RESUMO
Confocal Raman spectroscopy is being assessed as a tool with which to quantify the rate and extent of drug uptake to and its clearance from target sites of action within the viable epidermis below the skin's stratum corneum (SC) barrier. The objective of this research was to confirm that Raman can interrogate drug disposition within the living layers of the skin (where many topical drugs elicit their pharmacological effects) and to identify procedures by which Raman signal attenuation with increasing skin depth may be corrected and normalized so that metrics descriptive of topical bioavailability may be identified. It was first shown in experiments on skin cross-sections parallel to the skin surface that the amide I signal, originating primarily from keratin, was quite constant with depth into the skin and could be used to correct for signal attenuation when confocal Raman data were acquired in a "top-down" fashion. Then, using 4-cyanophenol (CP) as a model skin penetrant with a strong Raman-active C≡N functionality, a series of uptake and clearance experiments, performed as a function of time, demonstrated clearly that normalized spectroscopic data were able to detect the penetrant to at least 40-80 µm into the skin and to distinguish the disposition of CP from different vehicles. Metrics related to local bioavailability (and potentially bioequivalence) included areas under the normalized C≡N signal versus depth profiles and elimination rate constants deduced post-removal of the formulations. Finally, Raman measurements were made with an approved dermatological drug, crisaborole, for which delivery from a fully saturated formulation into the skin layers just below the SC was detectable.
Assuntos
Absorção Cutânea , Análise Espectral Raman , Análise Espectral Raman/métodos , Pele/metabolismo , Epiderme/metabolismo , Disponibilidade Biológica , Microscopia Confocal/métodosRESUMO
Evaluation of the bioavailability of drugs intended to act within the skin following the application of complex topical products requires the application of multiple experimental tools, which must be quantitative, validated, and, ideally and ultimately, sufficiently minimally invasive to permit use in vivo. The objective here is to show that both infrared (IR) and Raman spectroscopies can assess the uptake of a chemical into the stratum corneum (SC) that correlates directly with its quantification by the adhesive tape-stripping method. Experiments were performed ex vivo using excised porcine skin and measured chemical disposition in the SC as functions of application time and formulation composition. The quantity of chemicals in the SC removed on each tape-strip was determined from the individually measured IR and Raman signal intensities of a specific molecular vibration at a frequency where the skin is spectroscopically silent and by a subsequent conventional extraction and chromatographic analysis. Correlations between the spectroscopic results and the chemical quantification on the tape-strips were good, and the effects of longer application times and the use of different vehicles were clearly delineated by the different measurement techniques. Based on this initial investigation, it is now possible to explore the extent to which the spectroscopic approach (and Raman in particular) may be used to interrogate chemical disposition deeper in the skin and beyond the SC.
Assuntos
Pele , Vibração , Animais , Suínos , Pele/metabolismo , Epiderme , Absorção Cutânea , Análise Espectral RamanRESUMO
Stimulated Raman scattering (SRS) microscopy provides rapid label-free 3D chemical imaging with wide-ranging applications including histology, pharmacokinetic studies, and materials characterisation. SRS microscopy has seen a steady increase in utilisation since the early 2000s and has become more accessible due to the increase in availability of facilities, and the development of user-friendly instrumentation. Although some complete SRS systems are now commercially available, many instruments are home-built with highly varied laser sources, optics, and detection mechanisms. Signal intensity is also dependent on the effective spatiotemporal overlap of two (or more) laser beams, thus any drift in alignment can result in variable performance. Currently there is a lack of standard procedures or reference materials for SRS, which has important implications on reproducibility and consistency. These concerns are particularly relevant to the comparison of data from the same instrument at different times and instrument settings as well as between different instruments and laboratories. This tutorial-style review presents the most important practical considerations for sample preparation, instrument set-up, image acquisition and data analysis to obtain reproducible SRS measurements.
Assuntos
Microscopia Óptica não Linear , Análise Espectral Raman , Reprodutibilidade dos Testes , Microscopia Óptica não Linear/métodos , Análise Espectral Raman/métodos , Microscopia , LasersRESUMO
Four different label-free, minimally invasive, live single cell analysis techniques were applied in a quantitative comparison, to characterize embryonic stem cells and the hepatocytes into which they were differentiated. Atomic force microscopy measures the cell's mechanical properties, Raman spectroscopy measures its chemical properties, and dielectrophoresis measures the membrane's capacitance. They were able to assign cell type of individual cells with accuracies of 91% (atomic force microscopy), 95.5% (Raman spectroscopy), and 72% (dielectrophoresis). In addition, stimulated Raman scattering (SRS) microscopy was able to easily identify hepatocytes in images by the presence of lipid droplets. These techniques, used either independently or in combination, offer label-free methods to study individual living cells. Although these minimally invasive biomarkers can be applied to sense phenotypical or environmental changes to cells, these techniques have most potential in human stem cell therapies where the use of traditional biomarkers is best avoided. Destructive assays consume valuable stem cells and do not characterize the cells which go on to be used in therapies; whereas immunolabeling risks altering cell behavior. It was suggested how these four minimally invasive methods could be applied to cell culture, and how they could in future be combined into one microfluidic chip for cell sorting. © 2016 International Society for Advancement of Cytometry.
Assuntos
Hepatócitos/ultraestrutura , Células-Tronco Embrionárias Humanas/ultraestrutura , Gotículas Lipídicas/ultraestrutura , Análise de Célula Única/métodos , Biomarcadores/análise , Diferenciação Celular , Linhagem Celular , Espectroscopia Dielétrica/instrumentação , Espectroscopia Dielétrica/métodos , Eletroforese/instrumentação , Eletroforese/métodos , Hepatócitos/fisiologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Gotículas Lipídicas/fisiologia , Microscopia de Força Atômica/instrumentação , Microscopia de Força Atômica/métodos , Análise de Célula Única/instrumentação , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodosRESUMO
We used three label-free minimally invasive methods to characterize individual cells derived from primary and secondary tumours from the same patient, and of the same type colorectal. Raman spectroscopy distinguished cells by their biochemical 'fingerprint' in a vibrational spectrum with 100% accuracy, and revealed that the primary cell line contains more lipids and alpha-helix proteins, whereas the secondary cell line contains more porphyrins and beta-sheet proteins. Stimulated Raman scattering (SRS) microscopy distinguished cells in chemically-specific images of CH2 bonds which revealed lipid droplets in secondary tumour cells. Atomic force microscopy (AFM) was used to distinguish cells with 80% accuracy by measuring their elasticity secondary tumour cells (SW620) are around 3 times softer than primary ones (SW480). As well as characterizing the physical and biochemical differences between cell lines in vitro, these techniques offer three novel methods which could potentially be used for diagnosis to assign a tumour as primary or secondary.
Assuntos
Gotículas Lipídicas/patologia , Lipídeos/análise , Neoplasias/patologia , Porfirinas/análise , Proteínas/análise , Análise Espectral Raman/métodos , Linhagem Celular Tumoral , Humanos , Gotículas Lipídicas/química , Microscopia/métodos , Microscopia de Força Atômica , Neoplasias/química , Estrutura Secundária de Proteína , Células Tumorais CultivadasRESUMO
The formulation of paediatric medicines faces significant challenges to meet the requirements for safe and accurate administration, while maintaining a suitable taste. Multiparticulate formulations have a strong potential to address these challenges because they combine dose flexibility with ease of administration. Understanding the stability of multiparticulate formulations over storage as a function of time and environmental parameters, such as humidity and temperature, is important to manage their commercialisation and use. In this work, we have expanded the toolkit of available techniques for studying multiparticulates beyond those such as scanning electron microscopy (SEM) and confocal laser scanning microscopy. We include advanced methods of environmentally-controlled SEM to monitor temperature- and humidity-induced changes in-situ, and a variety of Raman spectroscopies including stimulated Raman scattering microscopy to identify and localise the different ingredients at the surface and inside the multiparticulates. These techniques allowed unprecedented monitoring of specific changes to the particulate structure and distribution of individual ingredients due to product aging. These methods should be considered as valuable novel tools for in-depth characterisation of multiparticulate formulations to further understand chemical changes occurring during their development, manufacturing and long-term storage. We envisage these techniques to be useful in furthering the development of future medicine formulations.
RESUMO
Tracking drug disposition in the skin in a non-destructive and at least semi-quantitative fashion is a relevant objective for the assessment of local (cutaneous) bioavailability. Confocal Raman spectroscopy has been shown potentially useful in this regard and, importantly, recent advances have enabled the presence of applied chemicals in the viable epidermis below the stratum corneum (SC) to be determined without ambiguity and having addressed the challenges of (a) background signals from endogenous species and noise and (b) signal attenuation due to absorption and scattering. This study aimed to confirm these observations using a different vibrational spectroscopy approach - specifically, stimulated Raman scattering (SRS) microscopy - and the more conventional in vitro skin penetration test (IVPT). SRS is a nonlinear optical imaging technique which enables more precise location of the skin surface and enhanced skin depth resolution relative to confocal Raman microscopy. The method can also probe larger areas of the sample under investigation and identify the localization of the permeating chemical in specific structural components of the skin. Here, SRS was shown capable of tracking the uptake and distribution of 4-cyanophenol (CP), the same model compound used in the recent confocal Raman investigation, at depths beyond the SC following skin treatment with different vehicles and for different times. The SRS results correlated well with those from the confocal Raman experiments, and both were consistent with independent IVPT measurements. Acquired images clearly delineated CP preference for the intercellular lipid layers of the SC relative to the corneocytes. The stage is now set to apply these and other correlative techniques to examine commercial drug products.
Assuntos
Epiderme , Pele , Pele/metabolismo , Epiderme/metabolismo , Absorção Cutânea , Microscopia Confocal/métodos , Microscopia Óptica não Linear , Análise Espectral Raman/métodosRESUMO
The mechanistic understanding of skin penetration underpins the design, efficacy and risk assessment of many high-value products including functional personal care products, topical and transdermal drugs. Stimulated Raman scattering (SRS) microscopy, a label free chemical imaging tool, combines molecular spectroscopy with submicron spatial information to map the distribution of chemicals as they penetrate the skin. However, the quantification of penetration is hampered by significant interference from Raman signals of skin constituents. This study reports a method for disentangling exogeneous contributions and measuring their permeation profile through human skin combining SRS measurements with chemometrics. We investigated the spectral decomposition capability of multivariate curve resolution - alternating least squares (MCR-ALS) using hyperspectral SRS images of skin dosed with 4-cyanophenol. By performing MCR-ALS on the fingerprint region spectral data, the distribution of 4-cyanophenol in skin was estimated in an attempt to quantify the amount permeated at different depths. The reconstructed distribution was compared with the experimental mapping of CN, a strong vibrational peak in 4-cyanophenol where the skin is spectroscopically silent. The similarity between MCR-ALS resolved and experimental distribution in skin dosed for 4 h was 0.79 which improved to 0.91 for skin dosed for 1 h. The correlation was observed to be lower for deeper layers of skin where SRS signal intensity is low which is an indication of low sensitivity of SRS. This work is the first demonstration, to the best of our knowledge, of combining SRS imaging technique with spectral unmixing methods for direct observation and mapping of the chemical penetration and distribution in biological tissues.
Assuntos
Microscopia Óptica não Linear , Pele , Humanos , Análise Multivariada , Análise dos Mínimos Quadrados , Microscopia Óptica não Linear/métodos , Análise Espectral Raman/métodosRESUMO
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.