and 80 mg of atorvastatin). Pre- and post- (36 h) USPIO-enhanced MRI were performed at baseline, and at 6 and 12 weeks. A linear mixed-effects model was applied to account for the inherent correlation of multiple-plaque measurements from the same patient and to assess dose-response differences to statin therapy. In the phantom study, the T(2)*-weighted sequence demonstrated an initial increase (T(1) effect), followed by a decrease (T(2)* effect), in relative signal intensity with increasing concentrations of USPIO. The qT(2)* values decreased exponentially with increasing concentrations of USPIO. In the patient study, there was a highly significant difference in post-USPIO T(2)* measurements in plaques between the low- and high-dose statin groups. This was observed for both the difference in qT(2)* measurements (post-USPIO minus pre-USPIO) (p < 0.001) and for qT(2)* post-USPIO only (p < 0.001). The post-USPIO qT(2)* values were as follows: baseline: low dose, 13.6 ± 5.5 ms; high dose, 12.9 ± 6.2 ms; 6 weeks: low dose, 13.3 ± 6.7 ms; high dose, 14.3 ± 7.7 ms; 12 weeks: low dose, 14.0 ± 7.6 ms; high dose, 18.3 ± 11.2 ms. It can be concluded that qT(2)* measurements provide an alternative method of quantifying USPIO uptake. These results also demonstrate that changes in USPIO uptake can be measured using post-USPIO imaging only.

Spherical bioactive glass particles and their interaction with human mesenchymal stem cells in vitro.

Sub-micron particles of bioactive glass (SMBGs) with composition 85 mol% SiO(2) and 15 mol% CaO were synthesised and characterised. Bioactivity was demonstrated by the formation of calcium apatite following 5 days immersion in simulated body fluid (SBF). The effect of a 24 h exposure of SMBGs (100 µg/ml, 150 µg/ml, 200 µg/ml) to human mesenchymal stem cells (hMSCs) on cell viability, metabolic activity and proliferation were determined using the LIVE/DEAD, MTT, total DNA and LDH assays after 1, 4 and 7 days of culture. None of the SMBG concentrations caused significant cytotoxicity at 1 and 4 days, but the doses of 150 and 200 µg/ml significantly decreased hMSC metabolic activity after 7 days of culture. Cell proliferation decreased as SMBG concentration increased; however none of the SMBGs tested had a significant effect on DNA quantity compared to the control. Confocal microscopy confirmed cellular uptake and localisation of the SMBGs in the hMSC cytoskeleton. Transmission electron microscopy revealed that the SMBGs localised inside the cell cytoplasm and cell endosomes. These findings are important for assessing the toxicity of sub-micron particles that may either be used as injectables for bone regeneration or generated by wear or degradation of bioactive glass scaffolds.

pH-dependent toxicity of high aspect ratio ZnO nanowires in macrophages due to intracellular dissolution.

High-aspect ratio ZnO nanowires have become one of the most promising products in the nanosciences within the past few years with a multitude of applications at the interface of optics and electronics. The interaction of zinc with cells and organisms is complex, with both deficiency and excess causing severe effects. The emerging significance of zinc for many cellular processes makes it imperative to investigate the biological safety of ZnO nanowires in order to guarantee their safe economic exploitation. In this study, ZnO nanowires were found to be toxic to human monocyte macrophages (HMMs) at similar concentrations as ZnCl(2). Confocal microscopy on live cells confirmed a rise in intracellular Zn(2+) concentrations prior to cell death. In vitro, ZnO nanowires dissolved very rapidly in a simulated body fluid of lysosomal pH, whereas they were comparatively stable at extracellular pH. Bright-field transmission electron microscopy (TEM) showed a rapid macrophage uptake of ZnO nanowire aggregates by phagocytosis. Nanowire dissolution occurred within membrane-bound compartments, triggered by the acidic pH of the lysosomes. ZnO nanowire dissolution was confirmed by scanning electron microscopy/energy-dispersive X-ray spectrometry. Deposition of electron-dense material throughout the ZnO nanowire structures observed by TEM could indicate adsorption of cellular components onto the wires or localized zinc-induced protein precipitation. Our study demonstrates that ZnO nanowire toxicity in HMMs is due to pH-triggered, intracellular release of ionic Zn(2+) rather than the high-aspect nature of the wires. Cell death had features of necrosis as well as apoptosis, with mitochondria displaying severe structural changes. The implications of these findings for the application of ZnO nanowires are discussed.

Generation and manipulation of magnetic multicellular spheroids.

Multicellular spheroids have important applications in tumour studies, drug screening and tissue engineering. To enable simple manipulation of spheroids, magnetically labelled HeLa cells were cultured in hanging drops to generate magnetic spheroids. HeLa cells were labelled by biotinylating their cell membrane proteins and then binding streptavidin paramagnetic particles onto the biotinylated cell surface. Spheroids of different sizes were obtained by varying the seeding cell concentrations within the hanging drops and the spheroids had good cell viability. Characterisation of the F-actin distribution within the spheroids indicated a three dimensional reorganisation of the cellular cytoskeleton compared to monolayer cultures. The magnetic moment of the spheroids was measured and showed a superparamagnetic response in an applied field. Transmission electron microscopy analysis indicated that the paramagnetic particles were still present in the spheroids even after 21 days of culture. These spheroids could be easily and quickly separated magnetically without the need for centrifugation. The magnetic spheroids were also successfully manipulated and patterned using magnetic fields within a few seconds. The patterned spheroids then fused together to form a larger tissue construct.

Toxicity and imaging of multi-walled carbon nanotubes in human macrophage cells.

Multi-walled carbon nanotubes (MWNTs) have been proposed for use in many applications and concerns about their potential effect on human health have led to the interest in understanding the interactions between MWNTs and human cells. One important technique is the visualisation of the intracellular distribution of MWNTs. We exposed human macrophage cells to unpurified MWNTs and found that a decrease in cell viability was correlated with uptake of MWNTs due to mainly necrosis. Cells treated with purified MWNTs and the main contaminant Fe(2)O(3) itself yielded toxicity only from the nanotubes and not from the Fe(2)O(3). We used 3-D dark-field scanning transmission electron microscopy (DF-STEM) tomography of freeze-dried whole cells as well as confocal and scanning electron microscopy (SEM) to image the cellular uptake and distribution of unpurified MWNTs. We observed that unpurified MWNTs entered the cell both actively and passively frequently inserting through the plasma membrane into the cytoplasm and the nucleus. These suggest that MWNTs may cause incomplete phagocytosis or mechanically pierce through the plasma membrane and result in oxidative stress and cell death.

Iron oxide particles for atheroma imaging.

The selection of patients for vascular interventions has been solely based on luminal stenosis and symptomatology. However, histological data from both the coronary and carotid vasculature suggest that other plaque features such as inflammation may be more important in predicting future thromboembolic events. Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation in humans. It has reached the stage of development to have been recently used in an interventional drug study to not only assess inflammatory progression but also select patients at high risk. This article reviews the basic science behind the use of USPIO contrast agents in atheroma MR imaging, experimental work in animals, and how this has led to the emergence of this promising targeted imaging platform for assessment of high risk carotid atherosclerosis in humans.