Raman spectroscopy of urinary extracellular vesicles to stratify patients with chronic kidney disease in type 2 diabetes.

In this study, we verified the hypothesis that Raman signature of urinary extracellular vesicles (UEVs) can be used to stratify patients with diabetes at various stages of chronic kidney disease (CKD). Patients with type 2 diabetes diagnosed with different stages of CKD and healthy subjects were enrolled in the study. UEVs were isolated using low-vacuum filtration followed by ultracentrifugation. Correlation analysis, multiple linear regression and principal component analysis were used to find differences between spectral fingerprints of UEVs derived from both groups of patients. Electron microscopy and nanoparticle tracking analysis were applied to characterize the size and morphology of UEVs. We observed significant correlations between selected Raman bands measured for UEVs and clinical parameters. We found significant differences in the area under the specific bands originating mainly from proteins and lipids between the study groups. Based on the tryptophan and amide III bands, we were able to predict the estimated glomerular filtration rate (eGFR). Principal component analysis, partial least squares regression (PLSR) and correlation analysis of the UEV Raman spectra supported the results obtained from the direct analysis of Raman spectra. Our analysis revealed that PLSR and a regression model including tryptophan and amide III bands allows to estimate the value of eGFR.

Congo Red as a Supramolecular Carrier System for Doxorubicin: An Approach to Understanding the Mechanism of Action.

The uptake and distribution of doxorubicin in the MCF7 line of breast-cancer cells were monitored by Raman measurements. It was demonstrated that bioavailability of doxorubicin can be significantly enhanced by applying Congo red. To understand the mechanism of doxorubicin delivery by Congo red supramolecular carriers, additional monolayer measurements and molecular dynamics simulations on model membranes were undertaken. Acting as molecular scissors, Congo red particles cut doxorubicin aggregates and incorporated them into small-sized Congo red clusters. The mixed doxorubicin/Congo red clusters were adsorbed to the hydrophilic part of the model membrane. Such behavior promoted transfer through the membrane.

In search of the correlation between nanomechanical and biomolecular properties of prostate cancer cells with different metastatic potential.

Nanomechanical properties of living cells, as measured with atomic force microscopy (AFM), are increasingly recognized as criteria that differentiate normal and pathologically altered cells. Locally measured cell elastic properties, described by the parameter known as Young's modulus, are currently proposed as a new diagnostic parameter that can be used at the early stage of cancer detection. In this study, local mechanical properties of normal human prostate (RWPE-1) cells and a range of malignant (22Rv1) and metastatic prostate cells (LNCaP, Du145 and PC3) were investigated. It was found that non-malignant prostate cells are stiffer than cancer cells while the metastatic cells are much softer than malignant cells from the primary tumor site. Next, the biochemical properties of the cells were measured using confocal Raman (RS) and Fourier-transform infrared (FT-IR) spectroscopies to reveal these cells' biochemical composition as malignant transformation proceeds. Nanomechanical and biochemical profiles of five different prostate cell lines were subsequently analyzed using partial least squares regression (PLSR) in order to identify which spectral features of the RS and FT-IR spectra correlate with the cell's elastic properties. The PLSR-based model could predict Young's modulus values based on both RS and FT-IR spectral information. These outcomes show not only that AFM, RS and FT-IR techniques can be used for discrimination between normal and cancer cells, but also that a linear correlation between mechanical response and biomolecular composition of the cells that undergo malignant transformation can be found. This knowledge broadens our understanding of how prostate cancer cells evolve thorough the multistep process of tumor pathogenesis.

Nanoscale AFM-IR spectroscopic imaging of lipid heterogeneity and effect of irradiation in prostate cancer cells.

The recent development of the AFM-IR technique, which combines nanoscale imaging with chemical contrast through infrared spectroscopy, opened up new fields for exploration, which were out of reach for other modalities, e.g. Raman spectroscopy. Lipid droplets (LDs) are key organelles, which are associated with stress response mechanisms in cells and their size falls into that niche. LDs composition is heterogeneous and varies depending on cancer cell type and the tumor microenvironment. Prostate cancer cells show a unique lipid metabolism manifested by an increased requirement for lipid accumulation in cytosolic LDs. In the current work, AFM-IR nanoimaging was undertaken to analyze lipids in untreated and x-ray irradiated PC-3 prostate cancer cells. Cells poor in LDs showed slightly increased lipid signal in cytoplasm close to the nucleus. On the other hand, high lipid signal coming from LDs accumulation could be found in any part of the cytoplasmic region. The observed behavior was found to be independent from irradiation and its dose. According to the band assignment of the collected AFM-IR spectra, the main components of LDs were assigned to cholesteryl esters. The size of LDs present in cells poor in lipids was found to be of less than 1 µm, whereas LDs aggregates spread out over a few microns. Analysis of AFM-IR spectra shows relative homogeneity of LDs composition in single cells and heterogeneity of LDs content within the PC-3 cell population.

Raman spectral signatures of urinary extracellular vesicles from diabetic patients and hyperglycemic endothelial cells as potential biomarkers in diabetes.

Raman spectroscopy was applied to the measurement of urinary and in vitro endothelium-derived extracellular vesicles (EVs) isolated by hydrostatic filtration dialysis (HFD) method. Raman spectra obtained for urinary EVs (UEVs) showed distinct differences in the fingerprint region. In contrast, average Raman spectra of endothelium-derived EVs samples were almost identical. Cluster Analysis of UEVs significantly discriminated diabetic samples from control, moreover endothelium-derived EVs revealed stronger similarity between long hyperglycemia and normoglycemia samples compared to short hyperglycemia. Results obtained from Partial Least Squares analysis corresponded well with integral intensities of selected bands. Our proof-of-concept approach demonstrates the potential for Raman spectroscopy to be used both for identification of EVs molecular signatures in urine samples from patients with type 2 diabetes mellitus and good glycemic control and unsatisfactory glycemic control as well as for in vitro hyperglycemic model. This noninvasive technique may be useful in identifying new biomarkers of diabetes and renal complications.

High-definition FT-IR reveals a synergistic effect on lipid accumulation in prostate cancer cells induced by a combination of X-rays and radiosensitizing drugs.

Radiotherapy is one of the most commonly used cancer therapies with many benefits including low toxicity to healthy tissues. However, a major problem in radiotherapy is cancer radioresistance. To enhance the effect of this kind of therapy several approaches have been proposed such as the use of radiosensitizers. A combined treatment of radiotherapy and radiosensitizing drugs leads to a greater effect on cancer cells than anticipated from the addition of both responses (synergism). In this study, high-definition FT-IR imaging was applied to follow lipid accumulation in prostate cancer cells as a response to X-ray irradiation, radiosensitizing drugs, and a combined treatment of X-rays and the drugs. Lipid accumulation induced in the cells by an increasing X-ray dose and the presence of the drugs was analyzed using Principal Component Analysis and lipid staining. Finally, the synergistic effect of the combined therapy (X-rays and radiosensitizers) was confirmed by calculations of the integral intensity of the 2850 cm-1 band.

Spectroscopic imaging to assess biochemical alterations in liver carcinoma cells exposed to transition metals.

Despite the invaluable role of transition metals in every living organism, it should be remembered that failure to maintain the proper balance and exceed the appropriate dose may have the opposite effect. In the era of such a popular and propagated need for supplementation in the media, one should bear in mind the harmful effects that may become the result of improper and excessive intake of transition metals. This article establishes the feasibility of Raman (RS) and Fourier-transform infrared (FT-IR) spectroscopic imaging at the single-cell level to investigate the cellular response to various transition metals. These two non-destructive and perfectly complementary methods allow for in-depth monitoring of changes taking place within the cell under the influence of the agent used. HepG2 liver carcinoma cells were exposed to chromium, iron, cobalt, molybdenum, and nickel at 1 and 2 mM concentrations. Spectroscopic results were further supported by biological evaluation of selected caspases concentration. The caspase- 3, 6, 8, 9, and 12 concentrations were determined with the use of the enzyme-linked immunosorbent assay (ELISA) method. This study shows the induction of apoptosis in the intrinsic pathway by all studied transition metals. Cellular metabolism alterations are induced by mitochondrial metabolism changes and endoplasmic reticulum (ER) metabolism variations. Moreover, nickel induces not only the intrinsic pathway of apoptosis but also the extrinsic pathway of this process.

Carotenoids contribution in rapid diagnosis of multiple sclerosis by Raman spectroscopy.

Rapid and accurate diagnosis of any illness determines the success of treatment. The same applies to multiple sclerosis (MS), chronic, inflammatory, and neurodegenerative diseases (ND) of the central nervous system (CNS). Unfortunately, the definitive diagnosis of MS is prolonged and involves mainly clinical symptoms observation and magnetic resonance imaging (MRI) of the CNS. However, as we previously reported, Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy shed new light on the minimally invasive, label-free, and rapid diagnosis of this illness through blood fraction. Herein we introduce Raman spectroscopy coupled with chemometric analysis to provide more detailed information about the biochemical changes behind MS. This pilot study demonstrates that mentioned combination may provide a new diagnostic biomarker and bring closer to rapid MS diagnosis. It has been shown that Raman spectroscopy provides lipid and carotenoid molecules as useful biomarkers which may be applied for both diagnosis and treatment monitoring.

Theoretical modeling of molecular spectra parameters of disubstituted diacetylenes.

Symmetrically disubstituted diacetylenes, X-C≡C-C≡C-X, were studied computationally by using the DFT B3LYP/aug-cc-pVDZ method. For more than 35 substituents the bond lengths, charge density and Laplacian in bond critical points, C≡C stretching vibrational frequencies, (13)C NMR chemical shifts and spin-spin CC coupling constants through diacetylene moiety were calculated and examined by using the substituent sEDA and pEDA descriptors. It is demonstrated that in disubstituted diacetylenes the triple bond length increases with the electron donating and decreases with the electron withdrawing properties of the substituents. The σ-electron repulsion is likely to be responsible for this phenomenon. The electron density of the C-X bond critical point decreases linearly with an increase of the σ-electron donating properties, whereas the Laplacian of electron density in the C≡C bond critical point increases as the sEDA descriptor i.e., σ-electron donating properties of the substituent, are increased. Thus, ρ(C≡C) is locally reduced with an increase of sEDA. The ν(as)(C≡C) and ν(s)(C≡C) mode frequencies decrease with the electron donating and increase with the electron withdrawing properties of the substituents. The calculated chemical shift of the C1-atom, to which the substituent is attached, does not correlate with the substituent descriptors, whereas the δ(C2) deshielding increases when the σ-electron donating properties and C≡C distance are increased. The calculated (1)J(CC) coupling constants decrease with an increase of the triple bond length, the sEDA parameter, and ρ(C-X), whereas they decrease with Laplacian in the C≡C BCP.

Spectroscopic studies on bioactive polyacetylenes and other plant components in wild carrot root.

Polyacetylenes and other common plant components, such as starch, pectin, cellulose, and lignin, were studied in roots of the wild carrot (Daucus carota) subspecies D. carota subsp. gummifer and D. carota subsp. maximus by Raman spectroscopy. The components were measured in situ, directly in the plant tissue and without any preliminary sample preparation. The analysis was performed on the basis of the intense and characteristic key bands observed in the Raman spectrum. The two main carrot polyacetylenes falcarinol (1) and falcarindiol (2) have similar molecular structures, but their Raman spectra exhibit a small band shift in the symmetric -C≡C-C≡C- mode from 2258 cm⁻¹ to 2252 cm⁻¹. Quantum chemical calculations confirmed that the differences observed between the samples may be due to conformational and environmental changes. The polyacetylenes were also detected by Raman mapping, which visualized the distribution of the compounds across sections of carrot roots. The mapping technique was also applied to assess the distribution of lignin and polysaccharide compounds. The results showed the tissue-specific accumulation of starch and cell wall components such as lignin, pectin, and cellulose.

Exploring subcellular responses of prostate cancer cells to clinical doses of X-rays by Raman microspectroscopy.

Modern techniques of radiotherapy such as fractioned radiotherapy require applications of low doses of ionizing radiation (up to 10 Gy) for effective patient treatment. It is, therefore, crucial to understand the response mechanisms in cancer cells irradiated with low (clinical) doses. The cell's response to irradiation depends on a dose and post-irradiation time. Both factors should be considered when studying the influence of ionizing radiation on cancer cells. Thus, in the present study, PC-3 prostate cancer cells were irradiated with clinical doses of X-rays to determine dose- and time-dependent response to the irradiation. Raman spectroscopy and biological methods (MTT and comet assays) were applied for the analysis of biochemical changes in the cells induced by low doses of X-ray irradiation at 0 h and 24 h post-irradiation timepoints. Due to a limited view of the biochemical changes at the subcellular level given by single spectrum Raman measurements, Raman mapping of the whole cell area was performed. The results were compared with those obtained for cell irradiation with high doses. The analysis was based on the Partial Least Squares Regression (PLSR) method for the cytoplasmic and nuclear regions separately. Additionally, for the first time, irradiation classification was performed to confirm Raman spectroscopy as a powerful tool for studies on cancer cells treated with clinical doses of ionizing radiation.

The Impact of Preprocessing Methods for a Successful Prostate Cell Lines Discrimination Using Partial Least Squares Regression and Discriminant Analysis Based on Fourier Transform Infrared Imaging.

Fourier transform infrared spectroscopy (FT-IR) is widely used in the analysis of the chemical composition of biological materials and has the potential to reveal new aspects of the molecular basis of diseases, including different types of cancer. The potential of FT-IR in cancer research lies in its capability of monitoring the biochemical status of cells, which undergo malignant transformation and further examination of spectral features that differentiate normal and cancerous ones using proper mathematical approaches. Such examination can be performed with the use of chemometric tools, such as partial least squares discriminant analysis (PLS-DA) classification and partial least squares regression (PLSR), and proper application of preprocessing methods and their correct sequence is crucial for success. Here, we performed a comparison of several state-of-the-art methods commonly used in infrared biospectroscopy (denoising, baseline correction, and normalization) with the addition of methods not previously used in infrared biospectroscopy classification problems: Mie extinction extended multiplicative signal correction, Eiler's smoothing, and probabilistic quotient normalization. We compared all of these approaches and their effect on the data structure, classification, and regression capability on experimental FT-IR spectra collected from five different prostate normal and cancerous cell lines. Additionally, we tested the influence of added spectral noise. Overall, we concluded that in the case of the data analyzed here, the biggest impact on data structure and performance of PLS-DA and PLSR was caused by the baseline correction; therefore, much attention should be given, especially to this step of data preprocessing.

Tracking of the biochemical changes upon pleomorphic adenoma progression using vibrational microspectroscopy.

Head and neck tumors can be very challenging to treat because of the risk of problems or complications after surgery. Therefore, prompt and accurate diagnosis is extremely important to drive appropriate treatment decisions, which may reduce the chance of recurrence. This paper presents the original research exploring the feasibility of Fourier transform infrared (FT-IR) and Raman spectroscopy (RS) methods to investigate biochemical alterations upon the development of the pleomorphic adenoma. Principal component analysis (PCA) was used for a detailed assessment of the observed changes and to determine the spectroscopic basis for salivary gland neoplastic pathogenesis. It is implied that within the healthy margin, as opposed to the tumoral tissue, there are parts that differ significantly in lipid content. This observation shed new light on the crucial role of lipids in tissue physiology and tumorigenesis. Thus, a novel approach that eliminates the influence of lipids on the elucidation of biochemical changes is proposed. The performed analysis suggests that the highly heterogeneous healthy margin contains more unsaturated triacylglycerols, while the tumoral section is rich in proteins. The difference in protein content was also observed for these two tissue types, i.e. the healthy tissue possesses more proteins in the anti-parallel ß-sheet conformation, whereas the tumoral tissue is dominated by proteins rich in unordered random coils. Furthermore, the pathogenic tissue shows a higher content of carbohydrates and reveals noticeable differences in nucleic acid content. Finally, FT-IR and Raman spectroscopy methods were proposed as very promising methods in the discrimination of tumoral and healthy tissues of the salivary gland.

Comparison between high definition FT-IR, Raman and AFM-IR for subcellular chemical imaging of cholesteryl esters in prostate cancer cells.

The family of vibrational spectroscopic imaging techniques grows every few years and there is a need to compare and contrast new modalities with the better understood ones, especially in the case of demanding biological samples. Three vibrational spectroscopy techniques (high definition Fourier-transform infrared [FT-IR], Raman and atomic force microscopy infrared [AFM-IR]) were applied for subcellular chemical imaging of cholesteryl esters in PC-3 prostate cancer cells. The techniques were compared and contrasted in terms of image quality, spectral pattern and chemical information. All tested techniques were found to be useful in chemical imaging of cholesterol derivatives in cancer cells. The results obtained from FT-IR and Raman imaging showed to be comparable, whereas those achieved from AFM-IR study exhibited higher spectral heterogeneity. It confirms AFM-IR method as a powerful tool in local chemical imaging of cells at the nanoscale level. Furthermore, due to polarization effect, p-polarized AFM-IR spectra showed strong enhancement of lipid bands when compared to FT-IR.

Physicochemical damage and early-stage biological response to X-ray radiation studied in prostate cancer cells by Raman spectroscopy.

Exposure to ionizing radiation significantly affects biochemistry of cancer cells. The effect of irradiation can be divided into two stages, that is, the physicochemical stage and the biological response. Both effects induce different biochemical changes in the cells and should be analyzed as two separate phenomena. Thus, in the current study, Raman spectroscopy of prostate cancer cells fixed before (the physicochemical damage model) and just after (the biological response model) irradiation was undertaken to compare biochemical composition of irradiated cancer cells at both stages. Spectroscopic analysis of the cells was performed separately for cytoplasmic and nuclear regions. Biochemical changes of irradiated cells were analyzed using partial least squares regression (PLSR) method on the basis of the collected Raman spectra. Regression coefficients were therefore used to describe differences and similarities between biochemical composition of cancer cells undergoing the physicochemical stage and biological response. Additionally, PLSR models of both phenomena were compared for linear dose-dependence and a cross prediction.

Lipid droplets in prostate cancer cells and effect of irradiation studied by Raman microspectroscopy.

Lipid droplets (LDs) are key organelles in cancer cells proliferation, growth, and response to stress. These nanometric structures can aggregate to reach the size of microns becoming important cell components. Although it is known that LDs contain various lipids, their chemical composition is still under investigation. Moreover, their function in cell's response to exogenous factors is also not fully understood. Raman spectroscopy, together with chemometrics, has been shown to be a powerful tool for analytical analyses of cancer cell components on the subcellular level. It provides the opportunity to analyse LDs in a label-free manner in live cells. In the current study, this method was applied to investigate LDs composition in untreated and irradiated with X-ray beams prostate cancer cells. Raman mapping technique proved lipids accumulation in PC-3 cells and allowed visualization of LDs spatial distribution in cytoplasm. A heterogeneous composition of LDs was revealed by detailed analysis of Raman spectra. Interestingly, PC-3 cells were found to accumulate either triacylglycerols or cholesteryl esters. Finally, effect of X-ray radiation on the cells was investigated using Raman spectroscopy and fluorescence staining. Significant influence of LDs in the process of cell response was confirmed and time dependence of this phenomenon was determined.

Exploring subcellular responses of prostate cancer cells to X-ray exposure by Raman mapping.

Understanding the response of cancer cells to ionising radiation is a crucial step in modern radiotherapy. Raman microspectroscopy, together with Partial Least Squares Regression (PLSR) analysis has been shown to be a powerful tool for monitoring biochemical changes of irradiated cells on the subcellular level. However, to date, the majority of Raman studies have been performed using a single spectrum per cell, giving a limited view of the total biochemical response of the cell. In the current study, Raman mapping of the whole cell area was undertaken to ensure a more comprehensive understanding of the changes induced by X-ray radiation. On the basis of the collected Raman spectral maps, PLSR models were constructed to elucidate the time-dependent evolution of chemical changes induced in cells by irradiation, and the performance of PLSR models based on whole cell averages as compared to those based on average Raman spectra of cytoplasm and nuclear region. On the other hand, prediction of X-ray doses for individual cellular components showed that cytoplasmic and nuclear regions should be analysed separately. Finally, the advantage of the mapping technique over single point measurements was verified by a comparison of the corresponding PLSR models.

Toxicity of silver nanoparticles towards tumoral human cell lines U-937 and HL-60.

The toxicity of three types of silver nanoparticles towards histiocytic lymphoma (U-937) and human promyelocytic cells (HL-60) was studied. The nanoparticles were synthesized in a chemical reduction method using sodium borohydride. Trisodium citrate and cysteamine hydrochloride were used to generate a negative and positive nanoparticle surface charge. The evaluation of cell viability, membrane integrity, antioxidant activity and the induction of inflammation were used to evaluate the difference in cellular response to the nanoparticle treatment. The results revealed that the cysteamine-stabilized (positively charged) nanoparticles (SBATE) were the least toxic although they exhibited a similar ion release profile as the unmodified (negatively charged) nanoparticles obtained using sodium borohydride (SBNM). Citrate-stabilized nanoparticles (SBTC) induced superoxide dismutase (SOD) activity in the HL-60 cells and total antioxidant activity in the U-937 cells despite their resistance to oxidative dissolution. The toxicity of SBNM nanoparticles was manifested in the disruption of membrane integrity, decrease in the mitochondrial functions of cells and the induction of inflammation. These findings allowed to conclude that mechanism of silver nanoparticle cytotoxicity is the combination of effects coming from the surface charge of nanoparticles, released silver ions and biological activity of stabilizing agent molecules.

Formation of positively charged gold nanoparticle monolayers on silica sensors.

Formation of positively charged gold nanoparticle monolayers on the Si/SiO2 was studied under in situ conditions using quartz microbalance (QCM). The gold nanoparticles were synthesized in a chemical reduction method using sodium borohydride as reducing agent. Cysteamine hydrochloride was applied to generate a positive surface charge of nanoparticles. The micrographs obtained from transmission electron microscopy (TEM) revealed that the average size of nanoparticles was equal to 12±3nm. The stability of nanoparticle suspensions under controlled pH and ionic strength was determined by dynamic light scattering (DLS). The electrophoretic mobility measurements showed that the zeta potential of nanoparticles was positive, decreasing with ionic strength and pH from 56mV at pH 4.2 and I=10-4M to 22mV at pH 8.3 and I=3×10-3M. The surface enhanced Raman spectroscopy (SERS) confirmed chemisorption of cysteamine on nanoparticles and the contribution of amine moieties in the generation of nanoparticle charge. The influence of suspension concentration, ionic strength and flow rate on the kinetics of nanoparticle deposition on the sensors was quantitatively determined. It was confirmed that the deposition for the low coverage regime is governed by the bulk mass transfer that results in a linear increase of the coverage with time. The significant increase in the maximum coverage of gold monolayers with ionic strength was interpreted as due to the decreasing range of the electrostatic interactions among deposited particles. Moreover, the hydratation of formed monolayers, their structure and the stability were determined by the comparison of the QCM results with those obtained by AFM and SEM. The experimental data were adequately interpreted in terms of the extended random sequential adsorption (eRSA) model that considers the bulk and surface transfer steps in a rigorous way. The obtained results are useful for a facile fabrication of gold nanoparticle-based biosensors capable to bind target molecules via available amine moieties.

Physicochemical properties and cytotoxicity of cysteine-functionalized silver nanoparticles.

The preparation of stable cysteine-capped silver nanoparticles (AgNPs), via the reduction of silver ions with sodium borohydride and modification of formed nanoparticles by l-cysteine, was developed. The micrographs from transmission electron microscopy (TEM) revealed that the spherical AgNPs exhibited an average size equal to 22±4nm. Surface enhanced Raman spectroscopy (SERS) and inductively coupled plasma optical emission spectrometry (ICP-OES) confirmed a chemisorption of cysteine molecules on the AgNPs. Additionally, dynamic light scattering (DLS) measurements showed that the AgNPs were stable for ionic strength lower than 5×10-3molL-1 and at 6.8