Bioactive Lavandula angustifolia essential oil-loaded nanoemulsion dressing for burn wound healing. In vitro and in vivo studies.

The aim of this study was to develop a dressing with bioactive lavender in a new form of nanoemulsion, and to verify its biosafety and effectiveness in burn wound healing. As part of this research, the composition of the bioactive carrier of lavender oil in the form of a nanoemulsion obtained using ultrasound was optimised. The mean particle size of the internal phase and polydispersity were determined using the dynamic light scattering method using a Zestasizer NanoZS by Malvern and using cryo-transmission electron microscopy (TEM). These studies confirmed that the selected formulation had a particle size of approximately 180 nm and remained stable over time. The preparation was also subjected to rheological analysis (viscosity approximately 480 mPa·s) and a pH test (approximately 6). A macroemulsion (ME) with the same qualitative composition was developed as a reference. Nanoformulations and MEs were tested for skin penetration using Raman spectroscopy in an in vitro model. Research has shown that both formulations deliver oil to living layers of the skin. Subsequently, studies were conducted to confirm the effect of lavender oil in emulsion systems on the mitigation of the inflammatory reaction and its pro-regenerative effect on the wound healing process in an in vitro cell culture model. The safe concentration of the oil in the emulsion preparation was also determined based on preliminary in vivo tests of skin sensitisation and irritation as well as an hemocompatibility test of the preparation.

Diversity among endothelial cell lines revealed by Raman and Fourier-transform infrared spectroscopic imaging.

Growing interest in the role of endothelium under physiological and pathological conditions has led to an increasing demand for its representative in vitro models especially suitable for drug tests and medical diagnostics. There are several endothelial cell lines commercially available whose biochemistry, and hence response to various stimuli, can be different. Recently, two vibrational techniques, Raman and Fourier-transform infrared microscopy, have been found to be potent tools for studying the biochemical composition of a single cell in an easy, rapid and label-free way. However, depending on the applied technique, the results may exhibit some divergence due to different selection rules as well as distinct experimental conditions. This paper presents the methodology of examination and characterization of three popular human endothelial cell lines: HAoEC (primary cells), HMEC-1 and EA.hy926 (immortalized cells). Based on high lateral resolution Raman imaging together with standard and high magnification Fourier-transform infrared measurements, the differences in spectral information and the distribution of biomolecules are presented and discussed.

FT-IR Spectroscopic Imaging of Endothelial Cells Response to Tumor Necrosis Factor-α: To Follow Markers of Inflammation Using Standard and High-Magnification Resolution.

Two endothelial cell lines were selected as models to investigate an effect of incubation with cytokine tumor necrosis factor type α (TNF-α) using Fourier transform infrared (FT-IR) imaging spectroscopy. Both cell lines are often used in laboratories and are typical lung vascular endothelial cells (HMLVEC) derived from the fusion of umbilical vein endothelial cells with lung adenocarcinoma cells (EA.hy926). This study was focused on alteration of spectral changes accompanying inflammation at the cellular level by applying two resolution systems of FT-IR microscopy. The standard approach, with a pixel size of ca. 5.5 µm2, determined the inflammatory state of the whole cell, while a high-magnification resolution (pixel size of ca. 1.1 µm2) provided information at the subcellular level. Importantly, the analysis of IR spectra recorded with different modes produced similar results overall and yielded unambiguous classification of inflamed cells. Generally, the most significant changes in the cells under the influence of TNF-α are related with lipids-their composition and concentration; however, segregation of cells into subcellular compartments provided an additional insight into proteins and nucleic acids related events. The observed spectral alterations are specific for the type of endothelial cell line.

A possible Fourier transform infrared-based plasma fingerprint of angiotensin-converting enzyme inhibitor-induced reversal of endothelial dysfunction in diabetic mice.

Angiotensin-converting enzyme inhibitors (ACE-I) display vasoprotective activity and represent the cornerstone in the treatment of cardiovascular diseases. In this study, we tested whether Fourier transform infrared (FTIR)-based analysis of blood plasma is sensitive to detect vasoprotective effects of treatment with perindopril including reversal of endothelial dysfunction in diabetes. For this purpose, plasma samples were collected from untreated db/db mice, db/db mice treated with 2 or 10 mg/kg perindopril and db+ mice. The effect of perindopril on endothelial function was examined in ex vivo aortic rings; 10 mg/kg but not 2 mg/kg of perindopril reversed endothelial dysfunction. In plasma of db/db mice, the balance between conformations of plasma proteins was noted, and treatment with perindopril at a high dose but not at a low dose reversed this effect. This was revealed by amide II/amide I ratio attributed to increased ß-sheet formation. Spectral markers at 3010, 1520/1238 cm-1 , representative for unsaturation degree of lipids and phosphorylation of tyrosine, respectively, were also affected by perindopril treatment. In conclusion, although metabolic abnormalities associated with type 2 diabetes mellitus such as hypertriglyceridemia and hyperglycemia strongly affected spectral FTIR profile of diabetic plasma, we identified FTIR features that seem to be associated with the vasoprotective activity of ACE-I.

Alterations in plasma biochemical composition in NO deficiency induced by L-NAME in mice analysed by Fourier Transform Infrared Spectroscopy.

Mouse model of nitric oxide deficiency, induced by prolonged treatment with NG -nitro-L-arginine methyl ester (L-NAME) was used for infrared spectroscopy (FTIR) analysis of plasma. L-NAME leads to increased peripheral resistance and systemic hypertension. Classification of spectral response was by principal component analysis (PCA) and linear discriminant analysis (LDA). PCA allowed to separate each animal group showing that FTIR spectra are sensitive to development of NO-deficiency on contrary to blood pressure values indicating hypertension. Globally, the most pronounced spectral alternations were observed in the second and third week of L-NAME treatment indicating that infrared signature of blood plasma can serve as indicator of early and late stages of the disease. The PLS-DA method provided >95% classification accuracy. Spectral features characteristic for L-NAME treatment were mainly associated with an elevated level of proteins accompanied by a decrease of a tyrosine content and changes in lipids/phospholipid concentration. In our work we discuss these changes for which statistically significant differences (p < 0.05 - 0.005) were observed between spectra collected for each time-point of the L-NAME treatment versus control subjects. We demonstrated for the first time that NO-deficiency and hypertension resulted in changes in biochemical profile of plasma that was detected by FTIR spectroscopy.

Raman scattering or fluorescence emission? Raman spectroscopy study on lime-based building and conservation materials.

This work presents an in-depth study on Raman spectra excited with 1064 and 532nm lasers of lime binders employed in the past as building materials and revealed today as valuable conservation materials. We focus our interest on the bands of strong intensity, which are present in the spectra of all binders acquired with laser excitation at 1064nm, but absent in the corresponding spectra acquired with laser excitation at 532nm. We suggest, that the first group of spectra represents fluorescence phenomena of unknown origin and the second true Raman scattering. In our studies, we also include two other phases of lime cycle, i.e. calcium carbonate (a few samples of calcite of various origins) and calcium oxide (quicklime) to assess how structural and chemical transformations of lime phases affect the NIR-Raman spectral profile. Furthermore, we analyse a set of carbonated limewashes and lime binders derived from old plasters to give an insight into their spectral characteristics after excitation with the 1064nm laser line. NIR-Raman micro-mapping results are also presented to reveal the spatial distribution of building materials and fluorescent species in the cross-section of plaster samples taken from a 15th century chapel. Our study shows that the Raman analysis can help identify lime-based building and conservation materials, however, a caution is advised in the interpretation of the spectra acquired using 1064nm excitation.

Raman microscopy as a novel tool to detect endothelial dysfunction.

Raman microscopy, a label-free method with high spatial resolution, shows growing potential in various fields of medical diagnostics. Several proof-of-concept studies related to the application of Raman microscopy to detect endothelial dysfunction are summarized in this work. Both ex vivo measurements of the tissues in the murine models of endothelial pathologies, as well as in vitro investigations of the cell cultures in the context of cellular transport, drug action and inflammation processes are discussed. The future directions in application of Raman spectroscopy-based methods in such studies are also described.

Complementary analysis of tissue homogenates composition obtained by Vis and NIR laser excitations and Raman spectroscopy.

Raman spectroscopy and four excitation lines in the visible (Vis: 488, 532, 633 nm) and near infrared (NIR: 785 nm) were used for biochemical analysis of rat tissue homogenates, i.e. myocardium, brain, liver, lung, intestine, and kidney. The Vis Raman spectra are very similar for some organs (brain/intestines and kidney/liver) and dominated by heme signals when tissues of lung and myocardium were investigated (especially with 532 nm excitation). On the other hand, the NIR Raman spectra are specific for each tissue and more informative than the corresponding ones collected with the Vis excitations. The spectra analyzed without any special pre-processing clearly illustrate different chemical composition of each tissue and give information about main components e.g. lipids or proteins, but also about the content of some specific compounds such as amino acid residues, nucleotides and nucleobases. However, in order to obtain the whole spectral information about tissues complex composition the spectra of Vis and NIR excitations should be collected and analyzed together. A good agreement of data gathered from Raman spectra of the homogenates and those obtained previously from Raman imaging of the tissue cross-sections indicates that the presented here approach can be a method of choice for an investigation of biochemical variation in animal tissues. Moreover, the Raman spectral profile of tissue homogenates is specific enough to be used for an investigation of potential pathological changes the organism undergoes, in particular when supported by the complementary FTIR spectroscopy.

Transmission versus transflection mode in FTIR analysis of blood plasma: is the electric field standing wave effect the only reason for observed spectral distortions?

Fourier transform infrared (FTIR) microspectroscopy is assessed in terms of two techniques (i.e., transmission and transflection) as a method for rapid measurements of blood plasma. Apart from the expected effect of the electric field standing wave (EFSW), we also noticed that second-derivative IR spectra recorded in transflection mode exhibited a significant shift in the amide I band (up to 1667 cm(-1)) in comparison to the one recorded in transmission (1658 cm(-1)). This has not been reported thus far in studies of the EFSW distortion of IR spectra of biological material. The thinner the sample deposited on the low-e microscope slide, the lower the position of the amide I band found in FTIR spectra, suggesting various plasma compositions after stratification or certain changes in secondary protein conformations due to chemical and/or physical effects. There are potentially several phenomena that can occur at the surface of both IR substrates affecting the protein profile, including changes in optical properties (refractive index), variation in water content in the sample, and segregation of plasma components. All three hypotheses are discussed here, with the help of atomic force microscopy (AFM).

Plasma biomarkers of pulmonary hypertension identified by Fourier transform infrared spectroscopy and principal component analysis.

The main goal of this study was to find specific plasma spectral markers associated with pulmonary arterial hypertension (PAH) induced by monocrotaline injection in rats. FTIR was used to monitor biochemical changes in plasma caused by PAH as compared with the systemic hypertension induced by partial ligation on the left artery and with the control group. Both pathologies, systemic and pulmonary hypertension, induced a unique response in the biochemical content of plasma, mainly related to the composition and secondary structure of plasma proteins. For PAH, ß-pleated sheet components of plasma proteins were identified whereas the protein composition in systemic hypertension was dominated by unordered structures. In addition, a higher concentration of tyrosine-rich proteins was found in plasma in PAH than in systemic hypertension. The differences between both pathologies were identified also in terms of lipid composition/metabolism as well as in the content of RNA and glucose, suggesting that lipid peroxidation appears upon pulmonary hypertension development. In summary, this work demonstrates that FTIR spectroscopy supported by principal component analysis (PCA) has the potential to become a fast and non-destructive method for biochemical characterization of plasma that consequently could have a diagnostic significance in pulmonary hypertension.