Lc-ms/ms method development for quantitation of nicotine in toenails as a biomaker for secondhand smoke and standard lipoprotein mimetic models

Passive smoke or (secondhand smoke) is defined as when a non-smoker is unintentionally exposed to a smoking environment from cigarettes, cigars, or pipes. Passive smoke can result in adverse health effects leading to heart disease, asthma attacks, lung cancer, and other major diseases. Smoke from active smokers has been extensively investigated by a number of researchers. These studies have examined methods for the analysis of nicotine and its metabolites. In contrast, the development of methods to follow nicotine and its metabolites in those exposed to passive or secondhand smoke, is lacking. Here we present a method developed for the determination of nicotine in toenails. We will describe a method that involves the pretreatment of toenails, followed by a liquid-liquid extraction. The extract is then analyzed by reverse phase high performance liquid chromatography (HPLC) – ion trap mass spectrometry. Some of the figures of merit for this method include quantification of the nicotine concentration level, standard curve linearity (R2 > 0.99), limit of detection (LOD = 0.005 ng/mg at m/z 163), and limit of quantitation (LOQ = 0.08 ng/mg), over the concentration range of 0.08 to 20 ng/mg. Toenail samples were individually collected for research purposes, including a non-smoker never exposed to secondhand smoke, non-smoker exposed to secondhand smoke, and an active smoker. The results indicted mean of nicotine content in non-exposed, exposed, and active smoker toenails samples are 0.103, 0.415, and 1.75 ng/mg respectively. This study also compared a solid phase extraction method. As a complex of globular proteins, lipoproteins, plays an essential role in the transport and metabolism of cholesterol. The level of several metabolites in blood are controlled by several mechanisms due to its profile. Development of common assays for lipoproteins have resulted in detection of abnormalities and can help physicians assess tissue injuries and disordering in early stages. Natural lipoprotein analysis related to cardiovascular disease is challenging even when utilizing modern analytical instrumentation. In this study, we developed mimetic lipoprotein models and characterize them using UV-Vis and fluorescence spectrophotometry to gain a better understanding of lipoproteins. An independent assay, the Amplex Red Cholesterol Assay, was also performed and used to support the mimetic lipoprotein model used in this study. Cardiovascular health is associated with different classes of lipoproteins and the composition of each component in lipoproteins. This study demonstrated that the carbon-carbon double bond of cholesterol (1668 cm-1) and the peptide backbone of tyrosine resonance are enhanced in deep ultraviolet resonance Raman (dUVRR) spectra (851, 1171, 1205, 1266, 1596, and 1615 cm-1). The excitation of wavelength 197 nm characterized features of mimetic lipoprotein models. Other measurements, such as circular dichroism (CD), UV-vis, and fluorescence spectroscopy provided spectroscopic information to identity and characterize the mimetic lipoprotein models

Doctor of Philosophy

dissertationKnowledge of physical and chemical properties such as size, diffusivity, concentration and stability of an analyte in a sample is critical in science and engineering. When working in a small size range (0.1-100 nm), which includes short-chain molecules (e.g., ethanol) and nanoparticles, the number of methods that can be used for their characterization becomes sparse and each one has its limitation such as accuracy, resolution, cost of instrumentation and time needed for sample preparation and analysis which introduces more complexity to the problem being solved. This became the motivation and focus of the presented work. The goal of the conducted research was to explore the available methods as well as develop new methods that can be used for characterization of different analytes. The first half of the dissertation introduces the use of interfacial tension for estimating stability of nanobubbles that can be applied as contrast agents for ultrasound imaging or as vehicles for drug delivery. Use of interfacial tension is then shown to be applicable in a new setup and conditions to determine the diffusion coefficient and concentration of an analyte in any given location. Adsorption of perfluorocarbon vapor to the water surface is then explored. The second half of the dissertation focuses on characterization of endogenous nanovesicles called exosomes. This section continues the first section by presenting a novel finding of surface activity of exosomes which provides a potential mechanism of their adsorption to the cell membrane as well as application of dynamic interfacial tension for measuring exosome concentration in a sample. This section continues by comparing techniques that were previously used by others to determine size and shape of exosomes but were not compared to each other either due to unavailability of instrumentation or focus of the study being unrelated to size and shape. This study not only allowed examining the advantages and disadvantages of each technique but also lead to new findings about the biophysical properties of exosomes. This section ends with an application of quartz crystal microbalance method for measuring average mass and concentration of exosomes in a sample

Biological reference materials for extracellular vesicle studies

LR: 20161010; CI: Copyright (c) 2016; JID: 9317982; OTO: NOTNLM; 2016/05/13 [received]; 2016/09/06 [revised]; 2016/09/06 [accepted]; aheadofprintExtracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories.Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (137), larger diameter (70% of the NanoE are over 200 nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories. (C) 2016 The Authors. Published by Elsevier B.V.Peer reviewe

Recent advances in the label-free characterization of exosomes for cancer liquid biopsy: From scattering and spectroscopy to nanoindentation and nanodevices

Exosomes (EXOs) are nano-sized vesicles secreted by most cell types. They are abundant in bio-fluids and harbor specific molecular constituents from their parental cells. Due to these characteristics, EXOs have a great potential in cancer diagnostics for liquid biopsy and personalized medicine. Despite this unique potential, EXOs are not yet widely applied in clinical settings, with two main factors hindering their translational process in diagnostics. Firstly, conventional extraction methods are time-consuming, require large sample volumes and expensive equipment, and often do not provide high-purity samples. Secondly, characterization methods have some limitations, because they are often qualitative, need extensive labeling or complex sampling procedures that can induce artifacts. In this context, novel label-free approaches are rapidly emerging, and are holding potential to revolutionize EXO diagnostics. These methods include the use of nanodevices for EXO purification, and vibrational spectroscopies, scattering, and nanoindentation for characterization. In this progress report, we summarize recent key advances in label-free techniques for EXO purification and characterization. We point out that these methods contribute to reducing costs and processing times, provide complementary information compared to the conventional characterization techniques, and enhance flexibility, thus favoring the discovery of novel and unexplored EXO-based biomarkers. In this process, the impact of nanotechnology is systematically highlighted, showing how the effectiveness of these techniques can be enhanced using nanomaterials, such as plasmonic nanoparticles and nanostructured surfaces, which enable the exploitation of advanced physical phenomena occurring at the nanoscale level

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

© 2024 The Authors. Journal of Extracellular Vesicles, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.Peer reviewe

Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018): A Position Statement of the International Society for Extracellular Vesicles and Update of the MISEV2014 Guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points