Microdistribution and quantification of the boron neutron capture therapy drug BPA in primary cell cultures of human glioblastoma tumour by NanoSIMS.

The ability of secondary ion mass spectrometry (SIMS) to provide high sensitivity imaging of elements and small-medium mass molecules in biological tissues and cells, makes it a very powerful tool for drug distribution studies. Here we report on the application of a high-resolution dynamic SIMS instrument for the quantification and localisation of therapeutic levels of the BNCT agent l-para-(dihydroxyboryl)-phenylalanine (BPA) in primary cell cultures from human patients exhibiting glioblastoma multiform tumours. Boron uptake and distribution was determined quantitatively as a function of cell-sampling location and different treatment regimes. Importantly, BPA was found to accumulate in cancer cells invading the 'brain around tumour' tissue in addition to the main tumour site. Pre-treatment of samples with l-tyrosine was found not to increase the uptake of BPA, nor change the intracellular drug distribution. In cultured cells from the tumour core and brain around tumour, with and without l-tyrosine pre-treatment, normalised boron-related signals were higher from cell nuclei than from cytoplasm. An efflux treatment was found to reduce BPA levels, but at a rate slower than the original uptake, and did not affect the intracellular drug distribution. To the best of our knowledge, these data represent the first published study of BPA uptake and l-amino acid pre-treatment in cultured primary human cells using dynamic SIMS, and the most detailed, subcellular distribution study of a BNCT agent in any cellular system.

Quartz Crystal Microbalance Assay of Clinical Calcinosis Samples and Their Synthetic Models Differentiates the Efficacy of Chelation-Based Treatments.

This paper sets out in vitro protocols for studying the relative effectiveness of chelators used in the dissolution-based treatment of hard calcinosis. Pulverized hard calcinosis samples from human donors or synthetic hydroxyapatite nanoparticles were deposited by electrophoretic deposition on the surface of a quartz crystal microbalance sensor. Over 150 deposits of <20 µg were dissolved over the course of 1 h by aliquots of buffered, aqueous solutions of two calcium chelators, EDTA and citrate, with the surface-limited dissolution kinetics monitored with <1 s time resolution. There was no statistically significant difference in dissolution rate between the four synthetic hydroxyapatite materials in EDTA, but the dissolution rates in citrate were lower for hydroxyapatite produced by acetate or nitrate metathesis. Hard calcinosis and synthetic hydroxyapatites showed statistically identical dissolution behavior, meaning that readily available synthetic mimics can replace the rarer samples of biological origin in the development of calcinosis treatments. EDTA dissolved the hydroxyapatite deposits more than twice as fast as citrate at pH 7.4 and 37 °C, based on a first-order kinetic analysis of the initial frequency response. EDTA chelated 6.5 times more calcium than an equivalent number of moles of citrate. Negative controls using nonchelating N,N,N',N'-tetraethylethylenediamine (TEEDA) showed no dissolution effect. Pharmaceutical dissolution testing of synthetic hydroxyapatite tablets over 6 h showed that EDTA dissolved the tablets four to nine times more quickly than citrate.

Probing the action of a novel anti-leukaemic drug therapy at the single cell level using modern vibrational spectroscopy techniques.

Acute myeloid leukaemia (AML) is a life threatening cancer for which there is an urgent clinical need for novel therapeutic approaches. A redeployed drug combination of bezafibrate and medroxyprogesterone acetate (BaP) has shown anti-leukaemic activity in vitro and in vivo. Elucidation of the BaP mechanism of action is required in order to understand how to maximise the clinical benefit. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Synchrotron radiation FTIR (S-FTIR) and Raman microspectroscopy are powerful complementary techniques which were employed to probe the biochemical composition of two AML cell lines in the presence and absence of BaP. Analysis was performed on single living cells along with dehydrated and fixed cells to provide a large and detailed data set. A consideration of the main spectral differences in conjunction with multivariate statistical analysis reveals a significant change to the cellular lipid composition with drug treatment; furthermore, this response is not caused by cell apoptosis. No change to the DNA of either cell line was observed suggesting this combination therapy primarily targets lipid biosynthesis or effects bioactive lipids that activate specific signalling pathways.

Peptide structural analysis using continuous Ar cluster and C60 ion beams.

A novel application of time-of-flight secondary ion mass spectrometry (ToF-SIMS) with continuous Ar cluster beams to peptide analysis was investigated. In order to evaluate peptide structures, it is necessary to detect fragment ions related to multiple neighbouring amino acid residues. It is, however, difficult to detect these using conventional ToF-SIMS primary ion beams such as Bi cluster beams. Recently, C60 and Ar cluster ion beams have been introduced to ToF-SIMS as primary ion beams and are expected to generate larger secondary ions than conventional ones. In this study, two sets of model peptides have been studied: (des-Tyr)-Leu-enkephalin and (des-Tyr)-Met-enkephalin (molecular weights are approximately 400 Da), and [Asn(1) Val(5)]-angiotensin II and [Val(5)]-angiotensin I (molecular weights are approximately 1,000 Da) in order to evaluate the usefulness of the large cluster ion beams for peptide structural analysis. As a result, by using the Ar cluster beams, peptide molecular ions and large fragment ions, which are not easily detected using conventional ToF-SIMS primary ion beams such as Bi3 (+), are clearly detected. Since the large fragment ions indicating amino acid sequences of the peptides are detected by the large cluster beams, it is suggested that the Ar cluster and C60 ion beams are useful for peptide structural analysis.

TOF-SIMS with argon gas cluster ion beams: a comparison with C60+.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an established technique for the characterization of solid sample surfaces. The introduction of polyatomic ion beams, such as C(60), has provided the associated ability to perform molecular depth-profiling and 3D molecular imaging. However, not all samples perform equally under C(60) bombardment, and it is probably naïve to think that there will be an ion beam that will be applicable in all situations. It is therefore important to explore the potential of other candidates. A systematic study of the suitability of argon gas cluster ion beams (Ar-GCIBs) of general composition Ar(n)(+), where n = 60-3000, as primary particles in TOF-SIMS analysis has been performed. We have assessed the potential of the Ar-GCIBs for molecular depth-profiling in terms of damage accumulation and sputter rate and also as analysis beams where spectral quality and secondary ion yields are considered. We present results with direct comparison with C(60) ions on the same sample in the same instrument on polymer, polymer additive, and biomolecular samples, including lipids and small peptides. Large argon clusters show reduced damage accumulation compared with C(60) with an approximately constant sputter rate as a function of Ar cluster size. Further, on some samples, large argon clusters produce changes in the mass spectra indicative of a more gentle ejection mechanism. However, there also appears to be a reduction in the ionization of secondary species as the size of the Ar cluster increases.

Synchrotron FTIR analysis of drug treated ovarian A2780 cells: an ability to differentiate cell response to different drugs?

Recently a new di-gold(I) organometallic complex [1,3-(Ph(3)PAu)(2)-C(6)H(4)] (KF0101) has been synthesised and found to exhibit cytotoxic activity in vitro. Subsequently it has been demonstrated that KF0101 shows little or no cross-resistance against a number of the cisplatin resistant ovarian cancer cell lines in vitro suggesting a different mode of action for the drug. In this study, syncrotron radiation infrared microspectroscopy (SR-IRMS) has been used on drug treated single A2780 cells in order to determine if this different mode of action can be identified spectroscopically. The aim of the study was to establish: (i) if single cell SR-IRMS could be used to give insight into the cellular response on treatment with different cytotoxic agents relative to non-treated cells (control) and (ii) that if the cytotoxic drugs elicit a different biochemical response these responses could be distinguished from each other. The most striking features obtained after Principal Components Analysis (PCA) of Resonant Mie Scattering (RMieS) corrected single cell spectra of drug treated ovarian A2780 cells are: (i) The spectra obtained for the control are quite heterogeneous and several hundred spectra are required to adequately define the nature of the control; (ii) after drug treatment at the IC50 level for 24 h with cisplatin, KF0101, methotrexate, paclitaxel or 5-fluorouracil the cell spectra, as represented on a PCA scores plot, generally concentrate in certain well defined areas of the control, there are however a small number of spectra that fall outside of the area defined by the control; and (iii) a differentiation between cell spectra obtained on treatment with different drugs is observed which fits well with different in vitro cell culture behaviour and a flow cytometry cell cycle analysis of the control and drug treated cells. Inspection of the loading plots shows that PC1 is essentially the same for all plots and reflects changes in cell biochemistry related to the cell cycle. PC2, however, on comparison of the control versus cisplatin or cisplatin versus KF0101 is indicative of differences induced by drug treatment and has been termed as cell cycle-plus behaviour. These data are shown to be consistent with that obtained using bench-top IRMS by averaging a number of single cell spectra and carrying out a PCA, but SR-IRMS offers more insight into how the drug is affecting the cell population. More importantly, this approach enables the influence of the cell cycle on both the control and drug treated samples to be taken into consideration when evaluating the drug-cell interaction.

Effects of cryogenic sample analysis on molecular depth profiles with TOF-secondary ion mass spectrometry.

Although the benefits of decreased sample temperature for the molecular profiling of organic materials with time-of-flight secondary ion mass spectrometry (TOF-SIMS) have been established, the mechanism behind spectral changes observed at low temperature, particularly increased protonated molecular ion (M + H)(+) yields, have not been examined in detail. We have developed a procedure to investigate these effects by monitoring secondary ion yields under sustained primary ion bombardment as the sample temperature is cooled from room temperature down to 80 K. Examination of biomaterials such as an amino acid (arginine), a polypeptide (Gly-Gly-Tyr-Arg), a lipid (1,2 dipalmitoyl-sn-glycero-3 phosphatidylcholine), and a drug molecule (cyclosporine A) each provide evidence of ion yield enhancement at 80 K under either 20 keV C(60)(+) or 20 keV Au(3)(+) bombardment. For example, arginine shows a 2-fold increase in the steady-state intensity for the (M + H)(+) ion at 80 K compared to the steady state at 300 K. It is shown that there is a correlation between the yield enhancement and a reduction in the damage cross section, which for arginine under 20 keV Au(3)(+) bombardment decreases from 5.0 ± 0.4 × 10(-14) cm(2) at 300 K to 2.0 ± 0.3 × 10(-14) cm(2) at 80 K. The role of water as the facilitator for this reduction is explored through the use of H(2)O and D(2)O dosing experiments at 80 K.

Discrimination of prostate cancer cells and non-malignant cells using secondary ion mass spectrometry.

This communication utilises Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) combined with multivariate analysis to obtain spectra from the surfaces of three closely related cell lines allowing their discrimination based upon mass spectral ions.

A new dynamic in mass spectral imaging of single biological cells.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has unique capabilities in the area of high-resolution mass spectrometric imaging of biological samples. The technique offers parallel detection of native and non-native molecules at physiological concentrations with potentially submicrometer spatial resolution. Recent advances in SIMS technology have been focused on generating new ion sources that can in turn be used to eject more intact molecular and biological characteristic species from a sample. The introduction of polyatomic ion beams, particularly C60, for TOF-SIMS analysis has created a whole new application of molecular depth profiling and 3D molecular imaging. However, such analyses, particularly at high lateral resolution, are severely hampered by the accompanying mass spectrometry associated with current TOF-SIMS instruments. Hence, we have developed an instrument that overcomes many of the drawbacks of current TOF-SIMS spectrometers by removing the need to pulse the primary ion beam. The instrument samples the secondary ions using a buncher that feeds into a specially designed time-of-flight analyzer. We have validated this new instrumental concept by analyzing a number of biological samples generating 2D and 3D images showing molecular localization on a subcellular scale, over a practical time frame, while maintaining high mass resolution. We also demonstrate large area mapping and the MS/MS capability of the instrument.

Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy.

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover, CaP metastasizes to the bone marrow, which harbors a rich source of lipids stored within adipocytes. Here, we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique, we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition, larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction, the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes, indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction.

A correlation of FTIR spectra derived from prostate cancer biopsies with gleason grade and tumour stage.

OBJECTIVES: We introduce biochemistry as a second dimension to Gleason grading, using Fourier transform infrared (FTIR) microspectroscopy. For the first time, we correlate FTIR spectra derived from prostate cancer (pCA) tissue with Gleason score and the clinical stage of the tumour at time of biopsy. METHODS: Serial sections from paraffin-embedded pCA tissue were collected. One was stained with hematoxylin and eosin and Gleason scored; FTIR spectra were collected from malignant locations using a second unstained section. FTIR spectra, representing different Gleason grades, were used to construct a diagnostic classifier for pCA using linear discriminant analysis (LDA). This model was blind tested using 383 IR spectra from 36 biopsies. RESULTS: Using a three-band Gleason criteria, we obtained sensitivity of > or =70% for the FTIR-LDA model to predict Gleason <7,=7, and >7, with specificities of > or =81%. Using a threshold of Gleason/FTIR-LDA score of > or =8, we obtained a sensitivity and specificity of 71% and 67%, respectively, for the correlation with metastatic tumours using the FTIR-LDA system and 85% and 63%, respectively, for the correlation of metastatic tumours using the Gleason system. CONCLUSIONS: There is a correlation between tissue architecture using Gleason score with tissue biochemistry using FTIR-LDA. Both systems are similar in their performance in predicting metastatic behaviour in tumours from individual patients.

Urban PM2.5 surface chemistry and interactions with bronchoalveolar lavage fluid.

This study investigated the surface chemistry of urban fine particles (PM(2.5)), and quantified the adsorbed and desorbed species after exposure to bronchoalveolar lavage fluid (BALF). Urban background and roadside PM(2.5) samples of different mass concentration and total weight were collected in triplicate in the South Bronx region of New York City. Simultaneously, the concentrations of other atmospheric pollutants (CO, NO(x), SO(2), O(3), elemental carbon) were measured, and weather conditions were recorded. The collected PM(2.5) samples underwent one of three treatments: no treatment, treatment in vitro with BALF, or treatment in a saline solution (control). The surfaces of untreated, saline-treated, and BALF-treated PM(2.5) samples were analyzed using x-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). These results were then compared with ambient air pollutant concentrations, weather variables, selected BALF characteristics, and results from a previous London study conducted using identical preparation methods by XPS analysis only. Both XPS and ToF-SIMS detected PM(2.5) surface species and observed changes in surface concentrations after treatment. XPS analysis showed the surface of untreated urban PM(2.5) consisted of 79 to 87% carbon and 10 to 16% oxygen with smaller contributions of N, S, Si, and P in the samples from both background and roadside locations. A wider variety of other inorganic and organic species (including metals, aliphatic and aromatic hydrocarbons, and nitrogen-containing molecules) was detected with ToF-SIMS. Surface characteristics of particles from the roadside and background sites were very similar, except for higher (p <.05) nitrate concentrations at the roadside, which were attributable to higher roadside NO(x) concentrations. Comparable species and quantities were identified in a previous study of London PM(2.5), where PM(2.5) surface chemistry differed considerably depending on the source, particularly in surface concentrations of oxygen and trace species. After treatment with BALF the N-C signal detected by XPS analysis increased in the average by 372 +/- 203%, indicating significant surface adsorption of protein or other N-containing biomolecules. Lower (nonsignificant) N-C signals were observed for smoker BALF, compared to nonsmoker BALF. ToF-SIMS data confirmed protein adsorption after BALF treatment--smoker BALF resulted in lower levels of adsorbed proteins compared to nonsmoker BALF. ToF-SIMS also indicated an adsorption of phospholipid on the treated PM(2.5) surfaces. The primary phospholipid in BALF is dipalmitoylphospatidylcholine (DPPC), although positive identification was not possible due to low concentrations at the PM(2.5) surface. Oxygen content of PM(2.5) surfaces was the most significant determinant of both N-C and phospholipid adsorption. The XPS signal of the soluble species NH(+)(4), NO(2-)(3), Si, and S decreased in both saline- and BALF-treated samples, showing that these species may be bioavailable in the lung. Similarly, ToF-SIMS analysis suggests the bioavailability of Na(+) and Al(+) as well as NH(+)(4) and Si(+).