Physico-chemical properties and digestibility of native and citrate starches change in different ways by synchrotron radiation.

The physico-chemical properties and digestibility of native and citrate cassava starches changed as a result of synchrotron radiation treatment. In this study, the native and citrate starch samples were exposed to radiation doses of 0.1, 0.4, 0.8 and 3.9 kGy. The granular morphology revealed that all samples were rupture and damage after radiation. As increasing radiation doses, the relative crystallinity as determined by WAXS and the ratio of 1047/1015 cm-1 from FTIR result decreased while the degree of degradation and solubility increased for all samples. The swelling power of radiated native starches decreased with higher radiation doses indicating that the cross-linking of starch was induced by synchrotron radiation which was related to an increase in the resistant starch content. On the contrary, for radiated citrate samples, the FTIR peak at 1724 cm-1 was observed. The ratio of 1724/2900 cm-1 and total esterified citric acid did not change. The swelling and degree of di-esterification were reduced while the degree of mono-esterification increased with higher doses. It implied that the cross-linking by ester bonds was broken into mono-ester bonds. This work demonstrated that synchrotron radiation changed the physical and chemical properties of native and citrate starches in different ways.

Oral Administration of Melatonin or Succinyl Melatonin Niosome Gel Benefits 5-FU-Induced Small Intestinal Mucositis Treatment in Mice.

Mucositis is one of the most adverse effects of 5-fluorouracil (5-FU) and had no standard drug for treatment. Melatonin is a neurohormone, and can ameliorate radiotherapy-induced small intestinal mucositis. Melatonin encapsulated in niosomes improved its poor bioavailability. Succinyl melatonin, a melatonin derivative, showed prolonged release compared with melatonin. This study investigated the efficacy of melatonin niosome gel (MNG) and succinyl melatonin niosome gel (SNG) in 5-FU-induced small intestinal mucositis treatment in mice. MNG and SNG with particle sizes of 293 and 270 nm were shown to have mucoadhesive potentials. The effect of a daily oral application of MNG, SNG, or fluocinolone acetonide gel (FAG, positive control) was compared to that of the normal group. The body weight, food consumption, histology, Fourier transform infrared (FTIR) spectroscopy, inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-1ß), and malondialdehyde (MDA) in the small intestine were monitored. The results showed decreased %body weight and food consumption in all 5-FU-injected groups compared with the normal group. The MNG and SNG treatments maintained the food consumption and the normal integrity of the small intestines, as evidenced by villus length and crypt depth, similar to the observations in the normal groups. The FTIR spectra showed no change in lipids of the MNG and SNG groups compared with the normal group. Moreover, SNG could reduce IL-1ß content to a level that was not different from the level in the normal groups. Therefore, the oral application of MNG and SNG could protect against 5-FU-induced small intestinal mucositis in mice.

Characteristic Evaluation of Gel Formulation Containing Niosomes of Melatonin or Its Derivative and Mucoadhesive Properties Using ATR-FTIR Spectroscopy.

Chitosan or polyvinyl pyrrolidone (PVP) were used in combination with hydroxypropyl methylcellulose (HPMC) and poloxamer 407 (P407) as gelling agents for oral drug delivery. The performance interaction with mucin of chitosan-composed gel (F1) and PVP-composed gel (F2) was compared using attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectroscopy at controlled temperatures of 25 and 37 °C for 1 and 5 min. F1 containing niosome-entrapped melatonin or its derivatives was investigated for mucoadhesive interaction on mucosa by ATR-FTIR spectroscopy under the same conditions. The results showed that F1-treated mucin gave a significantly lower amide I/amide II ratio than untreated mucin and F2-treated mucin did within 1 min, suggesting improved rapid affinity between mucin and chitosan. The spectra of mucosa treated with F1 incorporating niosomes of melatonin or its derivatives showed peak shifts at C=O (amide I), N-H (amide II), and carbohydrate regions and an associated decrease in the amide I/amide II ratio and increase in the carbohydrate/amide II ratio. These results indicated electrostatic interaction and hydrogen bonding between chitosan and mucin on the mucosa. In conclusion, the molecular interaction between gels and mucin/mucosa detected at amide I and amide II of proteins and the carbohydrate region could lead to an improved mucoadhesive property of the gel on the mucosa.

Topical Melatonin Niosome Gel for the Treatment of 5-FU-Induced Oral Mucositis in Mice.

BACKGROUND: Oral mucositis, one of the most common complications of 5-Fluorouracil (5-FU) treatment, leads to several problems, including pain, diarrhea and malnutrition, and reduces the quality of life and subsequent treatments. Melatonin, a neurohormone with anti-inflammatory and antioxidant activities, was encapsulated in niosomes and embedded in a mucoadhesive gel formulation as a Melatonin Niosome Gel (MNG) to perform oral mucositis treatment. OBJECTIVE: This study aimed to investigate the effectiveness of MNG for the treatment of 5-FU-induced oral mucositis in mice. METHODS: Oral mucositis was induced in ICR mice by 5-FU and randomly assigned to receive daily applications of the topical oral MNG, a fluocinolone acetonide gel, a blank niosome gel, or no treatment for 5 days in comparison with a normal group. Average body weights, food consumption, and behaviors of the mice as well as microscopic histopathology, Fourier-Transform Infrared Spectroscopy (FTIR) analysis, proinflammatory cytokine levels, and oxidative stress markers of the tongues were monitored and collected after sacrifice. RESULTS: In comparison to the normal group, the average body weights of the 5-FU-MNG mice did not deviate from that of the normal group, nor was there a significant difference in the time to sleep or licking (p>0.05 for both parameters). In addition, the mice treated with MNG and fluocinolone acetonide did not show significantly different histopathological, FTIR, interleukin-1ß or malondialdehyde (MDA) results in the tongues used as the oral tissue samples. CONCLUSION: Topical MNG potentially inhibits inflammation and lipid oxidative stress in 5-FU-induced oral mucositis.

Synchrotron FTIR microspectroscopy revealed apoptosis-induced biomolecular changes of cholangiocarcinoma cells treated with ursolic acid.

BACKGROUND: Ursolic acid (UA) is a natural triterpenoid which possesses anti-cancer activity. However, little is known regarding the activity and molecular mechanism of UA in cholangiocarcinoma (CCA). Thus, we investigated the effects of UA on growth inhibition and apoptosis induction through biomolecular changes in KKU-213 and KKU-055 CCA cell lines. METHODS: The anti-proliferative effect of UA against CCA cells was evaluated using SRB assay. Changes in biomolecules were assessed by SR-FTIR microspectroscopy combined with PCA and conventional methods (i.e., Annexin V-FITC/PI staining for lipid alteration and apoptosis induction; Western blot analysis and caspase-3/7 activity assay for apoptotic protein detection). RESULTS: UA suppressed the proliferation of CCA cells in a dose- and time-dependent manner. SR-FTIR data revealed a significant alteration in lipids attributable to changes in apoptotic cell membranes, confirmed by Annexin V-FITC/PI staining. SR-FTIR data showed that UA promoted changes in the protein secondary structure. Elevated expression of Bax and decreased expression of Bcl-2 and survivin/BIRC5 along with augmented caspase-3/7 activity supported alterations in apoptosis-related proteins. CONCLUSIONS: SR-FTIR microspectroscopy was successfully used as a label-free technique to monitor apoptosis-induced biomolecular changes in UA-treated CCA cells. UA exerted the cytotoxic and apoptotic activities in CCA cells through alterations in membrane lipids and apoptotic proteins. UA could be a potential anti-CCA candidate and a chemical starting point for the discovery of novel anti-cancer agents. SIGNIFICANCE: Our present study showed the first evidence that UA exhibited the anti-proliferative and pro-apoptotic activities toward CCA cells through changes in biomolecules, notably lipids and proteins.

Structural Studies of Epithelial Mesenchymal Transition Breast Tissues.

At the supramolecular level, the proliferation of invasive ductal carcinoma through breast tissue is beyond the range of standard histopathology identification. Using synchrotron small angle x-ray scattering (SAXS) techniques, determining nanometer scale structural changes in breast tissue has been demonstrated to allow discrimination between different tissue types. From a total of 22 patients undergoing symptomatic investigations, different category breast tissue samples were obtained in use of surgically removed tissue, including non-lesional, benign and malignant tumour. Structural components of the tissues were examined at momentum transfer values between q = 0.2 nm-1 and 1.5 nm-1. From the SAXS patterns, axial d-spacing and diffuse scattering intensity were observed to provide the greatest discrimination between the various tissue types, specifically in regard to the epithelial mesenchymal transition (EMT) structural component in malignant tissue. In non-lesional tissue the axial period of collagen is within the range 63.6-63.7 nm (formalin fixed paraffin embedded (FFPE) dewaxed) and 63.4 (formalin fixed), being 0.9 nm smaller than in EMT cancer-invaded regions. The overall intensity of scattering from cancerous regions is a degree of magnitude greater in cancer-invaded regions. Present work has found that the d-spacing of the EMT positive breast cancer tissue (FFPE (dewaxed)) is within the range 64.5-64.7 nm corresponding to the 9th and 10th order peaks. Of particular note in regard to formalin fixation of samples is that no alteration is observed to occur in the relative differences in collagen d-spacing between non-lesional and malignant tissues. This is a matter of great importance given that preserved-sample and also retrospective study of samples is greatly facilitated by formalin fixation. Present results indicate that as aids in tissue diagnosis SAXS is capable of distinguishing areas of invasion by disease as well as delivering further information at the supramolecular level.

Attenuated total reflection Fourier transform infrared as a primary screening method for cancer in canine serum.

Cancer is a major cause of death in dogs worldwide, and the incidence of cancer in dogs is increasing. The attenuated total reflection Fourier transform infrared spectroscopic (ATR-FTIR) technique is a powerful tool for the diagnosis of several diseases. This method enables samples to be examined directly without pre-preparation. In this study, we evaluated the diagnostic value of ATR-FTIR for the detection of cancer in dogs. Cancer-bearing dogs (n = 30) diagnosed by pathologists and clinically healthy dogs (n = 40) were enrolled in this study. Peripheral blood was collected for clinicopathological diagnosis. ATR-FTIR spectra were acquired, and principal component analysis was performed on the full wave number spectra (4,000-650 cm-1). The leave-one-out cross validation technique and partial least squares regression analysis were used to predict normal and cancer spectra. Red blood cell counts, hemoglobin levels and white blood cell counts were significantly lower in cancer-bearing dogs than in clinically healthy dogs (p < 0.01, p < 0.01 and p = 0.03, respectively). ATR-FTIR spectra showed significant differences between the clinically healthy and cancer-bearing groups. This finding demonstrates that ATR-FTIR can be applied as a screening technique to distinguish between cancer-bearing dogs and healthy dogs.

Analysis of xanthyletin and secondary metabolites from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 against Pythium insidiosum.

BACKGROUND: Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing pythiosis, an infectious disease with high morbidity and mortality rates. Antifungal agents are ineffective as pythiosis treatments because ergosterol, the target site of most antifungal agents, is not found in the P. insidiosum cytoplasmic membrane. The best choice for treatment is surgical removal of the infected organ. While natural plant products or secretory substances from bacterial flora have exhibited in vitro anti-P. insidiosum activity, their mechanism of action remains unknown. Therefore, this study hypothesized that the mechanism of action could be related to changes in P. insidiosum biochemical composition (such as lipid, carbohydrate, protein or nucleic acid) following exposure to the inhibitory substances. The biochemical composition of P. insidiosum was investigated by Synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy. RESULTS: Fraction No.6 from the crude extract of P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501 and xanthyletin were used as anti-P. insidiosum substances, with MFCs at 3.125, 1.57-1.91, 0.003 mg/ml, respectively. The synchrotron FTIR results show that the deconvoluted peak distributions in the amide I, amide II, and mixed regions were significantly different between the treatment and control groups. CONCLUSIONS: Xanthyletin and the secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501 exerted anti-P. insidiosum activity that clearly changed the proteins in P. insidiosum. Further study, including proteomics analysis and in vivo susceptibility testing, should be undertaken to develop a better understanding of the mechanism of anti-P. insidiosum activity.

Classification of Gemcitabine resistant Cholangiocarcinoma cell lines using synchrotron FTIR microspectroscopy.

Cholangiocarcinoma (CCA), a cancer of bile duct epithelium, is a major health problem in Thailand especially in the northeast. Overall treatment outcomes have not shown much improvement because the disease is usually detected at an advanced stage and often shows chemotherapeutic resistance. High-throughput Fourier Transform Infrared (FTIR) microspectroscopy can be used for cell classification and has the potential to diagnose cancer and possibly predict chemo-response. This study was aimed to differentiate gemcitabine-sensitive and gemcitabine-resistant induction in two CCA cell lines (KKU-M139 and KKU-M214) and xenograft tissues using synchrotron-FTIR microspectroscopy. Partial Least Squares Discriminant Analysis (PLS-DA) could discriminate between chemo-sensitive and chemo-resistant cells in the FTIR fingerprint spectral region (1800-1000 cm-1 ) with more than 90% sensitivity and specificity. The chemo-resistant and chemo-sensitive phenotypes were different in protein (amide I, amide II), lipids (carbonyl group and CH3 deformation) and phosphodiester from nucleic acids. Additionally, spectra from xenograft tissues showed similar results to the cell line study with marked differences between chemo-resistant and chemo-sensitive CCA tissues, and PLS-DA could discriminate the chemotherapeutic response with 98% sensitivity and specificity. This is the first study to demonstrate the use of FTIR microspectroscopy to assess chemo-response both in vitro and in vivo.

Gallic acid conjugated with gold nanoparticles: antibacterial activity and mechanism of action on foodborne pathogens.

Foodborne pathogens, including Plesiomonas shigelloides and Shigella flexneri B, are the major cause of diarrheal endemics worldwide. Antibiotic drug resistance is increasing. Therefore, bioactive compounds with antibacterial activity, such as gallic acid (GA), are needed. Gold nanoparticles (AuNPs) are used as drug delivery agents. This study aimed to conjugate and characterize AuNP-GA and to evaluate the antibacterial activity. AuNP was conjugated with GA, and the core-shell structures were characterized by small-angle X-ray scattering and transmission electron microscopy. Antibacterial activity of AuNP-GA against P. shigelloides and S. flexneri B was evaluated by well diffusion method. AuNP-GA bactericidal mechanism was elucidated by Fourier transform infrared microspectroscopic analysis. The results of small-angle X-ray scattering showed that AuNP-GA conjugation was successful. Antibacterial activity of GA against both bacteria was improved by conjugation with AuNP because the minimum inhibitory concentration value of AuNP-GA was significantly decreased (P<0.0001) compared to that of GA. Fourier transform infrared analysis revealed that AuNP-GA resulted in alterations of lipids, proteins, and nucleic acids at the bacterial cell membrane. Our findings show that AuNP-GA has potential for further application in biomedical sciences.

Gold Nanoparticles Enhance the Anticancer Activity of Gallic Acid against Cholangiocarcinoma Cell Lines.

Gold nanoparticles (GNPs) were conjugated with gallic acid (GA) at various concentrations between 30 and 150 µM and characterized using transmission electron microscopy (TEM) and UV-Vis spectroscopy (UV-VIS). The anticancer activities of the gallic acid-stabilized gold nanoparticles against well-differentiated (M213) and moderately differentiated (M214) adenocarcinomas were then determined using a neutral red assay. The GA mechanism of action was evaluated using Fourier transform infrared (FTIR) microspectroscopy. Distinctive features of the FTIR spectra between the control and GA-treated cells were confirmed by principal component analysis (PCA). The surface plasmon resonance spectra of the GNPs had a maximum absorption at 520 nm, whereas GNPs-GA shifted the maximum absorption values. In an in vitro study, the complexed GNPs-GA had an increased ability to inhibit the proliferation of cancer cells that was statistically significant (P<0.0001) in both M213 and M214 cells compared to GA alone, indicating that the anticancer activity of GA can be improved by conjugation with GNPs. Moreover, PCA revealed that exposure of the tested cells to GA resulted in significant changes in their cell membrane lipids and fatty acids, which may enhance the efficacy of this anticancer activity regarding apoptosis pathways.

Discrimination of micromass-induced chondrocytes from human mesenchymal stem cells by focal plane array-Fourier transform infrared microspectroscopy.

Rapid and sensitive methods for identifying stem cell differentiation state are required for facilitating future stem cell therapies. We aimed to evaluate the capability of focal plane array-Fourier transform infrared (FPA-FTIR) microspectroscopy for characterising the differentiation of chondrocytes from human mesenchymal stem cells (hMSCs). Successful induction was validated by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis for collagen and aggrecan expression as chondrocyte markers in parallel with the spectroscopy. Spectra derived from chondrocyte-induced cells revealed strong IR absorbance bands attributed to collagen near 1338 and 1234 cm(-1) and proteoglycan at 1245 and 1175-960 cm(-1) compared to the non-induced cells. In addition, spectra from control and induced cells are segregated into separate clusters in partial least squares discriminant analysis score plots at the very early stages of induction and discrimination of an independent set of validation spectra with 100% accuracy. The predominant bands responsible for this discrimination were associated with collagen and aggrecan protein concordant with those obtained from RT-PCR and Western blot techniques. Our findings support the capability of FPA-FTIR microspectroscopy as a label-free tool for stem cell characterization allowing rapid and sensitive detection of macromolecular changes during chondrogenic differentiation.

Toxicity and phototoxicity of Hypocrellin A on malignant human cell lines, evidence of a synergistic action of photodynamic therapy with Imatinib mesylate.

Photodynamic therapy combines a photosensitizer, localised preferentially in malignant cells with light activation. Hypocrellin A (HA), a lipid-soluble peryloquinone, is considered as a high potential photosensitizer. We report dose and light irradiation effects of HA on HeLa, Calu and K562 cell lines, the latter including a subclone resistant to Imatinib mesylate (IM, Gleevec). All cell lines and subclones tested are sensitive to HA PDT. In the epithelial tumour cell lines, we observe a significant photosensitizing effect in the presence of HA. In the leukemic K562 cells, HA exposure led to an inhibitory effect, which was not seen in the K562 cells resistant to Imatinib mesylate. However, experiments using IM and HA led to a reversal of IM-resistant phenotype in this cell line, with evidence of a major sensitizing effect of photodynamic therapy. Overall our results suggest a phototoxicity of HA in epithelial cell lines and demonstrate for the first time, a synergy between IM and photodynamic therapy to circumvent IM-resistance.

Spectroscopic signatures of single, isolated cancer cell nuclei using synchrotron infrared microscopy.

Single-cell studies have important implications in biomedicine. An accurate investigation of biochemical behaviour and status requires a biomolecular probe such as vibrational microscopy. Amongst other approaches, synchrotron infrared microspectroscopy is an appropriate analytical tool for single-cell investigation. However, it is important to understand the precise origin of spectral differences as they are directly related to the cell biochemistry. Beside biomolecular changes, physical properties can interfere in the resulting information, and the two effects need separating. Both cells and nuclei induce Mie scattering effects due to their equivalent size with the probe wavelength. This results in a large modification of the spectra, and its precise contribution has to be determined in order to extract the true spectral information. On this basis, we carried out this study in order to evaluate the exact contribution of cell nuclei to Mie scattering. To this purpose, we isolated whole cancer cell nuclei and obtained, for the first time, their FTIR spectra with good signal to noise ratio. The synchrotron-based FTIR (S-FTIR) spectra of nuclei showed changes in lipids, proteins, and DNA absorptions when compared to spectra of whole lung cancer cells. Importantly, we estimated the Mie scattering properties of single cells and single nuclei spectra and were consequently able to separate optical and chemical properties of single cells and nuclei. This is the first study which sheds new light on the identification of the precise spectral biomarkers of a whole cell and those of the cell nucleus.

Photosensitizer effects on cancerous cells: a combined study using synchrotron infrared and fluorescence microscopies.

Hypocrellin A (HA), a lipid-soluble peryloquinone derivative, isolated from natural fungus sacs of Hypocrella bambusae, has been reported to be a highly potential photosensitizer in photodynamic therapy (PDT). It has been studied increasingly because of its anticancer activities when irradiated with light. We have studied the interaction mechanisms of HA with HeLa cells as a function of incubation time. Fluorescence microscopy confirmed that HA localisation is limited in the cytoplasm before eventually concentrating in clusters around the nucleus. The IR spectra of HA-treated, PDT-treated and control HeLa cells were recorded at the ESRF Infrared beamline (ID21). Principal component analysis has been used to assess the IR spectral changes between the various HeLa cells spectral data sets (The Unscrambler software, CAMO). PCA revealed that there is a frequency shift of protein amide I and amide II vibrational bands, indicating changes in the protein secondary structures of the HA-treated and PDT-treated cancer cells compared to the control cells. In addition, the relative DNA intensity in HA-treated cells decreases gradually along the incubation time. The use of synchrotron infrared microscopy is shown to be of paramount importance for targeting specifically the biochemical modification induced in the cell nucleus.