Abdelilah Beljebbar

FT-IR, FT-Raman spectroscopic study of carotenoids from saffron (Crocus sativus L.) and some derivatives

Abstract The carotenoids of saffron, crocins (CRCs), were extracted and their derivatives, dimethylcrocetin (DMCRT) and crocetin (CRT) were prepared from the extract by alkaline hydrolysis in methanol (DMCRT) and by alkaline hydrolysis in water followed by acidification (CRT), respectively. FT-IR, FT-Raman spectroscopies were used to study these compounds. The FT-IR spectra of CRCs, DMCRT and CRT have characteristic absorbance bands between 1706 and 1664 cm−1 (νCO) and in the region between 1243 and 1228 cm−1 (νC–O). Two main Raman lines were observed near 1540 and 1166 cm−1 which are respectively assigned to (νCC) and (νC–C) stretching modes.

Modeling and quantifying biochemical changes in C6 tumor gliomas by Fourier transform infrared imaging.

The purpose of the study was to investigate molecular changes associated with glioma tissues using FT-IR microspectroscopic imaging (FT-IRM). A multivariate statistical analysis allowed one to successfully discriminate between normal, tumoral, peri-tumoral, and necrotic tissue structures. Structural changes were mainly related to qualitative and quantitative changes in lipid content, proteins, and nucleic acids that can be used as spectroscopic markers for this pathology. We have developed a spectroscopic model of glioma to quantify these chemical changes. The model constructed includes individual FT-IR spectra of normal and glioma brain constituents such as lipids, DNA, and proteins (measured on delipidized tissue). Modeling of FT-IR spectra yielded fit coefficients reflecting the chemical changes associated with a tumor. Our results demonstrate the ability of FT-IRM to assess the importance and distribution of each individual constituent and its variation in normal brain structures as well as in the different pathological states of glioma. We demonstrated that (i) cholesterol and phosphatidylethanolamine contributions are highest in corpus callosum and anterior commissure but decrease gradually towards the cortex surface as well as in the tumor, (ii) phosphatidylcholine contribution is highest in the cortex and decreases in the tumor, (iii) galactocerebroside is localized only in white, but not in gray matter, and decreases in the vital tumor region while the necrosis area shows a higher concentration of this cerebroside, (iv) DNA and oleic acid increase in the tumor as compared to gray matter. This approach could, in the future, contribute to enhance diagnostic accuracy, improve the grading, prognosis, and play a vital role in therapeutic strategy and monitoring.

Comparative FT SERS, resonance Raman and SERRS studies of doxorubicin and its complex with DNA

Abstract Fourier transform surface enhanced Raman scattering (FT SERS) coupled with a microscope has been used as a probe to obtain information on the interaction of a drug and of its complex with DNA. Micro-FT SERS spectra of the antitumour agent doxorubicin (DOX) at 10 −5 M and of this complex with DNA have been recorded in aqueous silver hydrosol and compared with the corresponding resonance Raman (RR) and SERS spectra at concentrations of 5 × 10 −4 M and 5 × 10 −8 M, respectively. The interactions between the drug and calf thymus DNA induced identical effects in the RR and visible SERS spectra. The data show that the adsorption of the drug-DNA complex on the silver hydrosol does not induce detectable perturbations of the molecular interactions within the complex. Micro-FT SERS spectra were found to be partially different from those obtained in visible SERS spectra. These differences concern the relative enhancement of some vibrational modes which could hardly be observed when resonance excitation was used. The FT SERS approach enables further information to be obtained and additional details on the geometry of the drug-DNA interaction to be revealed. An analysis of the FT SERS spectra of the drug and of its complex with DNA not only confirms the model of interaction proposed using RR and SERS data in the visible, but brings about new information, especially on the vibrations of ring A of the molecule, which are usually masked by the vibrations of rings B and C dominant in the visible SERS spectra.

Characterization of island films as surface-enhanced Raman spectroscopy substrates for detecting low antitumor drug concentrations at single cell level

Gold and silver vacuum-deposited island films were characterized by studying deposition variables such as film thickness, evaporation rate, and substrate temperature. For both metals, these parameters were correlated with the surface-enhanced Raman spectroscopy (SERS) effect and an increase in film thickness and low evaporation rates were shown to upshift the wavelength at maximum optical density (lambda max) and increase the optical density of the substrates. In contrast, pre- and postdeposition annealing of gold films led to the formation of substrates that exhibited a downshift of lambda max. Our spectral data also indicated that silver films are substrates that are more suited for SERS applications where high frequency visible excitations are used. Measurements on gold films classified them into two groups: thin Au films (10-50 A) well adapted for red excitations and thicker ones that are operative in the near infrared. SERS results, which were obtained from a single HL60 cell treated with micromolar drug quantities, placed on thin gold island films indicated that these island films could be future promising substrates for SERS imaging at the cellular level.

Monitoring of Biochemical Changes through the C6 Gliomas Progression and Invasion by Fourier Transform Infrared (FTIR) Imaging

We have investigated the spatial distribution of molecular changes associated with C6 glioma progression using Fourier transform infrared (FT-IR) microspectro-imaging in order to determine spectroscopic markers for early diagnosis of tumor growth. Our results showed that at day 7 after tumor implantation, FTIR investigations displayed a very small abnormal zone associated with the proliferation of C6 cells in the caudate putamen. From this day, rats developed solid and well-circumscribed tumors and invasive areas. The volume of peritumoral areas increased rapidly until day 19. The maturation of the tumor was accompanied by a diminution in its proliferative and invasive area. The presence of necrotic areas was visible from day 15. A non-negative least-squares algorithm was used to quantify spatial distribution of molecular changes in tissues (lipids, nucleic acids, and proteins) associated with glioma progression. Compared to those in normal brain, statistical tests on fit coefficients showed that the concentrations of sphingomyelin (SMY), nucleic acids, phosphatidylserine (PS), and galactocerebroside (GalC) were significantly affected during C6 glioma development. These constituents can be used as spectroscopic markers for C6 glioma progression. Indeed, the concentration of DNA decreased significantly from tumor to invasion, to normal brain tissues, the necrotic area has higher concentrations of the Galc than other areas. The PS content was significantly higher in the peritumoral zone and decreased in the tumor zones matter.

Coupling FT Raman and FT SERS microscopy with TLC plates for in situ identification of chemical compounds

Abstract Direct analysis of sub-femtogram quantities of chemical compounds on thin layer chromatography plates has been made possible by associating Fourier transform Raman microspectroscopy with SERS spectroscopy. The interfacing elements of the FT Raman microscope system are discussed and optimised such that a lateral resolution on the micron scale is achieved in the sample plane. Micro-FT SERS results obtained from a model biological molecule indicate preservation of molecular conformation upon adsorption at the SERS active surface. With NIR radiation it is thus possible to analyse plates with or without fluorescence indicators.

Screening of biochemical/histological changes associated to C6 glioma tumor development by FTIR/PCA imaging

Principal components analysis (PCA) combined to Fourier transform infrared (FTIR) microspectroscopic imaging was used to screen biochemical changes associated to C6 glioma tumor from 7 to 19 days growth. Normal brain and tumor obtained at 7, 12, 19 days after C6 cell injection were used to develop a diagnostic model of brain glioma based on PCA analysis. This classification model was validated using extra-measurements on normal and tumor at 9 and 15 days post-implantation. The spatial and biochemical information obtained from FTIR/PCA maps can be used to improve the discrimination between normal and grading human glioma. The first 4 PCs which account for more than 93.6% of total spectral variance were used to construct pseudo-color scores maps and compared each map to the corresponding hematoxylin and eosin (H&E) staining. Our results reported that by correlating pseudocolor map scores with H&E staining it was possible to screen histological changes associated with tissue transformation. In fact, PC1 and PC4 were associated to the tumor, surrounding tumor and necrosis. Indeed, at day 7 after tumor implantation, FTIR investigations displayed a very small abnormal zone associated with the proliferation of C6 cells in the caudate putamen (CP). PC2 and PC3 described normal brain structures such as white matter (corpus callosum (CC) and commissura anterior (CA)) and some cortex layers (grey matter). After delipidation of the tissues, we were still able to differentiate between different tissue features based on nucleic acid and protein content. By comparing the patterns of the PC loads with the spectra of lipids extracted from white and gray matters, and DNA, we have identified some biochemical changes associated with tissue transformation. This work demonstrated that our classification model provides a successful histological classification of different brain structures.

Molecular and Cellular Interaction of the Differentiating Antitumour Agent Dimethylcrocetin with Nuclear Retinoic Acid Receptor as Studied by Near-Infrared and Visible SERS Spectroscopy

Fourier transform (FT) Raman and surface enhanced Raman microspectroscopy (SERS) were used as a vibrational probe for investigating, in vitro and at the cellular level, the molecular interaction of dimethylcrocetin (DMCRT) with the retinoic acid nuclear receptor RAR-γ. FT-SERS results obtained in vitro with silver colloids demonstrated that DMCRT interacts specifically with RAR-γ. Comparison between the spectra of DMCRT in HL60 cancer cells and the in vitro DMCRT–RARγ complex showed practically the same shift in wavenumber of the band at 1210 cm-1 and the same intensity ratios I1541/I1165 and I1541/I1210. The similarity between the signal of DMCRT in K562 cells and in the free form is explained by either an absence of nuclear receptor or an absence of any interaction between the drug and receptor. These results seem to indicate that carotenoids could possibly activate the nuclear receptor in a similar manner to retinoids.

Rapid identification of the multidrug resistance in the human leukemic cells by near-infrared Fourier transform Raman microspectroscopy

In this work, we have studied the cancer cell lines sharing a common feature: the multi-drug resistance where P- glycoprotein is responsible for the active efflux of the drug out of the cell. For this, we have used two types of cells, MDR-human leukemic K562 cells and non-MDR acute promyelocytic leukemic HL60 cells. The comparison between normalized micro FT-Raman spectra of resistant and sensitive K652 cells shows a decrease in the intensity of the amide I and III bands and a down shift of the amide I band. On the other hand, control experiments with HL60 cells do not show any remarkable changes. Analysis of micro-FT-Raman spectra by resolution enhancement methods and by chemometrics tools reveal further information concerning the conformational changes of the cell constituents involved in the expression of the MDR-phenotype.

Surface-enhanced Raman scattering and fluorescence spectroscopy reveal molecular interactions of all-trans retinoic acid and RARγ ligand-binding domain

Surface-enhanced Raman scattering and fluorescence were used to investigate the interactions of all-trans retinoic acid with the gamma-type retinoic acid receptor. Raman data revealed a significant attenuation in intensity of the bands originating from the retinoic acid polyenic chain upon receptor binding, with the spectrum being dominantly that of the β-ionone ring. Fluorescence measurements supported the hydrophobic character of the ligand binding. These novel spectroscopic results are fully consistent with the published X-ray crystallographic data and suggest that these techniques may be valuable additional tools to characterize the interactions of agonists and antagonists with residues in the ligand-binding pockets of retinoid receptor homo- and heterodimers.