In vivo Raman spectroscopy in the diagnostics of colon cancer.

Early detection and accurate diagnosis of colorectal carcinoma are crucial for successful treatment, yet current methods can be invasive and even inaccurate in some cases. In this work, we present a novel approach for in vivo tissue diagnostics of colorectal carcinoma using Raman spectroscopy. This almost non-invasive technique allows for fast and accurate detection of colorectal carcinoma and its precursors, adenomatous polyps, enabling timely intervention and improved patient outcomes. Using several methods of supervised machine learning, we were able to achieve over 91% accuracy in distinguishing colorectal lesions from healthy epithelial tissue and more than 90% classification accuracy for premalignant adenomatous polyps. Moreover, our models enabled the discrimination of cancerous and precancerous lesions with a mean accuracy of almost 92%. Such results demonstrate the potential of in vivo Raman spectroscopy to become a valuable tool in the fight against colon cancer.

Voltammetric Detection of Vanillylmandelic Acid and Homovanillic Acid Using Urea-Derivative-Modified Graphite Electrode.

Vanillylmandelic acid (VMA) and homovanillic acid (HVA) are diagnostic markers of neuroblastoma. The purpose of this study was to understand the reason for the discrimination of structural analogues (VMA and HVA) onto a graphite electrode coated with an electrochemically oxidized urea derivative. Density functional theory calculations (DFT), FTIR spectroscopic measurements, and electrochemical impedance spectroscopic measurements were used in this work. Density functional theory calculations (DFT) were used to identify the most suitable binding sites of the urea derivative and to describe possible differences in its interaction with the studied analytes. The FTIR measurement indicated the enhancement and disappearance of NH vibrations on graphite and platinum surfaces, respectively, that could be connected to a different orientation and thus provide accessibility of the urea moiety for the discrimination of carboxylates. Additionally, the higher the basicity of the anion, the stronger the hydrogen-bonding interaction with -NH-groups of the urea moiety: VMA (pKb = 10.6, KAds = (5.18 ± 1.95) × 105) and HVA (pKb = 9.6, KAds = (4.78 ± 1.58) × 104). The differential pulse voltammetric method was applied to detect VMA and HVA as individual species and interferents. As individual analytes, both HVA and VMA can be detected at a concentration of 1.99 × 10-5 M (RSD ≤ 0.28, recovery 110-115%).

The potential of chiroptical and vibrational spectroscopy of blood plasma for the discrimination between colon cancer patients and the control group.

Colorectal cancer is one of the most abundant causes of cancer deaths in the world. At an early stage, the established clinical procedures have low reliability and sensitivity. Therefore, we tested a novel approach based on chiroptical methods such as electronic circular dichroism (ECD) and Raman optical activity (ROA). These methods are suitable for detecting slight changes in the 3D structure of chiral biomolecules, some of which may be caused by pathological processes occurring during cancer growth. Fifty-five blood plasma samples were analyzed using the combination of ECD and ROA supplemented by conventional Raman and FT-IR spectroscopy. All obtained spectra were evaluated together by linear discriminant analysis. The accuracy of sample discrimination reached 100% and the subsequent leave-one-out cross-validation resulted in 93% sensitivity and 81% specificity. The achieved results indicate that chiroptical methods supplemented by Raman and FT-IR spectroscopy might be new supporting and minimally invasive tools in the clinical diagnosis of colon cancer.

The minimizing of fluorescence background in Raman optical activity and Raman spectra of human blood plasma.

Raman optical activity (ROA) is inherently sensitive to the secondary structure of biomolecules, which makes it a method of interest for finding new approaches to clinical applications based on blood plasma analysis, for instance the diagnostics of several protein-misfolding diseases. Unfortunately, real blood plasma exhibits strong background fluorescence when excited at 532 nm; hence, measuring the ROA spectra appears to be impossible. Therefore, we established a suitable method using a combination of kinetic quenchers, filtering, photobleaching, and a mathematical correction of residual fluorescence. Our method reduced the background fluorescence approximately by 90%, which allowed speedup for each measurement by an average of 50%. In addition, the signal-to-noise ratio was significantly increased, while the baseline distortion remained low. We assume that our method is suitable for the investigation of human blood plasma by ROA and may lead to the development of a new tool for clinical diagnostics.

Automated classification pipeline for real-time in vivo examination of colorectal tissue using Raman spectroscopy.

Colorectal cancer is the third most common malignancy worldwide and one of the leading causes of death in oncological patients with its diagnosis typically involving confirmation by tissue biopsy. In vivo Raman spectroscopy, an experimental diagnostic method less invasive than a biopsy, has shown great potential to discriminate between normal and cancerous tissue. However, the complex and often manual processing of Raman spectra along with the absence of a suitable instant classifier are the main obstacles to its adoption in clinical practice. This study aims to address these issues by developing a real-time automated classification pipeline coupled with a user-friendly application tailored for non-spectroscopists. First, in addition to routine colonoscopy, 377 subjects underwent in vivo acquisitions of Raman spectra of healthy tissue, adenomatous polyps, or cancerous tissue, which were conducted using a custom-made microprobe. The spectra were then loaded into the pipeline and pre-processed in several steps, including standard normal variate transformation and finite impulse response filtration. The quality of the pre-processed spectral data was checked based on their signal-to-noise ratio before the suitable spectra were decomposed and classified using a combination of principal component analysis and a support vector machine, respectively. After five-fold cross-validation, the developed classifier exhibited 100% sensitivity toward adenocarcinoma and adenomatous polyps. The overall accuracy was 96.9% and 79.2% for adenocarcinoma and adenomatous polyps respectively. In addition, an application with a graphical user interface was developed to facilitate the use of our data pipeline by medical professionals in a clinical environment. Overall, the combination of supervised and unsupervised machine learning with algorithmic pre-processing of in vivo Raman spectra appears to be a viable way of reducing the relatively large number of biopsies currently needed to definitively diagnose colorectal cancer.

Raman spectroscopy in lung cancer diagnostics: Can an in vivo setup compete with ex vivo applications?

Lung carcinoma remains the leading cause of cancer death worldwide. The tactic to change this unfortunate rate may be a timely and rapid diagnostic, which may in many cases improve patient prognosis. In our study, we focus on the comparison of two novel methods of rapid lung carcinoma diagnostics, label-free in vivo and ex vivo Raman spectroscopy of the epithelial tissue, and assess their feasibility in clinical practice. As these techniques are sensitive not only to the basic molecular composition of the analyzed sample but also to the secondary structure of large biomolecules, such as tissue proteins, they represent suitable candidate methods for epithelial cancer diagnostics. During routine bronchoscopy, we collected 78 in vivo Raman spectra of normal and cancerous lung tissue and 37 samples of endobronchial pathologies, which were subsequently analyzed ex vivo. Using machine learning techniques, namely principal component analysis (PCA) and support vector machines (SVM), we were able to reach 87.2% (95% CI, 79.8-94.6%) and 100.0% (95% CI, 92.1-100.0%) of diagnostic accuracy for in vivo and ex vivo setup, respectively. Although the ex vivo approach provided superior results, the rapidity of in vivo Raman spectroscopy might become unmatchable in the acceleration of the diagnostic process.

Analysis of human blood plasma and hen egg white by chiroptical spectroscopic methods (ECD, VCD, ROA).

Chiroptical methods are widely used in structural and conformational analyses of biopolymers. The application of these methods to investigations of biofluids would provide new avenues for the molecular diagnosis of protein-misfolding diseases. In this work, samples of human blood plasma and hen egg white were analyzed using a combination of conventional and chiroptical methods: ultraviolet absorption/electronic circular dichroism (UV/ECD), Fourier transform infrared absorption/vibrational circular dichroism (FTIR/VCD), and Raman scattering/Raman optical activity (Raman/ROA). For comparison, the main components of these substances--human serum albumin (HSA) and ovalbumin (Ova)--were also analyzed by these methods. The ultraviolet region of the ECD spectrum was analyzed using the CDNN CD software package to evaluate the secondary structures of the proteins. The UV/ECD, FTIR/VCD, and Raman/ROA spectra of the substances were quite similar to those of the corresponding major proteins, while some differences were also detected and explained. The conclusions drawn from the FTIR/VCD and Raman/ROA data were in good agreement with the secondary structures calculated from ECD. The results obtained in this work demonstrate that the chiroptical methods used here can be applied to analyze not only pure protein solutions but also more complex systems, such as biological fluids.

Evaluation of IR and Raman spectroscopic markers of human collagens: Insides for indicating colorectal carcinogenesis.

Vibrational spectroscopic methods are widely used in the molecular diagnostics of carcinogenesis. Collagen, a component of connective tissue, plays a special role as a biochemical marker of pathological changes in tissues. The vibrational bands of collagens are very promising to distinguish between normal colon tissue, benign and malignant colon polyps. Differences in these bands indicate changes in the amount, structure, conformation and the ratio between the individual structural forms (subtypes) of this protein. The screening of specific collagen markers of colorectal carcinogenesis was carried out based on the FTIR and Raman (λex 785 nm) spectra of colon tissue samples and purified human collagens. It was found that individual types of human collagens showed significant differences in their vibrational spectra, and specific spectral markers were found for them. These collagen bands were assigned to specific vibrations in the polypeptide backbone, amino acid side chains and carbohydrate moieties. The corresponding spectral regions for colon tissues and colon polyps were investigated for the contribution of collagen vibrations. Mentioned spectral differences in collagen spectroscopic markers could be of interest for early ex vivo diagnosis of colorectal carcinoma if combine vibrational spectroscopy and colonoscopy.

Modification of chitosan-methylcellulose composite films with meso-tetrakis(4-sulfonatophenyl)porphyrin.

Polysaccharide films containing chitosan, methylcellulose, and a mixture of these polysaccharides in various ratios were prepared and modified with meso-tetrakis(4-sulfonatophenyl)porphyrin in an aqueous medium at pH 7. The modified films were compared with the initial films using spectroscopic methods and microscopic imaging. Electronic (UV-vis absorption, electronic circular dichroism (ECD)) and vibrational (FTIR and Raman) spectra showed that the porphyrin macrocycles had a strong affinity toward chitosan and did not interact with the methylcellulose. The total porphyrin uptake depended on the chitosan: methylcellulose ratio and pure methylcellulose films did not retain porphyrin macrocycles. ECD measurements detected the presence of optically active porphyrin species bound to the films. SEM and AFM images confirmed that the porphyrin macrocycles caused structural changes on the film surface and within the film layer.

Ex Vivo Vibration Spectroscopic Analysis of Colorectal Polyps for the Early Diagnosis of Colorectal Carcinoma.

Colorectal cancer is one of the most common and often fatal cancers in humans, but it has the highest chance of a cure if detected at an early precancerous stage. Carcinogenesis in the colon begins as an uncontrolled growth forming polyps. Some of these polyps can finally be converted to colon cancer. Early diagnosis of adenomatous polyps is the main approach for screening and preventing colorectal cancer, and vibration spectroscopy can be used for this purpose. This work is focused on evaluating FTIR and Raman spectroscopy as a tool in the ex vivo analysis of colorectal polyps, which could be important for the early diagnosis of colorectal carcinoma. Multivariate analyses (PCA and LDA) were used to assist the spectroscopic discrimination of normal colon tissue, as well as benign and malignant colon polyps. The spectra demonstrated evident differences in the characteristic bands of the main tissue constituents, i.e., proteins, nucleic acids, lipids, polysaccharides, etc. Suitable models for discriminating the three mentioned diagnostic groups were proposed based on multivariate analyses of the spectroscopic data. LDA classification was especially successful in the case of a combined set of 55 variables from the FTIR, FT Raman and dispersion Raman spectra. This model can be proposed for ex vivo colorectal cancer diagnostics in combination with the colonoscopic extraction of colon polyps for further testing. This pilot study is a precursor for the further evaluation of the diagnostic potential for the simultaneous in vivo application of colonoscopic Raman probes.

Hydrogels based on low-methoxyl amidated citrus pectin and flaxseed gum formulated with tripeptide glycyl-l-histidyl-l-lysine improve the healing of experimental cutting wounds in rats.

Hydrogels based on natural and modified polysaccharides represent growing group of suitable matrices for the construction of effective wound healing materials. Bioactive tripeptide glycyl-l-histidyl-l-lysine and amino acid α-l-arginine are known to accelerate wound healing and skin repair. In this study, hydrogels based on low-methoxyl amidated citrus pectin or flaxseed gum were prepared and used for the transport of these healing agents to the experimental cutting wounds affected by extensive skin damage. Fourier-transform infrared spectroscopy, rheology, differential scanning calorimetry, scanning electron microscopy, swelling and release tests confirmed that these hydrogels differed in structure and physical properties. The cationic tripeptide was found to bind to carboxylic groups in LMA pectin, and the C3OH hydroxyl and ring oxygen O5 are involved in this interaction. The pectin hydrogel showed high viscosity and strong elastic properties, while the flaxseed gum hydrogel was characterised as a viscoelastic system of much lower viscosity. The former hydrogel released the drugs very slowly, while the latter hydrogel demonstrated zero order releasing kinetics optimal for drug delivery. In the in vivo wound healing testing on rats, both polysaccharide hydrogels improved the healing process mediated by the mentioned biomolecules. The tripeptide applied in the hydrogels showed significantly higher healing degree and lower healing time than in the control animals without treatment and when it was applied in an aqueous solution. Despite the absence of a synergistic effect, the mixture of the tripeptide and α-l-arginine in the hydrogels was also quite effective in wound healing. According to histological analysis, complete healing was achieved only when using the tripeptide in the flaxseed gum hydrogel. These observations might have an important prospect in clinical application of polysaccharide hydrogels.

pH-controlled self-assembling of meso-tetrakis(4-sulfonatophenyl)porphyrin-chitosan complexes.

Solid meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS(4))-chitosan supramolecular complexes were prepared by addition of porphyrin to an aqueous solution of chitosan at pH values. The precipitates obtained were assigned as 1 (pH 6.8) and 2 (pH 2.5) and characterized by spectroscopic, thermal, and microscopic methods. Spectroscopic investigation confirmed the presence of TPPS(4) and chitosan in both products and that the porphyrin is highly self-associated. H-type (stacked) of TPPS(4) aggregation was proposed for 1 and J-type (tilted) for 2. Thermal analysis revealed different pyrolysis routes of the complexes depending on their structural diversity. Light microscopic analysis indicated fibrous and lamellar microstructures, respectively, for 1 and 2. SEM and AFM analysis showed that both complexes consist of compact nanostructures; their size and interconnection is different for 1 and 2. Based on structural inferences, self-assembling hierarchy models were proposed for both of the TPPS(4)-chitosan supramolecular complexes.

Interaction of meso-tetrakis(4-sulphonatophenyl)porphine with chitosan in aqueous solutions.

Interaction of meso-tetrakis(4-sulphonatophenyl)porphine (TPPS4) with chitosan (Mr approximately 400 kDa, N-acetyls approximately 20 mol.%) was studied in aqueous solutions. UV-vis absorption and circular dichroism (CD) spectroscopic titration of 10 micromol l-1 TPPS4 with chitosan demonstrated that an addition of the polysaccharide at appropriate concentrations and pH values induce and support self-aggregation of the macrocycles. The mode of aggregation was strongly dependent on pH: stacking (H-type) aggregates predominated at weak acidic conditions (pH 4.8-6.8) and tilted (J-type) aggregates at pH 2.5. At the intermediate pH value (3.6) both types of TPPS4 aggregates were detected. High amount of chitosan (>0.05 mmol l-1 of GlcN) disrupts H-aggregates forming monomeric porphyrin-chitosan complexes (pH 3.6-6.8), while J-aggregates (pH 2.5) are stable even at very high chitosan concentrations. CD titration experiments confirmed the formation of optically active species of TPPS4 in the presence of chitosan. The complex nature of CD bands assigned to both types of porphyrin aggregates indicated the occurrence of several chiral macrocyclic species dependently on pH value and chitosan concentration.

Novel porphyrin conjugates with a potent photodynamic antitumor effect: differential efficacy of mono- and bis-beta-cyclodextrin derivatives in vitro and in vivo.

In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(beta-CD)1 exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(beta-CD)2. However, in vivo P(beta-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm(-2)). In contrast, P(beta-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(beta-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.

In vitro interaction of macrocyclic photosensitizers with intact mitochondria: a spectroscopic study.

Six water-soluble macrocyclic photosensitizers, the members of two groups of expanded porphyrins (metallotexaphyrins and free-base sapphyrins) containing hydrophilic substituents and meso-tetra(4-sulfonatophenyl)-porphyrin, were tested by UV-Vis absorption and resonance Raman spectroscopy in the in vitro binding experiments with intact mitochondria isolated from swine liver. Studied macrocycles showed markedly different affinity to mitochondria. The highest uptake was observed for sapphyrin-sugar conjugate and metallotexaphyrins. Sapphyrin-polyamine conjugates exhibit something less affinity to mitochondria, while the porphyrin of anionic character showed very low mitochondrial uptake. Obtained spectroscopic results confirm that the binding process altered the self-aggregation degree of expanded porphyrins.

Synthesis and biolocalization of water-soluble sapphyrins.

The synthesis, characterization, and biolocalization properties of new, fully water-soluble sapphyrins is reported. These systems, which display light absorbing features that make them of potential interest as photodynamic therapy (PDT) sensitizers, were found to localize selectively in pancreatic carcinoma tissue in a xenographic murine model. In this model, the concentration ratios for malignant tissue to surrounding muscle tissue were found to be as high as 280 in the most favorable case. The concentrations of the water-soluble sapphyrins reported here, as well as a porphyrin control system, were determined as a function of time and tissue type.

Biodistribution assessment of a lutetium(III) texaphyrin analogue in tumor-bearing mice using NIR Fourier-transform Raman spectroscopy.

The use of near-infrared (NIR)-excited Fourier-transform (FT) Raman spectroscopy as a technique for evaluating the extent of photosensitizer localization in tumor (human pancreatic adenocarcinomas)-bearing mice has been tested using lutetium(III) texaphyrin analogue Lu-T2B2Tex. The complex was injected subcutaneously in the form of three injections given during the course of 3 days. The kinetics of biodistribution were then followed over a time scale of 1-6 days. The NIR-FT-Raman spectra of tissue samples obtained from the xenographic tumor, muscle, heart, brain, liver, spleen, kidney and blood were recorded and used to identify the presence of Lu-T2B2Tex in these tissues. Five Raman sensitizer markers were used to estimate the relative content of Lu-T2B2Tex in tumor at various postinjection times. UV-Visible (Vis) absorption spectroscopic detection of this sensitizer in tissue extracts was applied as a conventional method. Both spectroscopic methods were in good agreement with each other and confirm that Lu-T2B2Tex localizes well in tumor tissue. Maximal drug content was observed 3 days after the final injection. This time delay seems to be optimal for tumor irradiation in photodynamic therapy.

The complexation of metal cations by D-galacturonic acid: a spectroscopic study.

Solid complexes of D-galacturonic acid (GalA) with cobalt(II), copper(II), nickel(II) and oxovanadium(IV) (1-4) were prepared and characterised. The metal-to-ligand molar ratio was 1:2 for complexes 1-3 and 1:1 for complex 4. The alpha- and beta-anomers of GalA were detected in all the complexes in solid state and in solutions. An addition of small amounts of the paramagnetic complexes to the D2O solution of pure ligand led to NMR line broadening of some 1H and 13C nuclei. This broadening was sensitive to the anomeric state of GalA in the case of complexes 1 and 4. NMR and vibrational spectroscopic data indicate the formation of carboxylate complexes of all the cations, while noncarboxylic oxygens are also involved into the metal bonding in some cases. VCD spectra of complexes 1-4 in D2O and Me2SO-d6 solutions confirm that GalA carboxylic group may participate in the formation of optically active species around the metal cation. Possible ways of GalA coordination by metal cations of this study were proposed and discussed.

Biolocalisation and photochemical properties of two novel macrocyclic photosensitisers: a spectroscopic study.

Biolocalisation and photochemical properties of novel macrocyclic photosensitisers, guanidiniocarbonyl-substituted tetraphenylporphyrin (1) and sugar-substituted sapphyrin (2) were investigated by spectroscopic methods. Both photosensitisers absorb in far visible region and showed good tumour localisation. Photosensitiser 2 demonstrated significantly larger absolute and relative to normal tissue (T/N) amount in tumour (330 microg g(-1) wet tissue, T/N=19.0) than photosensitiser 1 did (13 microg g(-1) wet tissue, T/N=2.1). According to iodometric and uric acid assays, compound 1 produced large amount of 1O2 (phidelta=0.60-0.68), while compound 2 showed non-significant 1O2 production (phidelta=0.04). The electronic spectroscopic study confirms that only photosensitiser 1 is able to mediate photooxidation of model compounds (BSA, poly(Trp), Tyr, Trp, and GMP) after light irradiation. Pour photochemical activity of compound 2 was explained by its self-aggregation. Raman spectroscopic study indicated that monomerised photosensitiser 2 effectively damaged BSA and calf thymus DNA after light excitation at the conditions of high excess of these macromolecules.

Mekabu fucoidan: structural complexity and defensive effects against avian influenza A viruses.

Fucoidan from the sporophyll (Mekabu) of brown seaweed Undaria pinnatifida (wakame) is interesting due to its various biological activities. Mekabu fucoidan (Mw ∼ 9 kDa) of this study (MF) was previously isolated and characterized by chemical and separation methods including GPC and methylation analysis (Lee, Hayashi, Hashimoto, Nakano, & Hayashi, 2004). It was found that this fucoidan composed of partially sulphated (DS ∼ 0.72) fucose and galactose at approximately equal amounts. Methylation analyses revealed complex structure of MF. However, it has been still unclear about the linkages between units and substitution patterns. To solve these structural tasks, spectroscopic methods (FTIR, FT Raman and NMR) were used in the analysis of native MF and its deesterified derivatives. According to obtained results, this polysaccharide was defined as O-acetylated sulphated fucogalactan. The defensive effects of MF were evaluated on mice infected with avian influenza A viruses (H5N3 and H7N2 subtypes); its efficacy was determined in reducing viral replication and increasing antibody production. Oral administration of MF resulted in suppressing virus yields. In addition, the production of neutralizing antibodies and mucosal IgA in the animals inoculated with the avian influenza A viruses was significantly increased. These results suggested that MF could be used for the prevention of viral infection.