Visualisation of drug distribution in skin using correlative optical spectroscopy and mass spectrometry imaging.

A correlative methodology for label-free chemical imaging of soft tissue has been developed, combining non-linear optical spectroscopies and mass spectrometry to achieve sub-micron spatial resolution and critically improved drug detection sensitivity. The approach was applied to visualise the kinetics of drug reservoir formation within human skin following in vitro topical treatment with a commercial diclofenac gel. Non-destructive optical spectroscopic techniques, namely stimulated Raman scattering, second harmonic generation and two photon fluorescence microscopies, were used to provide chemical and structural contrast. The same tissue sections were subsequently analysed by secondary ion mass spectrometry, which offered higher sensitivity for diclofenac detection throughout the epidermis and dermis. A method was developed to combine the optical and mass spectrometric datasets using image registration techniques. The label-free, high-resolution visualisation of tissue structure coupled with sensitive chemical detection offers a powerful method for drug biodistribution studies in the skin that impact directly on topical pharmaceutical product development.

VAMAS interlaboratory study for desorption electrospray ionization mass spectrometry (DESI MS) intensity repeatability and constancy.

A VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study for desorption electrospray ionization mass spectrometry (DESI MS) measurements has been conducted with the involvement of 20 laboratories from 10 countries. Participants were provided with an analytical protocol and two reference samples: a thin layer of Rhodamine B and double-sided adhesive tape, each on separate glass slides. The studies comprised acquisition of positive ion mass spectra in predetermined m/z ranges. No sample preparation was required. Results for Rhodamine B show that very consistent craters may be generated. However, inadequacies of the spray and sample stage designs often lead to variable crater shapes. The average repeatability for Rhodamine B is 50%. Yet, repeatabilities better than 20% can be achieved. Rhodamine B proved to be an excellent reference sample to check the sample erosion crater, the sample stage movement and memory effects. Adhesive tape samples show that their average absolute intensity repeatability is 30% and the relative repeatability is 9%. The constancy of these spectra from relative intensities gives day-to-day average relative repeatabilities of 31%, three times worse than the short-term repeatability. Significant differences in the spectra from different laboratories arise from the different adventitious adducts observed or from contaminants that may cause the higher day-to-day variations. It is thought that this may be overcome by allowing some 20 ppb of sodium to be always present in the solvent, to be the dominating adduct. Repeatabilities better than 5% may be achieved with adequate control.

Three-dimensional cell morphometry for the quantification of cell-substrate interactions.

The initial attachment of cells to biomaterials is an important indicator of longer term cell-substrate biocompatibility. To study and quantify this interaction, we have developed a protocol for measuring temporal changes in the three-dimensional (3D) morphology of mammalian cells seeded onto different substrates using fluorescence confocal laser scanning microscopy and image processing techniques. This method has been used to investigate how morphology parameters, such as cell thickness, volume, and the footprint area, change over time for osteosarcoma cells on uncoated glass control, fibronectin-coated glass, and titanium substrates. Consistent with other studies, our results show that the presence of a fibronectin coating significantly increases the rate of cell spreading, judged by an increase in the cell footprint area and a decrease in cell thickness, indicating enhanced biocompatibility. Using similar criteria, the same cell line was observed to spread faster on titanium than on uncoated glass. We propose that 3D cell morphometry is a valuable multiparametric tool for quantifying initial cell-substrate interactions providing data which has important applications in quality control for ensuring product/batch consistency and for developing tailored surface finishes.

Methodologies for assessing local surface texture features that are relevant to cell attachment.

In this paper, we describe techniques for extracting features from surface topography data, gathered by a 3D-microscopy system, on a length scale that is relevant for cell attachment. The feature parameters considered include standard surface roughness parameters applied to the complete surface as well as new feature parameters designed to quantify local variations in surface topography potentially influencing cell behaviour. Methodologies have been developed both to determine the degree of homogeneity or isotropy of a surface and to compare the topographies of different samples. The approaches followed include wavelet decomposition and linear and nonlinear filtering techniques. The analysis has been used to investigate the correlation between osteoblast cell attachment and structural features of titanium-coated surfaces representative of orthopaedic implants. The results confirm that there is a discernible correlation between cell orientation and the underlying surface lay.

Influence of collagen denaturation on the nanoscale organization of adsorbed layers.

Adsorption (at 37 degrees C) of type I collagen, in native and heat-denatured (30 min at 40 and 90 degrees C) forms, on polystyrene was studied using quartz crystal microbalance with energy dissipation monitoring (QCM-D), atomic force microscopy (AFM) in tapping mode and X-ray photoelectron spectroscopy (XPS). The significance of the parameters deduced from QCM-D data was examined by comparing different approaches. The adsorbed layer of native collagen has a complex organization consisting of a thin mat of molecules near the surface, in which fibrils develop depending on concentration and time, and of a thicker overlayer containing protruding molecules or bundles which modify noticeably the local viscosity. As a result of drastic denaturation, the ability of collagen to assemble into fibrils in the adsorbed phase is lost and the protrusion of molecules into the aqueous phase is much less pronounced. The adsorbed layer of denatured collagen appears essentially as a monolayer of flattened coils. At low concentration, this is easily displaced upon drying, leading to particular dewetting figures; at high concentration, aggregates add to the first layer. Moderate denaturation leads to an adsorbed phase which shows properties intermediate between those observed with native and extensively denatured collagen, regarding the ability to form fibrillar structures and the adlayer thickness and viscosity.

Factors and mechanisms determining the formation of fibrillar collagen structures in adsorbed phases.

The adsorption of collagen (type I from calf skin) was studied, comparing different collagen sources and using substrates which differ according to surface hydrophobicity (polystyrene, either native, with OH substitution of each repeat unit, with COOH substitution of a small fraction of repeat units, or surface modified by oxygen plasma discharge). The atomic force microscopy observation of the adsorbed layers showed that aggregation in the solution acts in competition with the formation of fibrils in the adsorbed phase; more aggregated solutions behave like less concentrated solutions regarding adsorption. The fibrils formed in the adsorbed phase are much smaller than the fibrils formed in the suspension, and, in contrast with the latter, do not show regular band pattern. It is confirmed that fibrils formation occurs more readily on more hydrophobic surfaces, which is tentatively attributed to a greater mobility of individual molecules adsorbed on more hydrophobic substrates. This interpretation is supported by previously published radiochemical measurements. However, the comparison of strongly different adsorption procedures (progressive on the one hand; quick and massive on the other hand) did not provide any additional clue.

Resolution of the vertical and horizontal heterogeneity of adsorbed collagen layers by combination of QCM-D and AFM.

Collagen (type I from calf skin) adsorption on polystyrene (PS) and plasma-oxidized polystyrene (PSox) was studied, using a quartz crystal microbalance with energy dissipation measurements (QCM-D) and atomic force microscopy (AFM) in tapping mode. Radio-labeled collagen was used to measure the adsorbed amount and the ability of adsorbed collagen to exchange with molecules in the solution. The results show that the collagen adlayer consists of two parts: a dense and thin sheet in which fibrils are formed (directly observed by AFM) and an overlying thick layer (up to 200 nm) containing protruding molecules or bundles which are in very low concentration but modify noticeably the local viscosity. The thickness and viscosity of the semi-liquid adlayer both increase with adsorption time and collagen concentration. Fibril formation near the surface also increases with time and collagen concentration and occurs more readily on PS compared to PSox. Radiochemical measurements show that this may be related to the larger mobility of molecules adsorbed on PS, presumably owing to a smaller number of binding points.