Advancing Vibrational Spectroscopy for Cellular and Sub Cellular Analysis: Raman Spectroscopy as a Novel In Vitro Nanotoxicological Assessment Protocol

This work is designed to establish a ‘High content Nanotoxicological Screening method’ using in vitro Raman microspectroscopy. The undeniable increase of nanotechnology based products has brought challenges in terms of determining their toxicological properties. Considering the total time and cost of screening nanomaterials by conventional methods, the need for a rapid, label-free technique which will provide a wide range of information on multiple parameters is unquestionable. This study investigated the applicability of Raman microspectroscopy as a High Content Screening technique to clarify cell-nanoparticle interaction by determining the localisation of the nanoparticles and consequent effects in these localised areas in terms of cyto- and geno- toxicity. For this purpose, in the first part of the study, the potential of Raman spectroscopy has been explored to monitor sequential trafficking of nanoparticles in cellular organelles and to determine the differing spectral signatures of the organelles. Human lung carcinoma cells (A549) were exposed to non-toxic carboxylate-modified and fluorescently-labelled polystyrene nanoparticles for 4, 12 and 24 hours and Raman spectral maps were acquired from the subcellular regions to determine their localisation. With the aid of multivariate analysis techniques, the study demonstrated the applicability of Raman microspectroscopy to provide information regarding localisation and to determine the local environment based on differing signatures of intracellular compartments such as endosomes, lysosomes and endoplasmic reticulum, in a completely label free manner. Aminated polystyrene nanoparticles (PS-NH2) and polyamidoamine (PAMAM) dendrimers are known to show acute toxicity and in order to observe this toxicity and corresponding responses, time and dose dependant Raman spectroscopic markers of cellular toxic events were systematically monitored upon nanoparticle exposure to A549 cells. Alamar Blue (AB) and 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromid (MTT) assays were employed to determine the mean effective concentration, EC50 and Raman spectroscopy was used to acquire multiple point spectra from the cytoplasm, nucleus and nucleolus. The most significant changes were observed in the cytoplasm for both time and dose dependent cases. The Raman spectral markers for lipidosis and oxidative stress were determined as a function of dose and time, and the responses were correlated with conventional cytotoxicity assays. With the aid of multivariate analysis techniques, the study showed the ability of Raman spectroscopy to provide information about cellular responses at different particle concentrations and exposure times. Following this, the potential of Raman microspectroscopy was analysed by comparing spectral marker evolution in non-cancerous cells (immortalized human bronchial epithelium) with cancerous cells (A549 and human lung epidermoid). Spectral markers of oxidative stress, cytoplasmic RNA aberrations and liposomal rupture were identified and cell-line dependent variations in these spectral markers were observed, and were correlated with cellular assays and imaging techniques. The findings from the comparison of spectral markers, especially in the low wavenumber region, have shown the applicability of Raman spectroscopy to identify different cell death pathways in cancerous and non-cancerous cell lines. Different cell death mechanisms were also identified based on common and/or differing spectral markers of cyto- and geno- toxicity upon PS-NH2 and PAMAM exposure. The results were correlated with flow cytometry and cytotoxicity assays. The study further demonstrated the potential of Raman microspectroscopy to iii differentiate apoptotic and necrotic cell death mechanisms, as a function of time (from 4 to 72 h) and applied dose (sub-lethal/lethal). Finally, in order to establish a toxicological assessment protocol based on Raman microspectroscopy, 3D graphs of biomarker intensities are plotted as a function of time and dose and also intensities are correlated with % viability values. The established 3D models can be used to predict nanotoxicity, which can also be applied to nanomedicine

Monitoring Stem Cell Differentiation Using Raman Microspectroscopy: Chondrogenic Differentiation, Towards Cartilage Formation

Mesenchymal Stem Cells (MSCs) have the ability to differentiate into chondrocytes, the only cellular components of cartilage and are therefore ideal candidates for cartilage and tissue repair technologies. Chondrocytes are surrounded by cartilage-like extracellular matrix (ECM), a complex network rich in glycosaminoglycans, proteoglycans, and collagen, which, together with a multitude of intracellular signalling molecules, trigger the chondrogenesis and allow the chondroprogenitor to acquire the spherical morphology of the chondrocytes. However, although the mechanisms of the differentiation of MSCs have been extensively explored, it has been difficult to provide a holistic picture of the process, in situ. Raman Micro Spectroscopy (RMS) has been demonstrated to be a powerful analytical tool, which provides detailed label free biochemical fingerprint information in a non-invasive way, for analysis of cells, tissues and body fluids. In this work, RMS is explored to monitor the process of Mesenchymal Stem Cell (MSC) differentiation into chondrocytes in vitro, providing a holistic molecular picture of cellular events governing the differentiation. Spectral signatures of the subcellular compartments, nucleolus, nucleus and cytoplasm were initially probed and characteristic molecular changes between differentiated and undifferentiated were identified. Moreover, high density cell micromasses were cultured over a period of three weeks, and a systematic monitoring of cellular molecular components and the progress of the ECM formation, associated with the chondrogenic differentiation, was performed. This study shows the potential applicability of RMS as a powerful tool to monitor and better understand the differentiation pathways and process

Vibrational Spectroscopy for In Vitro Monitoring Stem Cell Differentiation

Stem cell technology has attracted considerable attention over recent decades due to its enormous potential in regenerative medicine and disease therapeutics. Studying the underlying mechanisms of stem cell differentiation and tissue generation is critical, and robust methodologies and different technologies are required. Towards establishing improved understanding and optimised triggering and control of differentiation processes, analytical techniques such as flow cytometry, immunohistochemistry, reverse transcription polymerase chain reaction, RNA in situ hybridisation analysis, and fluorescence-activated cell sorting have contributed much. However, progress in the field remains limited because such techniques provide only limited information, as they are only able to address specific, selected aspects of the process, and/or cannot visualise the process at the subcellular level. Additionally, many current analytical techniques involve the disruption of the investigation process (tissue sectioning, immunostaining) and cannot monitor the cellular differentiation process in situ, in real-time. Vibrational spectroscopy, as a label-free, non-invasive and non-destructive analytical technique, appears to be a promising candidate to potentially overcome many of these limitations as it can provide detailed biochemical fingerprint information for analysis of cells, tissues, and body fluids. The technique has been widely used in disease diagnosis and increasingly in stem cell technology. In this work, the efforts regarding the use of vibrational spectroscopy to identify mechanisms of stem cell differentiation at a single cell and tissue level are summarised. Both infrared absorption and Raman spectroscopic investigations are explored, and the relative merits, and future perspectives of the techniques are discussed

A Performance Measurement Framework and Solution Approach for the Integrated Facility Layout Problem With Uncertain Demand

The integrated facility layout problem (IFLP) focuses on the simultaneous determination of the relative locations of multiple copies of capacitated equip- ment or machinery in a facility, as well as the material ow between these units. In this paper, we consider the IFLP in the existence of uncertain demand for the products of the facility. Motivated by the framework for next generation facility layouts by Benjaafar et al. (2002), we extend the approaches in the lit- erature for distributed facility layouts to the case of dynamic demand and the possibility of relayouts, and propose a heuristic solution approach to minimize the expected total material handling cost over the planning horizon. We also analyze the performance of the resulting solutions in terms of empty travel of the material handling equipment and waiting time. Our computational results reveal that when demand is dynamic and stochastic, the relationship between the level of uncertainty and relayout cost plays an important role in determin- ing layout performance, and therefore a priori assumption of using a certain layout type may lead to detrimental results

Determination of Spectral Markers of Cytotoxicity and Genotoxicity Using in vitro Raman Microspectroscopy: Cellular Responses to Polyamidoamine Dendrimer Exposure

Although consumer exposure to nanomaterials is ever increasing, with potential increased applications in areas such as drug and/or gene delivery, contrast agents and diagnosis, determination of cyto- and geno- toxic effect of nanomaterials on human health and the environment still remains challenging. Although many techniques have been established and adapted to determine the cytotoxicity and genotoxicity of nano-sized materials, these techniques remain limited by the number of assays required, total cost, use of labels and they struggle to explain the underlying interaction mechanisms. In this study, Raman microspectroscopy is employed as an in vitro label free high content screening technique to observe toxicological changes within the cell in a multi-parametric fashion. The evolution of spectral markers as a function of time and applied dose has been used to elucidate the mechanism of action of polyamidoamine (PAMAM) dendrimers associated with cytotoxicity and their impact on nuclear biochemistry. PAMAM dendrimers are chosen as a model nanomaterial due to their widely studied cytotoxic and genotoxic properties and commercial availability. Point spectra were acquired from cytoplasm to monitor the cascade of toxic events occurring in the cytoplasm upon nanoparticle exposure, whereas the spectra acquired from nucleus and nucleolus were used to explore PAMAM-nuclear material interaction as well as genotoxic responses

Toxicological Assessment of Nanomaterials: The Role of In Vitro Raman Microspectroscopic Analysis

The acceleration of nanomaterials research has brought about increased demands for rapid analysis of their bioactivity, in a multi-parametric fashion, to minimise the gap between potential applications and knowledge of their toxicological properties. The potential of Raman microspectroscopy for the analysis of biological systems with the aid of multivariate analysis techniques has been demonstrated. In this study, an overview of recent efforts towards establishing a ‘label-free high content nanotoxicological assessment technique’ using Raman microspectroscopy is presented. The current state of the art for cellular toxicity assessment and the potential of Raman microspectroscopy are discussed, and the spectral markers of the cellular toxic responses upon exposure to nanoparticles, changes on the identified spectral markers upon exposure to different nanoparticles, cell death mechanisms and the effects of nanoparticles on different cell lines are summarised. Moreover, 3D toxicity plots of spectral markers, as a function of time and dose, are introduced as new methodology for toxicological analysis based on the intrinsic properties of the biomolecular changes, such as cytoplasmic RNA aberrations, protein and lipid damage associated with the toxic response. The 3D evolution of the spectral markers are correlated with the results obtained by commonly-used cytotoxicity assays and significant similarities are observed between band intensity and percentage viability obtained by the Alamar Blue assay, as an example. Therefore, the developed 3D plots can be used to identify toxicological properties of a nanomaterial and can potentially be used to predict toxicity which can provide rapid advances in nanomedicine

Antibiotic susceptibility patterns among respiratory isolates of Gram-negative bacilli in a Turkish university hospital

BACKGROUND: Gram-negative bacteria cause most nosocomial respiratory infections. At the University of Cumhuriyet, we examined 328 respiratory isolates of Enterobacteriaceae and Acinetobacter baumanii organisms in Sivas, Turkey over 3 years. We used disk diffusion or standardized microdilution to test the isolates against 18 antibiotics. RESULTS: We cultured organisms from sputum (54%), tracheal aspirate (25%), and bronchial lavage fluid (21%). The most common organisms were Klebsiella spp (35%), A. baumanii (27%), and Escherichia coli (15%). Imipenem was the most active agent, inhibiting 90% of Enterobacteriaceae and A. baumanii organisms. We considered approximately 12% of Klebsiella pneumoniae and 21% of E. coli isolates to be possible producers of extended-spectrum beta-lactamase. K. pneumoniae isolates of the extended-spectrum beta-lactamase phenotype were more resistant to imipenem, ciprofloxacin, and tetracycline in our study than they are in other regions of the world. CONCLUSIONS: Our results suggest that imipenem resistance in our region is growing

Label-Free, High Content Screening Using Raman Microspectroscopy: The Toxicological Response of Different Cell Lines to Amine-Modified Polystyrene Nanoparticles

Nanotoxicology has become an established area of science due to growing concerns over the production and potential use of nanomaterials in a wide-range of areas from pharmaceutics to nanomedicine. Although different cytotoxicity assays have been developed and are widely used to determine the toxicity of nanomaterials, the production of multi-parametric information in a rapid and non-invasive way is still challenging, when the amount and diversity of physicochemical properties of nanomaterials are considered. High content screening can provide such analysis, but is often prohibitive in terms of capital and recurrent costs in academic environments. As a label-free technique, the applicability of Raman microspectroscopy for the analysis of cells, tissues and bodily fluids has been extensively demonstrated. The multi-parametric information in the fingerprint region has also been used for the determination of nanoparticle localisation and toxicity. In this study, the applicability of Raman microspectroscopy as a \u27high content nanotoxicological screening technique\u27 is demonstrated, with the aid of multivariate analysis, on non-cancerous (immortalized human bronchial epithelium) and cancerous cell-lines (human lung carcinoma and human lung epidermoid cells). Aminated polystyrene nanoparticles are chosen as model nanoparticles due to their well-established toxic properties and cells were exposed to the nanoparticles for periods from 24-72 hours. Spectral markers of cellular responses such as oxidative stress, cytoplasmic RNA aberrations and liposomal rupture are identified and cell-line dependent systematic variations in these spectral markers, as a function of the exposure time, are observed using Raman microspectroscopy, and are correlated with cellular assays and imaging techniques