Potential of Raman Spectroscopy For the Analysis of Plasma/serum in the Liquid State: Recent Advances

There is compelling evidence in the literature to support the application of Raman spectroscopy for analysis of bodily fluids in their native liquid state. Naturally, the strategies described in the literature for Raman spectroscopic analysis of liquid samples have advantages and disadvantages. Herein, recent advances in the analysis of plasma/serum in the liquid state are reviewed. The potential advantages of Raman analysis in the liquid form over the commonly employed infrared absorption analysis in the dried droplet form are initially highlighted. Improvements in measurement protocols based on inverted microscopic geometries, clinically adaptable substrates, data preprocessing and analysis, and applications for routine monitoring of patient health as well as therapeutic administration are reviewed. These advances suggest that clinical translation of Raman spectroscopy for rapid biochemical analysis can be a reality. In the future, this method will prove to be highly beneficial to clinicians for rapid screening and monitoring of analytes and drugs in the biological fluids, and to the patients themselves, enabling early treatment, before the disease becomes symptomatic, allowing early recovery

A comparison of catabolic pathways induced in primary macrophages by pristine single walled carbon nanotubes and pristine graphene

Understanding the correlation between the physico-chemical properties of carbonaceous nanomaterials and how these properties impact on cells and subcelluar mechanisms is critical to their risk assessment and safe translation into newly engineered devices. Here the toxicity, uptake and catabolic response of primary human macrophages to pristine graphene (PG) and pristine single walled carbon nanotubes (pSWCNT) are explored, compared and contrasted. The nanomaterial toxicity was assessed using three complementary techniques (live-dead assay, real time impedance technique and confocal microscopic analysis), all of which indicated no signs of acute cytotoxicity in response to PG or pSWCNT. Transmission electron microscopy (TEM) demonstrated that PG was phagocytosed by the cells into single membrane lysosomal vesicles, whereas the primary macrophages exposed to pSWCNT contained many double membrane vesicles indicative of an autophagic response. These distinct catabolic pathways were further verified by biochemical and microscopic techniques. Raman spectroscopic mapping was used to explore the nanomaterial uptake and distribution. Based on the G-band, significant uptake and accumulation of the PG in discrete vesicles was recorded, whereas the pSWCNT were not taken up to the same extent. Thermogravimetric analysis (TGA) of the cells treated with PG revealed that ~ 20-30% of the remaining dry mass was made up of PG. No detectable amount of pSWCNT was recorded using TGA. TEM analysis confirmed that PG was still graphitic even after 24 hours of accumulation in the lysosomal compartments. In conclusion, these two nanomaterials with similar surface chemistries but unique geometries differ significantly in their uptake mechanisms and subsequently induced lysosomal and autophagic catabolic pathways in human primary macrophages

Quantitative Analysis of Human Blood Serum using Vibrational Spectroscopy.

Analysis of bodily fluids using vibrational spectroscopy has attracted increasing attention in recent years. In particular, infrared spectroscopic screening of blood products, particularly blood serum, for disease diagnostics has been advanced considerably, attracting commercial interests. However, analyses requiring quantification of endogenous constituents or exogenous agents in blood are less well advanced. Recent advances towards this end are reviewed, focussing on infrared and Raman spectroscopic analyses of human blood serum. The importance of spectroscopic analysis in the native aqueous environment is highlighted, and the relative merits of infrared absorption versus Raman spectroscopy are considered, in this context. It is argued that Raman spectroscopic analysis is more suitable to quantitative analysis in liquid samples, and superior performance for quantification of high and low molecular weight components, is demonstrated. Applications for quantitation of viral loads, and therapeutic drug monitoring are also discussed

Raman Spectroscopic Analysis of High Molecular Weight Proteins in Solution: Considerations for Sample Analysis and Data Pre-processing

This study explores the potential of Raman spectroscopy, coupled with multivariate regression techniques and a protein separation technique (ion exchange chromatography), to quantitatively monitor diagnostically relevant changes in high molecular weight proteins in liquid plasma. Measurement protocols to detect the imbalances in plasma proteins as an indicator of various diseases using Raman spectroscopy are optimised, such that strategic clinical applications for early stage disease diagnostics can be evaluated. In a simulated plasma protein mixture, concentrations of two proteins of identified diagnostic potential (albumin and fibrinogen) were systematically varied within physiologically relevant ranges. Scattering from the poorly soluble fibrinogen fraction is identified as a significant impediment to the accuracy of measurement of mixed proteins in solution, although careful consideration of pre-processing methods allows construction of an accurate multivariate regression prediction model for detecting subtle changes in the protein concentration. Furthermore, ion exchange chromatography is utilised to separate fibrinogen from the rest of the proteins and mild sonication is used to improve the dispersion and therefore quality of the prediction. The proposed approach can be expeditiously employed for early detection of pathological disorders associated with high or low plasma/serum proteins

Determination of Nanoparticle Localisation Within Subcellular Organelles in Vitro Using Raman Spectroscopy

Ease of sample preparation, narrow spectral bandwidth and minimal influence from water are features of Raman spectroscopy which make it a powerful, label-free way to study a wide range of biological structures and phenomena. In this context, given the concerns over their toxicology arising from their increased production and use, evaluation of nanoparticle uptake and localisation in biological systems and determination of the mechanisms of subcellular interaction and trafficking can provide long-term solutions for nanotoxicology, and potential strategies for nanomedicine. In this study, Raman spectroscopy is explored to monitor the sequential trafficking of nanoparticles through subcellular organelles in-vitro and to establish the spectroscopic signatures of those organelles. A549 human lung carcinoma cells were exposed to 40 nm carboxylate-modified and fluorescently-labelled polystyrene nanoparticles for 4, 12 and 24hrs. Raman spectroscopy was applied to nanoparticle exposed cells to determine the localisation within cellular compartments. Confocal laser scanning fluorescence microscopy (CLSM) with different organelle staining kits confirmed the localisation of the nanoparticles in organelles at the chosen exposure periods and co-localization was quantified using ImageJ with the JACoP colocalisation plugin. To confirm nanoparticle localisation and elucidate the spectroscopic signatures of the different subcellular organelles, a combination of K-means clustering (KMCA) and Principal components analysis (PCA) was applied to the Raman spectroscopic maps. The study showed the applicability of the techniques for elucidation of the localisation of polystyrene nanoparticles within the cell as well as determination of their local environment, differentiating the spectral signatures of intracellular compartments such as endosomes, lysosomes and endoplasmic reticulum, in a completely label free manner

Raman Spectroscopic Analysis of High Molecular Weight Proteins in Solution - Considerations for Sample Analysis and Data Pre-processing

This study explores the potential of Raman spectroscopy, coupled with multivariate regression techniques and a protein separation technique (ion exchange chromatography), to quantitatively monitor diagnostically relevant changes in high molecular weight proteins in liquid plasma. Measurement protocols to detect the imbalances in plasma proteins as an indicator of various diseases using Raman spectroscopy are optimised, such that strategic clinical applications for early stage disease diagnostics can be evaluated. In a simulated plasma protein mixture, concentrations of two proteins of identified diagnostic potential (albumin and fibrinogen) were systematically varied within physiologically relevant ranges. Scattering from the poorly soluble fibrinogen fraction is identified as a significant impediment to the accuracy of measurement of mixed proteins in solution, although careful consideration of pre-processing methods allows construction of an accurate multivariate regression prediction model for detecting subtle changes in the protein concentration. Furthermore, ion exchange chromatography is utilised to separate fibrinogen from the rest of the proteins and mild sonication is used to improve the dispersion and therefore quality of the prediction. The proposed approach can be expeditiously employed for early detection of pathological disorders associated with high or low plasma/serum proteins

Industrial grade 2D Molybdenum Disulphide (MoS2): An in-vitro exploration of the impact on cellular uptake, cytotoxicity, and inflammation

The recent surge in graphene research, since its liquid phase monolayer isolation and characterization in 2004, has led to advancements which are accelerating the exploration of alternative 2D materials such as molybdenum disulphide (MoS2), whose unique physico-chemical properties can be exploited in applications ranging from cutting edge electronic devices to nanomedicine. However, to assess any potential impact on human health and the environment, the need to understand the bio-interaction of MoS2 at a cellular and sub-cellular level is critical. Notably, it is important to assess such potential impacts of materials which are produced by large scale production techniques, rather than research grade materials. The aim of this study was to explore cytotoxicity, cellular uptake and inflammatory responses in established cell-lines that mimic different potential exposure routes (inhalation, A549; ingestion, AGS; monocyte, THP-1) following incubation with MoS2 flakes of varying sizes (50 nm, 117 nm and 177 nm), produced by liquid phase exfoliation. Using high content screening (HCS) and live/dead assays, it was established that 1 μg/ml (for the three different MoS2 sizes) did not induce toxic effects on any of the cell-lines. Confocal microscopy images revealed a normal cellular morphology in all cases. Transmission electron microscopy (TEM) confirmed the uptake of all MoS2 nanomaterials in all the cell-lines, the MoS2 ultimately locating in single membrane vesicles. At such sub-lethal doses, inflammatory responses are observed, however, associated, at least partially, with the presence of lipopolysaccharide endotoxin in nanomaterial suspensions and surfactant samples. Therefore, the inflammatory response of the cells to the MoS2 or endotoxin contamination was interrogated using a 10-plex ELISA which illustrates cytokine production. The experiments carried out using wild-type and endotoxin hyporesponsive bone marrow derived dendritic cells confirmed that the inflammatory responses result from a combination of endotoxin contamination, the MoS2 nanomaterials themselves, and the stabilizing surfactant

Label-free Screening of Biochemical Changes in Macrophage-like Cells Following MoS2 Exposure Using Raman Micro-spectroscopy

The emergence of large scale production techniques for 2D particulate materials has dramatically increased their applications potential. Understanding the interactions of biological cells with such particulate material is therefore of paramount importance, both for toxicological assessment and potential biomedical applications. Conventional in-vitro cytological assays commonly record only a single colorimetric end-point, and do not provide an in-depth analysis of how such materials are uptaken and processed within cells. To demonstrate its potential as an alternative, label free approach, confocal Raman micro-spectroscopy has been used to profile the cellular response of macrophage-like immune cells as a result of exposure to a sub-lethal dose of particulate MoS2, as an example novel 2D material. Particles were seen to be uptaken and trafficked in sub-cellular vesicles, and this sensitive technique allows differences in the biochemical composition of the vesicles to be assessed and monitored as a function of time. Untreated macrophage-like cells contain lipidic vesicles which are found to be relatively rich in the membrane lipid sphingomyelin, key to the process of cell membrane regeneration. After exposure to MoS2, the particulate material is seen to be invaginated in similar vesicles, the most prominent of which now, however, have spectroscopic signatures which are dominated by those of phosphatidyl family lipids, consistent with the phagocytotic pathway. The lipidic content of cells is seen to increase at all time-points (4, 24 and 72 h). although vesicles composed of sphingomyelin become more prominent again following a prolonged incubation of 72 h to a sub-lethal dose of MoS2, as the immune cell has processed the particulate material and initiates recovery to a normal/untreated state. This study reveals Raman micro-spectroscopy is an effective method for monitoring cellular responses and evolution of organelle compositions in response to MoS2 exposure. The additional benefit of using this technique is that cells can be monitored as a function of time, while it can also be used for screening other micro/nano materials for toxicology and/or establishing cell responses

In-vitro Localisation and Degradation of Few-layer MoS2 Submicrometric Plates in Human Macrophage-like Cells: a Label Free Raman Micro-spectroscopic Study

Monitoring the uptake, micro-environment and fate of micro or nano scaled particulate materials in cells is of paramount importance for the emerging fields of toxicology and medicine. Such particulate materials are known to interfere with colorimetric assays and many such assays record only a single end-point. Therefore, there is a need for a label-free, cost effective technique with little or no inference from the particulate materials. Raman micro-spectroscopy was used to simultaneously interrogate the integrity of few-layer MoS2 submicrometric plates in human macrophage-like cells, in-vitro, as well as the biochemical characteristics of the local micro-environment in which they are encompassed. Firstly, the degradation profile of MoS2 plates induced by hydrogen peroxidase was established using UV-Vis absorption and Raman micro-spectroscopy. Raman micro-spectroscopic maps interrogated all aspects of the cell, including the nucleus, cytoplasm and perinuclear region, and the location/distribution of MoS2 was monitored as a function of time (4, 24 and 72 h). Whereas only pristine MoS2 was detectable after 4 and 72 periods, degradation in-vitro was confirmed following a 24 h incubation. Analysis of the MoS2 micro-environments revealed the presence of both phosphatidyl lipidic vesicles and enzymatic regions containing lysozyme, the former being most associated with the MoS2 degradation. There was an increase and saturation of cytosolic neutral lipids detected following a 24 h incubation with MoS2, which reduces following a prolonged incubation of 72 h. This study reveals that macrophage-like cells perform degradation of the material in-vitro within lipidic vesicles subsequent to phagocytosis, which manifest as an increase in the production of lipid bodies as a mechanism of defense following exposure to industrial grade MoS2

Evaluation of cytotoxicity profile and intracellular localisation of doxorubicin-loaded chitosan nanoparticles

In the emerging field of nanomedicine, targeted delivery of nanoparticle encapsulated active pharmaceutical ingredients (API) is seen as a potential significant development, promising improved pharmacokinetics and reduced side effects. In this context, understanding the cellular uptake of the nanoparticles and subsequent subcellular distribution of the API is of critical importance. Doxorubicin (DOX) was encapsulated within chitosan nanoparticles to investigate its intracellular delivery in A549 cells in vitro. Unloaded (CS-TPP) and doxorubicin-loaded (DOX-CS-TPP) chitosan nanoparticles were characterised for size (473±41 nm), polydispersity index (0.3±0.2), zeta potential (34±4 mV), drug content (76±7 µM) and encapsulation efficiency (95±1%). The cytotoxic response to DOX-CS-TPP was substantially stronger than to CS-TPP, although weaker than that of the equivalent free DOX. Fluorescence microscopy showed a dissimilar pattern of distribution of DOX within the cell, being predominantly localised in the nucleus for free form and in cytoplasm for DOX-CS-TPP. Confocal microscopy demonstrated endosomal localisation of DOX-CS-TPP. Numerical simulations, based on a rate equation model to describe the uptake and distribution of the free DOX, nanoparticles and DOX loaded nanoparticles within the cells, and the subsequent dose and time dependent cytotoxic responses, were used to further elucidate the API distribution processes. The study demonstrates that encapsulation of the API in nanoparticles results in a delayed release of the drug to the cell, resulting in a delayed cellular response. This work further demonstrates the potential of mathematical modelling in combination with intracellular imaging techniques to visualise and further understand the intracellular mechanisms of action of external agents, both APIs and nanoparticles in cells