Applications of Raman spectroscopy in skin research--From skin physiology and diagnosis up to risk assessment and dermal drug delivery.

Advanced Drug Delivery Reviews 89 (2015) 91–104

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Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr

Applications of Raman spectroscopy in skin research — From skin physiology and diagnosis up to risk assessment and dermal drug delivery☆ Lutz Franzen a, Maike Windbergs a,b,c,⁎ a b c

Saarland University, Department of Biopharmaceutics and Pharmaceutical Technology, Saarbruecken, Germany Helmholtz Centre for Infection Research, Helmholtz Institute for Pharmaceutical Research Saarland, Department of Drug Delivery, Saarbruecken, Germany PharmBioTec GmbH, Saarbruecken, Germany

a r t i c l e

i n f o

Available online 11 April 2015 Keywords: Confocal Raman microscopy Dermal drug delivery Skin physiology Skin disease diagnosis In vitro skin models

a b s t r a c t In the field of skin research, confocal Raman microscopy is an upcoming analytical technique. Substantial technical progress in design and performance of the individual setup components like detectors and lasers as well as the combination with confocal microscopy enables chemically selective and non-destructive sample analysis with high spatial resolution in three dimensions. Due to these advantages, the technique bears tremendous potential for diverse skin applications ranging from the analysis of physiological component distribution in skin tissue and the diagnosis of pathological states up to biopharmaceutical investigations such as drug penetration kinetics within the different tissue layers. This review provides a comprehensive introduction about the basic principles of Raman microscopy highlighting the advantages and considering the limitations of the technique for skin applications. Subsequently, an overview about skin research studies applying Raman spectroscopy is given comprising various in vitro as well as in vivo implementations. Furthermore, the future perspective and potential of Raman microscopy in the field of skin research are discussed. © 2015 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. State-of-the-art analytics in skin research. . . . . . . . . . . . 1.2. Optical methods in skin research . . . . . . . . . . . . . . . The potential of Raman spectroscopy for skin research . . . . . . . . . 2.1. The Raman effect . . . . . . . . . . . . . . . . . . . . . . 2.2. Confocal Raman microscopy (CRM) . . . . . . . . . . . . . . 2.3. Analytical techniques based on the Raman effect . . . . . . . . 2.3.1. Surface and tip enhanced Raman spectroscopy . . . . . 2.3.2. Coherent anti-Stokes and stimulated Raman spectroscopy 2.4. Challenges for confocal Raman microscopy in skin research . . . Applications of Raman microscopy in skin research . . . . . . . . . . 3.1. In vitro applications for human skin . . . . . . . . . . . . . . 3.1.1. In vitro analysis of human skin physiology . . . . . . . 3.1.2. In vitro penetration studies on human skin . . . . . . . 3.1.3. In vitro skin diagnosis . . . . . . . . . . . . . . . . 3.2. In vitro skin models . . . . . . . . . . . . . . . . . . . . . 3.2.1. Snake skin models . . . . . . . . . . . . . . . . . . 3.2.2. Porcine skin models . . . . . . . . . . . . . . . . .

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Pharmaceutical applications of Raman spectroscopy — From diagnosis to therapeutics”. ⁎ Corresponding author at: Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, Campus A4.1, 66123 Saarbruecken, Germany. Tel.: +49 681 302 4763; fax: +49 681 302 4677. E-mail address: [email protected] (M. Windbergs).

http://dx.doi.org/10.1016/j.addr.2015.04.002 0169-409X/© 2015 Elsevier B.V. All rights reserved.

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L. Franzen, M. Windbergs / Advanced Drug Delivery Reviews 89 (2015) 91–104

3.2.3. Tissue engineered skin models. . . . . . . . . . 3.2.4. Lipid-based skin models . . . . . . . . . . . . 3.3. In vivo investigations on human skin . . . . . . . . . . . 3.3.1. Instrumental development for in vivo experiments 3.3.2. In vivo analysis of skin physiology . . . . . . . . 3.3.3. In vivo skin penetration studies . . . . . . . . . 3.3.4. In vivo skin diagnosis . . . . . . . . . . . . . . 4. Conclusions and future perspective . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction 1.1. State-of-the-art analytics in skin research The skin forms the outermost biological barrier between the human body and the external environment. It consists of different layers exhibiting individual composition and physiology. The main barrier function is constituted by the stratum corneum as the outermost skin layer, comprising cornified cells which are surrounded by a coherent lipid matrix. Deeper skin layers are constituted by the viable epidermis with the basal membrane as well as by the dermis, where appendices like sweat glands and hair follicle are located. Due to the complex assembly and diverse functions of the skin, skin related research is focused on a variety of different aspects. The scientific interest ranges from the elucidation of the biochemical composition and physiological functions up to the diagnosis of pathological skin states. Based on such basic research studies, the scientific interest proceeds towards the discovery of strategies to influence and manipulate physiological functions and processes in the skin. In this context, skin absorption and permeation processes are in the focus of interest. On the one hand, this interest stems from the necessity to evaluate the absorption of harmful substances for risk assessment and safety studies. On the other hand, skin absorption processes can be used to deliver substances via the skin for cosmetic, protective or therapeutic purposes. While the cosmetic delivery is mainly based on local treatment, the therapeutic application aims either for local delivery to the skin as a target organ or for systemic delivery by crossing the skin as a barrier to reach the systemic blood circulation. Rational development of drug delivery systems for targeted application of molecules to the skin requires accurate knowledge of absorption mechanisms and transport kinetics in skin tissue. Thus, for investigation and evaluation of novel skin therapeutics, suitable analytical techniques are essential prerequisites. The in vivo evaluation of rate and extent of the penetration behavior of a substance into the skin is usually performed by tape stripping. After application of a substance to the skin, the tape stripping procedure includes a layer-by-layer removal of the stratum corneum by means of adhesive tapes with subsequent extraction and quantification of the applied substance most commonly achieved by HPLC. In contrast, the in vivo skin permeation of a substance in vivo can directly be determined by its serum concentrations in the systemic blood circulation. However, this approach does not elucidate information about metabolism and permeation pathways within the skin tissue. Furthermore, in vivo studies with human volunteers are limited to therapeutic substances and are not suitable for risk assessment. Moreover, as in vivo diagnosis of skin related diseases solely based on visual inspection is often challenging, diagnostic procedures frequently depend on the removal of biopsies of the affected skin areas. Most commonly, the final diagnostic decision is made after subsequent histological in vitro analysis. For in vitro penetration studies, excised skin samples are subjected to tape stripping in combination with further tissue segmentation and extraction of the deeper skin layers allowing depot assessment of an applied substance or monitoring substance degradation in the viable epidermis and dermis. For in vitro permeation experiments, the Franz-

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diffusion cell is the most common analytical setup. The cell consists of a donor and an acceptor compartment which are separated by an excised skin sample. Substance permeation can be analyzed by investigating the donor compartment, whereas the remaining substance can subsequently be extracted from the excised skin sample. However, all these techniques are destructive as well as labor intense and therefore restricted. Additionally, the supply of excised mammal skin is limited and at the same time high inter- and intraindividual variability in skin samples demands high numbers of experiments. Against this background, there is a strong need for non-destructive analytical techniques to reduce sample size in vitro as well as to allow more comprehensive in vivo analysis. In this context, non-destructive optical methods with immediate analytical readout, such as fluorescence, Infrared (IR) and Raman spectroscopy show the potential to fill this scientific gap.

1.2. Optical methods in skin research Great progress in optics, computer sciences and electronic engineering facilitated the rise of optical methods in all fields of research. The immediate analytical readout and the contact-free implementation gave rise to the transfer of these techniques to skin research. Since the late 90s, several optical methods have been implemented in pathophysiological skin diagnosis, physiological tissue evaluation as well as permeation and penetration studies in vitro and in vivo [1,2]. In this context, optical coherence tomography (OCT), an interferometric technique based on infrared light scattering, provides images of the skin with micrometer resolution and has already widely been used in diagnosis of skin diseases [3,4]. However, the inadequate spatial resolution and the restriction to solely visualize the macroscopic skin structure constrain OCT. Recently, microscopy based techniques like confocal laser scanning microscopy (CLSM) and multiphoton microscopy (MPM) have been used to track substances within the skin and to explore in vivo and in vitro skin physiology [5,6]. These techniques provide information about dermal penetration depth and molecular substance interaction with the skin tissue with high spatial resolution. Although, MPM showed good results by monitoring the skin's autofluorescence and represents a powerful tool in dermatological imaging [7], most tracking procedures require labeling of the substances of interest with fluorescent dyes. Unfortunately, the labeling is prone to change the physicochemical properties of the substances, thus affecting transport within and interaction with biological tissues. Therefore, transferability of such data to the in vivo situation in the human body is impaired. In contrast, vibrational spectroscopy techniques can provide direct molecular information of a sample without labeling. Infrared spectroscopy (IR), an optical technique based on laser light absorption is the widest spread technology in this particular field. In skin research, near-IR spectroscopy has successfully been applied in vivo [8,9] and in vitro [10,11] to track substances within the skin by chemically selective depth profiling and mapping. Unfortunately, the resolution of IR measurements is limited to 5–10 μm and water interferes with IR


measurements, since asymmetric OH-vibrations are resulting in strong absorption bands. In this context, Raman spectroscopy as a complementary analytical technique to IR spectroscopy bears the advantage of spatial resolution below 1 μm and no interference by strong water signals. Additionally, Ali et al. [12] reported higher spectral details in Raman spectra compared to IR, independent of instrumentation factors. In summary, it can be concluded, that Raman spectroscopy provides a high potential for the analysis of biological tissues such as skin. 2. The potential of Raman spectroscopy for skin research

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molecules involve aromatic structures, they tend to be good scatterers, exhibiting strong characteristic Raman bands. This makes them easy to detect, even in small amounts. For biological tissues such as the skin, the contrary scenario applies. The high variability in chemical composition, and therefore in functional groups, leads to highly complex Raman patterns with strong background and weak characteristic peaks. As only 1 out of 107 scattered photons is considered as Raman scattering, utilizing the Raman effect for spectroscopic analysis of biological tissue demands highly potent detectors and sophisticated optical engineering. Only recently, technical progress led to an increase in sophisticated and commercially available Raman instrumentations and opened the door for broader application in skin research.

2.1. The Raman effect 2.2. Confocal Raman microscopy (CRM) The Raman effect is based on the scattering of light and was reported for the first time in 1928 by the Indian physicist Sir C.V. Raman [13]. When light interacts with matter, different optical phenomena can occur, among them are transmission, absorption and scattering as a rather weak effect. If the light–matter interaction is considered to be a collision on a molecular basis, this collision can be either elastic or inelastic. In case of an elastic collision, no energy changes occur and scattered photons are detected at the same frequency as incident photons. This type of scattering is called Rayleigh scattering. In contrast, Raman scattering involves inelastic collisions causing energy changes. If the scattered photons loose energy compared to the incident photons, Stokes scattering occurs, the contrary process is called anti-Stokes scattering. Fig. 1A depicts a diagram indicating the energy levels of molecular vibrations and the corresponding scattered photons. Compared to the incident light, Stokes scattering is detected at longer wave lengths (lower energy), whereas anti-Stokes scattering is detected at shorter wave lengths (higher energy). A Raman spectrum is generally displayed by the wave number shift from incident to scattered photons against the scattering intensity, bearing the advantage that the correlation of wave number, frequency and thus energy is linear. The patterns of wavelength shifts after irradiation of a sample with laser light are generally a unique characteristic for a specific chemical structure, comparable to a molecular fingerprint. Thus, Raman spectroscopy can be used as a chemically selective analytical technique without the necessity to use labels for detection. This entails a linear correlation of the scattering intensity and the quantity of excited functional groups, offering the possibility for quantitative measurements with Raman microscopy. Mostly, strong bands in a Raman spectrum correspond to non-polar functional groups or aromatic rings in the Raman active compound, because large changes in polarizability are generally connected with molecules having large delocalized electron systems. As most small drug

The combination of Raman spectroscopy and confocal microscopy opened up new possibilities for the acquisition of spatially resolved Raman information. Fig. 1B exhibits an exemplary schematic drawing of a conventional confocal Raman microscope. An analytical setup of a confocal Raman microscope usually consists of a laser source, a confocal microscope and a spectrometer with a camera as the detection unit. The laser beam is focused through the confocal microscope allowing the laser light to interact with the sample positioned on the microscopic stage. The scattered photons can likewise be collected by the objective and detected by a spectrometer. Cameras usually equipped with charge-coupled devices visualize scattering patterns of the sample as Raman spectra. The different components are connected via optical fibers enabling high flexibility in set-up assembling. In a confocal setup, a pinhole rejects signal from the out-of-focus region. Therefore, the laser beam can be focused on one particular focal plane, only gaining spectral information from one volumetric pixel. Combined with an automated sample positioner, spectra can reproducibly be obtained from any sample spot in all three dimensions. This allows a broad panel of different measurement modes. Single Raman spectra provide information about chemical composition at the exact measured spot. Assemblages of single spectra can be combined to gain insight in changes in spectral bands dependent on the spatial position. In a one dimensional approach, a series of single spectra is collected in different depths inside the sample. These depth profiles provide information about spectral changes with increasing depth. The corresponding Raman profiles display relative or absolute changes in specific peak intensities plotted against the depth. Two dimensional series of data acquisition can be assembled to spectral maps. Therefore, a defined plane in either x–y or x–z direction is measured step by step collecting single spectra. Depending on data processing, a Raman map can compile peak intensities

Fig. 1. A. Energy level diagram illustrating different types of scattering, line thickness corresponds to probability of appearance. B. Schematic of a confocal Raman microscopic setup, red lines indicate the way of excitation laser light and gathered Raman scattering.

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as pixel brightness and depict the distribution of different chemical components in different colors. These Raman images are an important visualization tool for sample composition and component distribution. Three dimensional analysis is possible by gathering multiple Raman maps generating a picture stack with spectral information from every spatial point. However, this technique is rarely used due to its time consuming measurements and the necessity for advanced data processing. Fig. 2 illustrates three examples of measurement modes and data visualization by Raman microscopy. In confocal Raman microscopic imaging, the spatial resolution is limited by the laser spot size. Derived from the point spread function, lateral resolution (LR) can be estimated as LR = 0.82λ/NA (NA = numerical aperture of the objective). For the axial resolution (AR), the refractive index of the immersion medium (n) has additional influence: AR = 2.2nλ/π(NA)2 [14]. In state-of-the-art Raman instruments, resolution can be down to 200 nm in lateral direction and about 500 nm in depth of field. After recording of Raman spectra, further data processing is necessary. A removal of cosmic ray related peaks is automated by most measurement software. In addition, adjusting the spectral baseline is required especially for samples with high background noise. As this is most likely the case for biological samples, the application of polynomic fitting functions to describe the baseline became an important task for user and software. 2.3. Analytical techniques based on the Raman effect 2.3.1. Surface and tip enhanced Raman spectroscopy Besides confocal Raman microscopy solely relying on spontaneous Raman scattering, a variety of related techniques is based on the

Raman effect. Another linear method most commonly applied is surface enhanced Raman spectroscopy (SERS). Using SERS, adsorbents on particular surfaces pronounce a higher Raman signal compared to conventional spontaneous Raman spectroscopy. In principle, the excitation frequency matches the plasmon resonance of the surface atoms of such adsorbents and therefore the intensity of incident as well as of scattered light is increased. The localized surface plasmon resonance is an electromagnetic wave, which propagates along a metal surface on the boundary to the external medium. This electromagnetic field enhances intensity of incident as well as retrieved light. With both effects contributing, the enhancement factor can be 1010 to 1011, therefore allowing even single molecule detection [15]. Since scattering can only occur when the collective plasmon oscillation is perpendicular to the surface, rough surfaces like aggregates of nanoparticles are preferential for SERS measurements. Most common surface materials are gold and silver, since their surface plasmon resonance frequency corresponds to visible and near infrared light [16]. An advancement of SERS is tip enhanced Raman spectroscopy (TERS). TERS minimizes the enhancing surface to a nano scaled area by attaching the surface material to a conventional scanning probe microscopy tip, as used for atomic force microscopy. The strongly enhanced Raman signal is only obtained from this area, which is significantly smaller than the laser spot. This special procedure allows overcoming the resolution limits of CRM and a lateral resolution down to 2 nm has been reported using TERS [17]. Despite the advantages in resolution and signal intensity, both techniques are rarely used in dermal research. The complex instrumentation and a contact requiring measurement mode restrain SERS and TERS mainly to chemistry and physics laboratories. However, the recent development of SERS fiber probes indicates their future potential for skin research [18].

Fig. 2. A. Depth profiling: Raman spectra are recorded along a z-directed line, peak intensities are depicted as intensity profiles against depth. B. y–z intensity mapping: in x–z direction acquired Raman spectra are presented by color coded peak intensities in a two dimensional picture creating a virtual cross section. C. Z-stack false color imaging: series of x–y maps are stacked to a three dimensional data set, different components are assigned to different colors.


2.3.2. Coherent anti-Stokes and stimulated Raman spectroscopy Stimulated Raman spectroscopy (SRS) and coherent anti-Stokes Raman spectroscopy (CARS) are non-linear imaging techniques, which employ the coupling of two laser beams to achieve resonance conditions between the exciting light and the targeted molecules. This allows fast data acquisition and imaging speed close to video rate [19,20]. SRS signals are detected as loss or gain in intensity of one of the exciting lasers. This leads to a linear dependence of concentration of probed molecules and signal intensity for SRS. In a CARS setup quantification is significantly more difficult. Molecule concentration and signal intensity of the distinctly excited light scattering, which is detected at a shifted frequency have a complex quadratic dependence. Nevertheless, sophisticated excitation and data interpretation arrangements are supposed to allow quantitative interpretation of CARS signals [21]. While CARS is already broadly implemented in biomedical research, the more recent instrumentation of SRS was only used for first pilot studies on skin samples. Most studies employing CARS focus on the imaging of lipids within the skin structure and especially the stratum corneum [22–24] and sebaceous glands [25]. Further, the combination with multiphoton imaging allows in vivo instrumentation, tackling clinical applications [26–29]. For SRS, first studies were conducted on skin penetration focusing on imaging of drugs or chemicals inside skin samples. Saar et al. [30] used SRS to image the penetration of ketoprofen in deuterated polyglycol down to 15 μm in mice skin. Furthermore, Belsey et al. [31] visualized the occurrence of drug crystals on the skin surface after application of a close-to-saturation solution of ibuprofen in propylene glycol. 2.4. Challenges for confocal Raman microscopy in skin research The application of confocal Raman microscopy in skin research bares many challenges. As biological tissue, skin composition and structure are highly complex and variable. Since Raman scattering is unique for particular chemical functional groups, the Raman spectra acquired from skin are equally complex and diverse. This tremendous intraand interindividual variability demands a sufficient number of repetitions for all skin related studies. To gain feasible spectral information, rather long acquisition times are needed. This prolongation of the skin exposure to the laser radiation increases the potential for thermal damage. Therefore, an accurately defined laser power balancing between signal intensity and the risk of burning is required [32]. During time consuming in vitro analysis of skin samples, ongoing diffusion, microbial growth and changes in the skin hydration state exacerbate Raman analysis. However, Franzen et al. [33] introduced the possibility to freeze dry excised skin samples overcoming these obstacles. Chemical changes in the skin due to the lyophilization could successfully be excluded and spectral similarity to Raman spectra acquired on human skin in vivo was proven. In addition, freeze drying of skin samples was evolved to an analytical option allowing the quantitative detection of a model drug within skin tissue by Raman microscopy [34]. Furthermore, the skin exhibits a certain autofluorescence covering the entire spectral width and therefore burying most Raman signals. Na et al. [35] reported, that excitation in the visible spectral range facilitates the occurrence of a highly fluctuating fluorescence. To collect reliable and reproducible Raman data, incitement of the skin's fluorescence needs to be avoided at all costs. Shifting the excitation from the visible to the near infrared wavelength region is currently considered the best option. With some background fluorescence remaining, adjustment of the spectral baseline becomes a necessary prerequisite for valid evaluations. Silveira et al. [36] compared background subtraction by discrete wavelet transform and polynomial fit. Both filtering methods did not alter the discrimination of basal cell carcinoma and normal skin. Ramirez-Elias et al. [37] characterized the contribution of fluorescence-to-noise removal algorithms in Raman spectroscopy of biological tissue. Wang

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et al. [38] described photobleaching as potential method to reduce tissue autofluorescence and increase signal-to-noise ratio in human skin. Bonnier et al. [39,40] pointed out the advantages of water immersion and evaluated experimental features such as photobleaching for background reduction. According to their results, they raised the question whether the skin's fluorescence is due to diffuse scattering rather than to intrinsic photoluminescence when using a wavelength of 785 nm. Near infrared light also exhibits best penetration depth in skin tissue, allowing data collection from within the skin while higher frequencies are limited to the surface region [41,42]. Everall et al. [43–45] summarized pitfalls and uncertainties in designation of depth resolution and depth determination in profiling with confocal Raman microscopy. They highlighted the influence of numerical aperture and refractive index matching and concluded a degradation of resolution for deep buried layers. Mendelssohn et al. [46] used polymer films to mimic the sequence of refractive indices in skins. Multivariate and univariate analyses led to an underestimation of exact depth determination by 11–16%. Based on the mathematical correction models of Everall [47], Batchelder [48] and Sourisseau [49], the group of Manfait corrected their depth profiling data towards accurate depth determination [50]. Besides exact depth determination, the signal attenuation in deeper layers represents a major challenge in depth profiling. When the laser spot enters a non-transparent sample in z-direction, incident and collected light intensity is weakened. Fig. 3 schematically depicts the effect of Raman signal attenuation on Raman peak intensity upon depth profiling. So far, this issue has been bypassed by correlating the drug related peak to a general skin derived peak. In a systematic investigation, Franzen et al. [51] reported a loss of the linear correlation between Raman signal intensity and substance concentration due to Raman signal attenuation. Furthermore, they tested the peak correlation method for its suitability for quantitative depth profiling in human skin. They discovered that only the most intense and unspecific Raman peak representing the C–H vibration is suitable as internal standard. An alternative solution to this challenge was offered by Franzen et al. [52], who developed a mathematical approach to correct depth profiling data for signal attenuation. A skin surrogate was constructed with homogenous composition and similar optical properties to human skin. The signal attenuation inside this surrogate was described and a correction algorithm could be successfully applied on a set of depth profiles. The described challenges represent key features for successful application of confocal Raman microscopy and are not only limited to skin research. Though some issues have already successfully been addressed, these basic problems still have to be considered upon reliable future deployment of CRM. 3. Applications of Raman microscopy in skin research 3.1. In vitro applications for human skin 3.1.1. In vitro analysis of human skin physiology For skin absorption testing, in vitro tests are often performed based on excised human skin, as the barrier function of such skin samples is similar compared to human skin in vivo. Two major sources for excised human skin are commonly used. The highest availability is given by cadaver skin, removed from deceased humans. Limited freshness and potential alterations by ongoing decomposition after death confines its suitability. Preferred source for research is human skin obtained from plastic surgery. Minimal changes compared to viable status are guaranteed by direct usage after excision and storage in frozen state. However, the gathered skin pieces are rather small and sources are limited. Fig. 4 depicts Raman spectra from human skin acquired in vivo and in vitro. The spectra were taken from the stratum corneum of a healthy female volunteer in vivo and excised female abdominal skin obtained from plastic surgery. The very few detectable differences highlighted in Fig. 4 are most likely related to lipid residues on the skin surface,

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Fig. 3. Schematic explanation of Raman signal attenuation. A. Basic principle of depth profiling in skin. B. Raman spectra recorded along the red line in A. C. Intensity depth profile of a selected Raman peak highlighted in B. With ongoing depth, the Raman signal decays due to a reduced yield of collected photons.

which are unavoidable during excision. This underlines the suitability of excised human skin as gold-standard in vitro model. First investigations of excised human skin by Raman microscopy where made in the early 90s by evaluating the physiology in comparison to IR spectroscopy in vitro. The group of Barry, Edwards and Williams [53,54] used excised human cadaver skin and isolated stratum corneum to prove the suitability of Raman analysis for skin research and to assign the individual bands of the acquired Raman spectra to the corresponding endogenous skin components. Table 1 sums up the most specific bands recognized in Raman spectra obtained from human skin. Based on these results, Williams, Edwards and Barry [55] analyzed the skin decomposition of the about 5200-year-old iceman, which was found in South Tyrol. The mummy's skin showed protein degradation, oxidized olefinic bonds but unaltered lipids compared to freeze dried contemporary skin samples.

Fig. 4. Raman spectra obtained from human skin in vivo (red) compared to excised human skin in vitro (black).

Table 1 Assignment of spectral bands in a typical Raman spectrum of healthy human skin [54]. Raman shift [cm−1]

Assignment

526 600 623 644 746 827 850 883 931 956 1002 1031 1062 1082 1126 1155 1172 1244 1274 1296 1385 1421 1438 1552 1585 1652 1743 1768 2723 2852 2883 2931 2958 3050 3280

ν (SS) ρ (H) ν (CS) ν (CS) amide 4 ρ (CH2) in phase δ (CCH) aliphatic δ (CCH) aromatic δ (CH2), ν (CC), ν (CN) ρ (CH3) terminal, ν (CC) α-helix ρ (CH3), δ (CCH) alkenic ν (CC) aromatic ring ν (CC) skeletal cis ν (CC) skeletal trans ν (CC) skeletal random ν (CC) skeletal trans ν (CC), δ (COH) ν (CC) δ (CH2) wagging, ν (CN)amide 3 disordered ν (CN), δ (NH) amide 3 α-helix δ (CH2) δ (CH3) symmetric δ (CH3) δ (CH2) scissoring δ (NH), ν (CN) amide 2 ν (C_C) alkenic ν (C_O) amide 1 α-helix ν (C_O) amide 1 lipid ν (COO) ν (CH) aliphatic ν (CH2) symmetric ν (CH2) asymmetric ν (CH3) symmetric ν (CH3) asymmetric ν (CH) alkenic ν (OH) of H2O

ν = stretch, ρ = rock, δ = deformation.


The same group made a comprehensive analysis of keratotic tissues: skin, callus, hair and nail. They were able to detect differences in the sulphur content and further structural differences attributable to variations in the amino acid [56]. Recently, Ali et al. [57] investigated the suitability of preparation techniques for human skin tissue sections. Unprocessed and formalinfixed, paraffin-processed and subsequently dewaxed skin samples from human cadaver hand and tight were compared. The study revealed that dewaxing also removed most of the stratum corneum lipids, impeding further differentiation between the anatomical sites. The skin hydration status was in focus of a study by Vyumvuhore et al. [58], who exposed isolated stratum corneum to atmospheres of different humidity. The influence of the ratio between bound and unbound water on lipid and protein organization indicated direct correlations of the skin barrier function and its hydration status. GasiorGlogowska et al. [59] combined mechanical testing of full thickness skin obtained from cadaver thighs and Raman spectroscopy to investigate structural changes during uniaxial stress applied on the skin samples. In another study, Vyumvuhore et al. [60] correlated exterior applied stretching forces to changes in protein and lipid conformation of isolated SC. The disorganization in skin structure reached a plateau after approximately 9% deformation. This again indicated an exterior influence on the SC barrier function detectable by Raman spectroscopy. The presented studies in this chapter show the versatility of different applications for in vitro skin analysis by Raman microscopy ranging from the first initial studies performed on human skin, on which numerous current studies are based on, up to valuable analytical insights into the skin of mummies in the field of archeology. 3.1.2. In vitro penetration studies on human skin As explained earlier, confocal Raman microscopy allows not only measurements on the skin sample surface, but also the acquisition of spectra series in z-direction. This option of depth profiling is of special interest in skin research, since it allows non-invasive evaluation of penetration behavior of substances. Anigbogu et al. [61] used this measurement mode to investigate the mechanism of skin penetration enhancement by dimethyl sulfoxide. They observed a transition from α-helical to β-sheet structure in stratum corneum keratin and an increased fluidity in lipid organization. Lately, Adlhart and Baschong [62] analyzed the penetration behavior of an active ingredient from sunscreens. The volar forearms of three volunteers were incubated with an organic UV absorber and tape stripping was performed after a certain incubation time. Raman imaging was performed on the surface of the tape strips, visualizing the sunscreen in the network of folds and furrows after removal from the skin surface. A study by Tfayli et al. [63] used excised human skin from plastic surgery to follow-up metronidazole absorption. The specific Raman bands of metronidazole in 2-(2-ethoxyethoxy)ethanol, a penetration enhancing formulation, could be detected down to 40 μm deep in the skin and relative penetration profiles correlated drug amount to depth. To verify the results obtained from the non-destructive depth profiling, tissue slices were acquired and metronidazole could be detected in similar depths with respect to the incubation time. In a subsequent study, Tfaili et al. [64] performed real time depth profiling of caffeine solution in excised human skin. Although experimental limitations like drug crystallization during measurements were exposed, Raman spectroscopy was successfully used to follow up caffeine down to 12 μm depth within the intact skin tissue in a non-destructive setup. Ashtikar et al. [65] used Raman imaging to visualize the penetration and distribution of deuterated water and beta-carotene. Although the acquisition of virtual x–z cuts was impeded by the skin's autofluorescence, a homogenous spreading of the labeled water became visible, while beta-carotene penetration was limited to the top 10 μm of the stratum corneum. In a systematic approach to assess CRMs' potential for direct quantitative measurements, Franzen and Windbergs [66] evaluated the biovariability of Raman signal intensities in skin samples obtained

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from different donors. They stated no detectable difference between intra- and interindividual variability. Furthermore, they evaluated different endogenous skin derived Raman peaks regarding their suitability as internal reference for substance depth profiling. In vitro depth profiling is an essential method for drug delivery evaluation and risk assessment. In this field of research, CRM provides many advantageous features like contact- and label-free measurement and minimized sample sizes. The studies mentioned above already indicate the versatile usage of CRM but also exhibit severe drawbacks that were partly addressed in the chapter on challenges for CRM. However, with the opportunity of quantification CRM might complement or even replace state-of-the-art in vitro depth profiling in the near future. 3.1.3. In vitro skin diagnosis Besides the exploration of skin physiology and substance penetration, Raman spectroscopy has gained considerable interest for investigation of pathophysiological states. In this context, an impressive panel of diagnostic applications has already been established [67]. Specifically, the in vitro evaluation of tissue biopsies taken from potential cancerous human skin has been deeply employed by several groups. Gniadecka et al. [68,69] compared the Raman spectra of melanoma, pigmented nevi, cell carcinoma, seborrheic keratoses, and normal skin. Less intense amid I and increased lipid Raman bands allowed the implementation of neural network analysis as an automated discrimination tool with 85% sensitivity and 99% specificity. Nijssen et al. [70,71] succeeded in discrimination of basal cell carcinoma (BCC) from their surrounding tissue. Raman mapping was compared to the state-of-the-art staining methods. Cluster analysis was able to differentiate between dermis and BCC, but was troubled to detect differences between epidermis and BCC. Furthermore, some findings indicated a reduction of collagen in nodular BCC surrounding dermis. This finding was later confirmed by Short et al. [72]. In their study they did not only detect lower collagen ratios in tumor neighboring dermis, but could also assign structural alterations in the remaining collagen. Moreover, different compositions in the nuclei of tumor cells and healthy epidermis cells could be observed by the means of Raman spectroscopy. Lieber et al. [73] was able to discriminate between the different cancer types, BCC, squamous cell carcinoma (SCC), melanoma and normal skin after analyzing a broad panel of biopsies. Larraona-Puy et al. [74] compared an automated Raman spectra based discrimination model between BCC and hair follicles with trained histopathologists. With 90% sensitivity and 85% specificity, the detection with the automated Raman system is in the same range as the histopathologic evaluation by human eye. Recently, Silveira et al. [75] refined a computerized discrimination model by inputting data from selected tissue compounds. This advanced classification model based on the single component spectra of collagen III, elastin, actin, triolein and melanin has proven its suitability for detection of BCC and melanoma. With a tight focus on skin cancer, Raman spectroscopy was implemented by various groups to detect and distinguish cancer cells in excised tissue. Hereby, the challenge was to establish and optimize an automated data evaluation algorithm. The abovementioned progress already allows clinical application, but further improvements in cost effectiveness and simplified handling are necessary to provide a serious alternative to established histophatologic methods. 3.2. In vitro skin models 3.2.1. Snake skin models The limited availability of excised human skin in quality and quantity requests suitable alternatives. In pharmaceutical research, animal models are usually the first choice to replace human testing. In the beginning of the 90s, snake skin was in the center of attention as a potential model for the human skin. With its wide availability and the absence of ethical and hazardous restrictions, shed snake skin bares many

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advantages for in vitro testing. Barry, Williams and Edwards [76–78] evaluated the Raman spectra of different snake skins and compared it to mammalian skin samples. Besides a significant autofluorescence, only little difference was detected between different snake species. Comparison to human and porcine skin pointed out the anatomical discrepancies and variability in permeation, due to less ordered lipids in snake skin. Edwards, Hunt and Sibley [79] compared the Raman spectra of mammalian and reptilian keratotic materials. Keratin spectra of bovine hoof, Texas Longhorn cattle horn, kudu horn, tortoiseshell and human finger nail were evaluated and differences indicated mainly α-helical formation in mammalian and predominant β-sheet structures in reptilian skin. These investigations highlighted the difference between human and reptile skin regarding composition and structure. This impedes its comparability to human skin and limits its applicability as in vitro model explaining the rare usage of reptile skin in today's skin research.

Xiao et al. [87,88] combined IR imaging with Raman spectroscopy to follow up liposomes made from different deuterated lipids into excised pig skin. IR imaging was performed on histological skin sections, while CRM provided spatial information by non-destructive depth profiling. One lipid could be detected in depth of 25–35 μm, whereas another lipid penetrated 15 μm into the skin in 24 h. Further investigations showed that vesicles made of other lipid components did not overcome the skin barrier in their intact structure. Mao et al. [89] applied IR and Raman spectroscopy on human and porcine skin to compare the penetration of deuterated sodium dodecyl sulfate. Sections were mapped by Raman and IR microscopy and the peaks representing the carbon–deuterium stretching vibrations could clearly be visualized. On hairless pig ribs, less ordered lipids are associated with a slightly deeper penetration than in the human abdominal skin.

3.2.2. Porcine skin models 3.2.2.1. In vitro physiological evaluation of porcine skin. The pig as a mammalian animal is considered as the closest animal model for human skin. Therefore, several studies already concentrated on Raman analysis of porcine skin for penetration and permeation testing [80,81]. Tfaili et al. [82] compared the Raman spectral features of excised human and porcine skin. Although notable differences were pointed out, a general comparability of human and porcine Raman spectra was concluded. Several Raman microscopic investigations evaluated the physiology of excised pig skin. Wu and Polefka [83] moistened porcine skin sheets in controlled humidity's and acquired water content depth profiles. The reference method Karl Fischer titration showed a good correlation with the Raman data. Finally, a panel of different cosmetic and cleaning products was evaluated regarding their moisturizing abilities. Forster et al. [84] evaluated the completeness of stratum corneum removal by tape stripping, cyanoacrylate biopsies and trypsinization. In confocal Raman depth profiles, the water content within the individual skin samples could be correlated to the stratum corneum–epidermis border. This allows a non-invasive estimation of the stratum corneum thickness. After the former mentioned procedures, the remaining stratum corneum was detected by Raman microscopy and verified by histological cuts. For the standard tape stripping procedure, an inhomogeneous and incomplete removal of the stratum corneum could be verified by both methods. Franzen et al. [85] utilized Raman imaging to visualize cross sections of porcine hair follicles as potential targets for drug delivery. The anatomical composition of the follicle could be illustrated without any markers or dyes and a spectral comparison hinted towards the suitability of porcine hair follicles to mimic the situation in humans. Furthermore, the standard analytical technique of cyanoacrylate biopsies was evaluated by combining confocal Raman microscopy and optical profilometry. Lyndgaard et al. [86] determined the quality and thickness of porcine adipose tissue by confocal Raman depth profiling. Loin cuts were evaluated regarding thickness and composition of the outer and inner fat layer. The quality of back fat represented by the degree of unsaturation to mono-, poly-saturated fatty acids was measured. The noted physiological investigations mainly focus on the comparison to human confirming the suitability of porcine skin as model system. Hereby, CRM is predominantly utilized as reference method to evaluate existing techniques. The exceptional application in food science demonstrates again the versatility of Raman spectroscopy regarding quality control and assurance. 3.2.2.2. In vitro penetration studies on porcine skin. As affordable and easy accessible resource, porcine skin represents an acknowledged substitute for human skin regarding penetration experiments.

Fig. 5. Illustration of the localization of drug (B, D) and prodrug (A, C) relative to endogenous skin proteins (phe) after penetration at two different temperatures (A, B and C, D, E). Several regions in the viable epidermis have a relatively high concentration of drug (red) corresponding to low prodrug concentrations relative to phe as marked by the asterisks in (C) [92].


Forster et al. [90] tracked retinol in different polyethylene glycol (PEG) emulsions and solutions in excised pig flank skin. The observed effect of penetration enhancement was explained by lipid fluidization by oily soluble PEG. Zhang et al. [91,92] monitored the transformation of different prodrugs upon skin penetration. Pure 5-fluoruracil could be detected down to 5 μm into skin, while a penetration enhancing prodrug led to detectable 5-fluoruracil amount in depth of 12 μm. Fig. 5 illustrates the localization of drug and prodrug in the skin by x–z Raman mapping. Another approach was tested with resveratrol-triphosphate which is clipped to the active form by skin enzymes. No free resveratrol could be detected in brewed skin samples which are known to lack enzyme activity. Again time dependence of penetration depth of prodrug and drug could be shown. The studies noted in this chapter highlight CRM as powerful tool in drug delivery and formulation development. Especially the investigation of drug–prodrug transformation takes advantages of CRM's unique features to elucidate simultaneous localization of drug and metabolite.

3.2.3. Tissue engineered skin models For ethical reasons and especially in the development of cosmetics and skin care products the use of animal models is severely restricted. Therefore, tissue engineered skin reconstructions or lipid-based models are equally in the center of attention. Thus, such models are also in focus for Raman analysis. For instance, Tfayli et al. [93] conducted a vibrational comparison of cultured adult human keratinocytes on a collagen substrate and excised human abdominal skin. Differences in the C–S bond vibration could be observed and the engineered skin model contained less carotenoids than the excised human tissue. Furthermore, oxidation of disulfide bonds to sulfoxide explained the appearance of S–O vibrations in the artificial skin model. Ali et al. [94] used another type of reconstructed human epidermis to monitor photo damage during simulated solar radiation. An eminent decrease of the DNA representing Raman spectral feature indicated a reduced viability of the skin model, even before changes are observed by cytotoxicity assays and histopathology. In commercially available skin models, immortalized human epithelial keratinocyte cell lines like HaCaT or HUKE are widely used. Besides, the primary material of either healthy or diseased skin is cultured for specific research interests. For advanced tissue engineering, different cell types need to be combined for artificial skin constructs. In this regard, Pudlas et al. [95] successfully used Raman microscopy to discriminate between primary human fibroblasts, keratinocytes and melanocytes, as well as HaCaT cells. Furthermore, they were able to detect potential chemical changes to assure quality of cell populations. Bernard et al. [96] investigated a psoriatic skin substitute derived from the primary material. Raman spectroscopy in particular was used to elucidate changes in the secondary structure of proteins in involved psoriatic cell lines. Perna et al. [97,98] studied the toxicity of HgCl2 and the pesticide chlorpyriphos on HUKE cells. Raman spectroscopy revealed several structural and biochemical alterations even by non-cytotoxic concentrations. Donfack et al. [99] were able to correlate alterations in amid and CH deformation bands with high passage number in HaCaT cells. Since the same alterations appeared in the cancerogenic A5RT3 cell line, influence on the tumorigenic development is assumed. As a novel analytical technique for investigation of reconstructed human epidermis models, CRM was able to detect differences which were not visible in histopathological analysis. This points out the complexity of tissue engineered constructs and makes CRM a valuable characterization tool for further development. The application of CRM on cultured cells regarding toxicity and development has already broadly been explored, although with a narrow overlap with skin related research topics, which are mentioned above.

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3.2.4. Lipid-based skin models Other than tissue engineering, lipid-coated membranes are also intended to mimic the barrier function of the human skin and gain insight into penetration processes. Percot und Lefluer [100] used Raman mapping to detect crystalline structures in lipid mixtures mimicking the stratum corneum lipid composition. Ochalek et al. [101] proved the homogeneity of their lipid coated model membranes by confocal Raman mapping as well. Neubert's group combined FTIR and Raman microscopy for a threedimensional evaluation of dithranol in artificial acceptor membranes [102]. Lateral drug distribution was detected by FTIR mapping and confocal Raman microscopy was utilized to determine the active ingredient down to 49 μm in the model membrane. Tfayli et al. [103] investigated the influence of penetration enhancers on ceramides as the main skin lipid component. Different effects could be reported by different penetration enhancers and assigned to specific spectral features of the ceramides like double-bonds affected by limonene. In the defined and controlled systems based on lipid membranes, CRM can not only provide information on substance localization, but also especially the elucidation of lipid orientation and organization makes it a valuable asset complementary to IR spectroscopy. 3.3. In vivo investigations on human skin 3.3.1. Instrumental development for in vivo experiments For in vivo application of Raman microscopy, different approaches in sample prearrangement and instrumental setup had to be conducted. In the 90s, optic technology advanced to a point, where Raman setups became versatile, applicable and cost effective. Researchers quickly discovered the advantages and possibilities for in vivo skin investigations. Since, human in vivo studies represent the gold standard method, innovators focused on this particular application. In the late 90s, Shim et al. [104,105] developed and evaluated fiber optic probes for in vivo application on human tissues. In an alternative approach, Caspers et al. implemented an inverted confocal setup and were able to collect data from deeper skin layers for the first time. Caspers et al. [106–108] provided the final proof of concept with their comprehensive evaluation of in vitro and in vivo spectra of human skin. Fig. 6A depicts a scheme of the most commonly used confocal Raman setup for in vivo applications, in Fig. 6B a photo of the commercially available instrument is shown. In subsequent studies, they were able to collect concentration depth profiles of physiological components and a combination with CLSM allowed the targeted acquisition of Raman spectra from skin appendices like the sweat and sebaceous glands and even blood vessels. After successful implementation of harmless and non-invasive use of Raman microscopy on humans, animal testing was subordinated. The availability of these easy-to-use instruments promoted versatile in vivo applications of CRM with special focus on dermatology and skin care. 3.3.2. In vivo analysis of skin physiology 3.3.2.1. In vivo investigations of spectral variability. After providing adequate instrumental setups, in vivo investigations on human volunteers became numerous. The high specificity of Raman spectroscopy hereby confirmed the high variability of skin as complex biological tissue. Chrit et al. [109] evaluated the inter- and intraindividual variability of Raman spectra acquired from stratum corneum. Differences in specific spectral regions were associated with alterations in protein, amino acid and lipid distribution. Knudsen et al. [110] focused on diurnal and day-to-day variability of Raman spectra by repeated measurements on the same spot. Slight variabilities of amid I and amid III bands could be detected. Additionally, Knudsen et al. stated that pigmentation did not influence the wave

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Fig. 6. A. Schematic drawing of the assembly of a confocal Raman microscope modified for in vivo use [107]. B. Commercially available in vivo confocal Raman instrumentation.

numbers of the main skin components, but enhanced spectral background noise. Huang et al. [111] acquired Raman spectra from human melanin in vivo and stated no spectral difference from extracted natural and synthetic eumelanins. Tfayli et al. [112] reported slight variability in skin lipids upon aging. The Raman spectral features of the skin lipids shifted in lateral packing with increasing age of the volunteers. Gonzalez et al. [113] were able to differentiate between chronological aging and photoinduced skin damage by principle component analysis of Raman spectra from sun-exposed and sun-protected body sites. Although the noted studies already elucidated the influence of factors like time and sun exposure on skin physiology, analysis of skin variability and its many influencing factors is still a challenging task. Furthermore, the obtained results are mainly limited to Caucasian volunteers and an all-encompassing comprehensive investigation using CRM is eagerly anticipated. 3.3.2.2. In vivo analysis of skin hydration state. Further physiological investigations focused on the hydration state of skin and its different layers. The hydration state of the skin is not only important for skin care, but it also influences skin barrier function and substance penetration and permeation. First of all, Caspers et al. [114] implemented depth profiling of the skin water content. They acquired Raman spectra stepwise down to 40 μm depth. To overcome the issue of Raman signal attenuation, the water representing peak intensity was correlated to the protein representing peak intensity, therefore, generating relative concentration depth profiles. Skin hydration experiments could clearly alter the skin hydration state. Egawa and Tagami [115] reported a high variability in the water content of skin in relation to season, body site and age in a group of 50 Japanese volunteers over one year. In a subsequent study, Egawa and Kajikawa [116] conducted a variety of hydration experiments based on the exposure of skin to water. Depth profiles of the stratum corneum water content and subsequent questionnaires of the volunteers associated different hydration states with cutaneous sensations. Nakagawa, Matsumoto and Sakai [117] tackled the challenge of spectral acquisition in the deeper skin layers. Adjustments of laser power and acquisition time enabled the collection of spectra from down to 175 μm. By this method, an increase in dermal water content with increasing age and daytime could be reported. Recently, Boncheva et al. [118] used the previously described Raman setup as a reference method to evaluate the suitability of combination of high-frequency conductance and trans-epidermal-water-loss to determine water amount depth profiles during tape stripping. Chrit et al. [119,120] monitored the skin hydration status after application of different moisturizing enhancers. They revealed an increase in skin hydration after application of a widely used glycerol based hydration cream. Thereupon, a novel moisturizing polymer was evaluated which exposed better hydration capability in a microcapsule formulation combined with hyaluronic acid.

Further physiological investigations were conducted by Egawa, Herao and Takahashi [121] who monitored water gradients in the human epidermis. At the depth where the water amount became constant the stratum corneum–viable epidermis border was assumed. The results obtained from different body sites correlated well with literature and confirmed the suitability of this technique. Bielfeldt et al. [122] successfully derived an automated algorithm based on Fick's law of diffusion to determine stratum corneum thickness from water content depth profiles. In a follow-up study, Boehling et al. [123] validated CRM and CLSM for accurate determination of stratum corneum thickness in vivo. The in vivo acquisition of water content depth profiles by CRM measurements has become a standardized procedure. It provides information on moisturizing effects, and in addition structural information like stratum corneum thickness can be derived. The non-invasive nature and its reliable performance make this measurement method one of most popular in vivo applications of CRM. 3.3.2.3. In vivo evaluation of antioxidative potential in human skin. A research cooperation led by the Charite, Berlin focused on the evaluation of the skin's antioxidative potential. This topic is hardly accessible by other analytical methods and bares a lot of potential insights in skin aging mechanisms. Darvin et al. [124] tested two Raman spectroscopy based methods to detect carotenoids in the human skin in vivo. Carotenoids are assumed to represent the skin's main protection system against free radicals. Depth profiles revealed no differences between conventional Raman microscopy and resonance Raman. Furthermore, all tested anatomical sites showed the same carotenoid distribution, with the highest amount in the upper stratum corneum. This led to the conclusion that carotenoids are secreted via eccrine glands. Lademann et al. [125] focused on the follow-up of carotenoids after external application. Naturally carotenoids were mainly located in the upper stratum corneum. Topical application of an anti-aging cream leads to a higher penetration rate, and carotenoids could be detected in the epidermis as well. Haag et al. [126] correlated the carotenoid content to the decrease of free radicals in the skin. The long living radical nitroxide was monitored by electron paramagnetic resonance spectroscopy and compared to the carotenoid amount determined by Raman spectroscopy. Scarmo et al. [127] compared single point with multiple measurements of carotenoids in the skin. Little differences indicated the validity of single measurements, if other influencing factors like season and patient nutrition are taken into account. Furthermore, Fluhr et al. [128] studied the influence of tissue tolerable plasma for wound disinfection. This included the observation of β-carotene and water content in the treated skin regions. A decrease in β-carotene indicated the involvement in reactive oxygen species in the antimicrobial process. This chapter illustrates the potential of in vivo CRM. Besides deeper insight in basic physiological mechanisms within human skin, the development and evaluation of a disinfections formulation were successfully


performed with the help of CRM. However, so far, CRM is predominantly found as a complementary method to accompany established techniques in day-to-day research. 3.3.3. In vivo skin penetration studies 3.3.3.1. In vivo investigation of penetration enhancers. Besides the water content and other natural skin components, topically applied substances can be monitored over depth by confocal Raman microscopy, evaluating penetration behavior in vivo. By co-application of penetration enhancing substances, uptake of drug formulations and skin care products can be optimized. Caspers et al. [129] followed the well-known penetration enhancer dimethyl sulfoxide (DMSO) into the stratum corneum over time and depth. In a subsequent study conducted by Mélot et al. [130], the penetration enhancer propylene glycol (PG) in ethanol, oleic acid and Triton X were evaluated regarding their suitability to facilitate retinol delivery through the skin. All penetration enhancer remarkably increased the penetration depth of the model drug. The PG/ethanol mixture revealed synergistic effects, enabling penetration with the highest efficiency. Besides, the lipid fluidization by oleic acid showed greater effect on the retinol penetration depth than the lipid extracting abilities of Triton X. Recently, Mateus et al. [131] followed-up a combination of the penetration enhancer propylene glycol and ibuprofen in vivo. The relative concentration depth profiles showed similar rate and extent compared to tape stripping results which were previously published. Mohammed et al. [132] compared the penetration of different niacinamide formulations analyzed by HPLC in vitro with results obtained by CRM in vivo. The combined application of penetration enhancers and drugs leads to complex effects and demands extensive analytical investigations. CRM provides the opportunity for simultaneous follow-up of different components inside the skin and can also provide information on the penetration altering mechanism like lipid disordering or protein degradation. 3.3.3.2. In vivo studies on sunscreen formulations. An example for local delivery by a skin formulation is presented by the application of sunscreens. The UV-protective agent is supposed to stay in the upper skin layers to absorb the harmful radiation. The possibility of nondestructive detection of UV-absorbers in the skin drew the attention to CRM as tool for in vivo evaluation of sunscreens. Furthermore, to evaluate the quality of UV-protective agents in sunscreen formulations, the amount of urocanic acid (UCA) can be measured. UCA is known to isomerize from trans- to cis-form upon UV exposure. Egawa and Iwaki [133] tracked the trans-UCA content in the stratum corneum of 27 volunteers throughout one year. The lowest detected amounts in summer on the temporarily UV exposed forearms indicated the feasibility of confocal Raman microscopy to evaluate UV protection. Therefore, a second group of volunteers was artificially exposed to UV radiation with and without sunscreen. No statistical difference between no-exposure and exposure to sunscreen treated skin proved the UV blocking capacity of the applied agent. Furthermore, Egawa, Nomura and Iwaki [134] determined the transition rate of cis- and trans-UCA simultaneously by confocal Raman microscopy. The detected isomerization upon UV exposure was confirmed by HPLC results after tape stripping and extraction of the stratum corneum. Further, Caspers, van der Pol and de Sterke [135] proved the excellent penetration of a commercially used UV absorber by Raman microscopy, as it was found in the deeper stratum corneum regions only minutes after application. Broding et al. [136] demonstrated the ability of confocal Raman spectroscopy to monitor the skin penetration of potentially hazardous substances. The flux of three chemicals could be calculated after application on the forearms of healthy volunteers and was in good accordance with literature. Since the main region of interest for the evaluation of sunscreen formulations is located in the upper skin regions, the intrusion depth as

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one of the major limitations of CRM becomes irrelevant. Furthermore, the detection of the active agent and the quantification of metabolites indicating the protective potential make CRM a valuable tool to gain new insights into skin absorption of sunscreens and to reevaluate existing formulations. 3.3.4. In vivo skin diagnosis Besides studies about the physiological state of human skin and evaluations of substance penetration, confocal Raman microscopy also bares the potential for clinical application in disease diagnosis in vivo and the follow-up of therapeutic effects. Egawa et al. [137] characterized the involved and uninvolved areas of lesional psoriatic skin. Confocal Raman microscopy and optical coherence tomography detected lower water contents and a thicker stratum corneum in the affected skin areas. Interestingly, after treatment all monitored skin components such as natural moisturizing factor and ceramides returned to levels of healthy skin. Bakker Schut et al. [138] developed a software package that allows automated real time discrimination between different human tissues during Raman analysis. The presented program allows spectra classification, not only for healthy, but also for pathophysiological states of human skin. Alda et al. [139] accessed changes of the skin structure and composition of patients suffering from nickel allergy. Principal component analysis of the spectra set could clearly distinguish between healthy and allergenic skin areas. O'Regan et al. [140] discriminated between fillagrin genotype determined atopic dermatitis by analyzing the different Raman signatures of natural moisturizing factor in human stratum corneum. Recently, Baclig et al. [141] even reduced the necessary input data and simplified applicability of Raman spectroscopy in clinical diagnosis of atopic dermatitis. Systemic urea levels are useful indicators for chronic kidney disease and for monitoring progress of hemodialysis. The existence of a skin reservoir of urea is assumed which needs to be overcome to correlate the outward flux of urea caused by reversed iontophoresis to blood concentration. Wascotte et al. [142] monitored urea depletion during reversed iontophoresis in the upper skin layers by CRM. A depletion of urea in the upper epidermis highlighted the existence of this skin reservoir and the necessity to overcome it to employ urea flux for monitoring systemic concentrations. Schallreuter et al. [143] discovered the ability of Dead Sea water to decompose H2O2. They followed the degradation of H2O2 in the skin of vitiligo patients after a bath in Dead Sea water and therefore stated a pseudocatalase activity of transition metal ions. Recently, Lui et al. [144] approached the challenge of non-invasive, in vivo cancer diagnosis in human skin by confocal Raman microscopy. A broad panel of Raman spectra from cancerous and non-cancerous tissue was evaluated. A principal component analysis allowed discrimination of malignant from benign skin lesions with good diagnostic specificity and sensitivity. Philipsen et al. [145] extended the applicability to the detection of malignant melanoma and basal cell carcinoma independent from skin pigmentation in a group of 72 patients. So far, the Raman spectroscopic investigation of diseased skin focuses on mechanistic investigations to gain deeper insight into disease cause and development. However, all studies report distinguishable divergence in the spectroscopic patterns of healthy and diseased skin. This implicates the potential for comprehensive characterization and classification of skin lesions by CRM in vivo. 4. Conclusions and future perspective Confocal Raman microscopy has already proven its versatile and beneficial application in natural sciences and medicines. In skin research, numerous proof-of-concept studies highlighted the potential of CRM and basic analytical hindrances and limitations have successfully been tackled. Based on this knowledge, more and more laboratories

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resort CRM as a tool to target various questions. CRM shows big impact for the evaluation of in vitro skin tests using human, porcine and even reptile skin, as well as for studies on tissue engineered and lipid-based models. Further, the special focus on physiological investigations and penetration studies points out the advantages of CRM as non-invasive and non-destructive method. Especially for depth profiling of substance penetration in vitro and in vivo, CRM has proven its value. For the in vivo application on humans, the high sample variability still represents a major challenge for investigators and computational data analysis. Considering upcoming improvements like the establishment of quantification routines, CRM exhibits the potential to become a standard instrumentation in dermatopharmaceutical and cosmetic laboratories for the evaluation of novel therapeutics and cosmetics. Another application with very bright perspective is diagnosis, classification and characterization of skin diseases. With modern Raman microscopic devices it is already possible to successfully detect skin cancer in excised skin samples. Even the identification of malignant areas in vivo without prior excision has been reported. However, handling of the instrument and evaluation of the spectral data sets still require trained and experienced personnel. Further development in automated measurement systems and computational analysis will lead to commercially available low-cost, easy-to-use instruments. In fact, the first devices dedicated for cancer diagnosis are already on the market. Complementary to conventional CRM, advanced Raman spectroscopic instrumentations like CARS and SRS will be more and more employed in dermatology and in the adjoining fields of research. Further advances in technology will provide the possibilities of commercial availability and liberate other Raman based techniques from the restriction to physic laboratories. Proceeding specialization and simplification of Raman spectroscopy based instruments and software solutions will generate a broad panel of user friendly analytical tools for many of the applications in the field of skin research and will emerge to a standard analytical technique in the repertoire of scientists and operators at bench and bedside. References [1] K. Cal, J. Stefanowska, D. Zakowiecki, Current tools for skin imaging and analysis, Int. J. Dermatol. 48 (2009) 1283–1289. [2] B. Gotter, W. Faubel, R.H.H. Neubert, Optical methods for measurements of skin penetration, Skin Pharmacol. Physiol. 21 (2008) 156–165. [3] G. Abignano, S. Aydin, C. Castillo-Gallego, D. Woods, A. Meekings, D. McGonagle, P. Emery, F. Del Galdo, Optical coherence tomography validation: a new quantitative imaging biomarker for affected skin in scleroderma, Rheumatology 51 (2012) 35. [4] M.A. Boone, S. Norrenberg, G.B. Jemec, V. del Marmol, High-definition optical coherence tomography: slice and en-face imaging of normal skin and basal cell carcinoma, J. Invest. Dermatol. 132 (2012) S83. [5] R. Alvarez-Roman, A. Naik, Y.N. Kalia, H. Fessi, R.H. Guy, Visualization of skin penetration using confocal laser scanning microscopy, Eur. J. Pharm. Biopharm. 58 (2004) 301–316. [6] M.S. Roberts, Y. Dancik, T.W. Prow, C.A. Thorling, L.L. Lin, J.E. Grice, T.A. Robertson, K. Konig, W. Becker, Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy, Eur. J. Pharm. Biopharm. 77 (2011) 469–488. [7] K. Koenig, I. Riemann, High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution, J. Biomed. Opt. 8 (2003) 432–439. [8] I. Notingher, R.E. Imhof, Mid-infrared in vivo depth-profiling of topical chemicals on skin, Skin Res. Technol. 10 (2004) 113–121. [9] L. Kilpatrick-Liverman, P. Kazmi, E. Wolff, T.G. Polefka, The use of near-infrared spectroscopy in skin care applications, Skin Res. Technol. 12 (2006) 162–169. [10] R. Mendelsohn, C.R. Flach, D.J. Moore, Determination of molecular conformation and permeation in skin via IR spectroscopy, microscopy, and imaging, Biochim. Biophys. Acta Biomembr. 1758 (2006) 923–933. [11] C.R. Flach, G.R. Mao, P. Saad, R.M. Walters, R. Mendelsohn, SDS distribution and interaction in human and porcine skin by IR spectroscopy and imaging, Int. J. Cosmet. Sci. 34 (2012) 368. [12] S.M. Ali, F. Bonnier, H. Lambkin, K. Flynn, V. McDonagh, C. Healy, T.C. Lee, F.M. Lyng, H.J. Byrne, A comparison of Raman, FTIR and ATR–FTIR micro spectroscopy for imaging human skin tissue sections, Anal. Methods 5 (2013) 2281–2291. [13] C.V. Raman, K.S. Krishnan, A new type of secondary radiation, Nature 121 (1928) 501–502. [14] C.B. Juang, L. Finzi, C.J. Bustamante, Design and application of a computer-controlled confocal scanning differential polarization microscope, Rev. Sci. Instrum. 59 (1988) 2399–2408.

[15] E.C. Le Ru, E. Blackie, M. Meyer, P.G. Etchegoin, Surface enhanced raman scattering enhancement factors: a comprehensive study, J. Phys. Chem. C 111 (2007) 13794–13803. [16] D. Cialla, A. Marz, R. Bohme, F. Theil, K. Weber, M. Schmitt, J. Popp, Surfaceenhanced Raman spectroscopy (SERS): progress and trends, Anal. Bioanal. Chem. 403 (2012) 27–54. [17] K.F. Domke, B. Pettinger, Studying surface chemistry beyond the diffraction limit: 10 years of TERS, ChemPhysChem 11 (2010) 1365–1373. [18] T. Suzuki, Y. Kitahama, Y. Matsuura, Y. Ozaki, H. Sato, Development of a flexible fiber surface-enhanced Raman scattering (SERS) probe using a hollow optical fiber and gold nanoparticles, Appl. Spectrosc. 66 (2012) 1022–1026. [19] A. Zumbusch, G.R. Holtom, X.S. Xie, Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering, Phys. Rev. Lett. 82 (1999) 4142–4145. [20] C.W. Freudiger, W. Min, B.G. Saar, S. Lu, G.R. Holtom, C.W. He, J.C. Tsai, J.X. Kang, X.S. Xie, Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy, Science 322 (2008) 1857–1861. [21] H.A. Rinia, M. Bonn, M. Muller, E.M. Vartiainen, Quantitative CARS spectroscopy using the maximum entropy method: the main lipid phase transition, ChemPhysChem 8 (2007) 279–287. [22] C.L. Evans, E.O. Potma, M. Puoris'haag, D. Cote, C.P. Lin, X.S. Xie, Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 16807–16812. [23] C.L. Evans, X.S. Xie, 883-909, Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine, Annu. Rev. Anal. Chem. Palo Alto (2008) 883–909. [24] S. Heuke, N. Vogler, T. Meyer, D. Akimov, F. Kluschke, H.J. Rowert-Huber, J. Lademann, B. Dietzek, J. Popp, Multimodal mapping of human skin, Br. J. Dermatol. 169 (2013) 794–803. [25] Y. Jung, J. Tam, H.R. Jalian, R.R. Anderson, C.L. Evans, Longitudinal, 3D in vivo imaging of sebaceous glands by coherent anti-Stokes Raman scattering microscopy: normal function and response to cryotherapy, J. Invest. Dermatol. 135 (2015) 39–44. [26] K. König, H.G. Breunig, R. Bückle, M. Kellner-Höfer, M. Weinigel, E. Büttner, W. Sterry, J. Lademann, Optical skin biopsies by clinical CARS and multiphoton fluorescence/SHG tomography, Laser Phys. Lett. 8 (2011) 465. [27] H.G. Breunig, R. Buckle, M. Kellner-Hofer, M. Weinigel, J. Lademann, W. Sterry, K. Konig, Combined in vivo multiphoton and CARS imaging of healthy and diseaseaffected human skin, Microsc. Res. Tech. 75 (2012) 492–498. [28] B. Hans Georg, W. Martin, B. Rainer, K.-H. Marcel, L. Jürgen, E.D. Maxim, S. Wolfram, K. Karsten, Clinical coherent anti-Stokes Raman scattering and multiphoton tomography of human skin with a femtosecond laser and photonic crystal fiber, Laser Phys. Lett. 10 (2013) 025604. [29] M. Weinigel, H.G. Breunig, M. Kellner-Höfer, R. Bückle, M.E. Darvin, M. Klemp, J. Lademann, K. König, In vivo histology: optical biopsies with chemical contrast using clinical multiphoton/coherent anti-Stokes Raman scattering tomography, Laser Phys. Lett. 11 (2014) 055601. [30] R.H. Guy, B.G. Saar, L.R. Contreras-Rojas, X.S. Xie, Imaging drug delivery to skin with stimulated Raman scattering microscopy, Mol. Pharm. 8 (2011) 969–975. [31] N.A. Belsey, N.L. Garrett, L.R. Contreras-Rojas, A.J. Pickup-Gerlaugh, G.J. Price, J. Moger, R.H. Guy, Evaluation of drug delivery to intact and porated skin by coherent Raman scattering and fluorescence microscopies, J. Control. Release 174 (2014) 37–42. [32] T. Dai, B.M. Pikkula, L.V. Wang, B. Anvari, Comparison of human skin opto-thermal response to near-infrared and visible laser irradiations: a theoretical investigation, Phys. Med. Biol. 49 (2004) 4861–4877. [33] L. Franzen, L. Vidlarova, K.H. Kostka, U.F. Schaefer, M. Windbergs, Freeze-drying as a preserving preparation technique for in vitro testing of human skin, Exp. Dermatol. 22 (2013) 54–56. [34] L. Franzen, J. Anderski, V. Planz, K.H. Kostka, M. Windbergs, Combining confocal Raman microscopy and freeze drying for quantification of substance penetration into human skin, Exp. Dermatol. 23 (2014) 942–944. [35] R. Na, I.M. Stender, M. Henriksen, H.C. Wulf, Autofluorescence of human skin is age-related after correction for skin pigmentation and redness, J. Invest. Dermatol. 116 (2001) 536–540. [36] L. Silveira, B. Bodanese, R.A. Zangaro, M.T.T. Pacheco, Discrete wavelet transform for denoising Raman spectra of human skin tissues used in a discriminant diagnostic algorithm, Instrum. Sci. Technol. 38 (2010) 268–282. [37] M.G. Ramirez-Elias, J. Alda, F.J. Gonzalez, Noise and artifact characterization of in vivo Raman spectroscopy skin measurements, Appl. Spectrosc. 66 (2012) 650–655. [38] H.Q. Wang, J.H. Zhao, A.M.D. Lee, H. Lui, H.S. Zeng, Improving skin Raman spectral quality by fluorescence photobleaching, Photodiagnosis Photodyn. 9 (2012) 299–302. [39] F. Bonnier, S.M. Ali, P. Knief, H. Lambkin, K. Flynn, V. McDonagh, C. Healy, T.C. Lee, F.M. Lyng, H.J. Byrne, Analysis of human skin tissue by Raman microspectroscopy: dealing with the background, Vib. Spectrosc. 61 (2012) 124–132. [40] F. Bonnier, A. Mehmood, P. Knief, A.D. Meade, W. Hornebeck, H. Lambkin, K. Flynn, V. McDonagh, C. Healy, T.C. Lee, F.M. Lyng, H.J. Byrne, In vitro analysis of immersed human tissues by Raman microspectroscopy, J. Raman Spectrosc. 42 (2011) 888–896. [41] J.M. Bjordal, C. Couppe, R.T. Chow, J. Tuner, E.A. Ljunggren, A systematic review of low level laser therapy with location-specific doses for pain from chronic joint disorders, Aust. J. Physiother. 49 (2003) 107–116. [42] S. Tfaili, G. Josse, C. Gobinet, J.F. Angiboust, M. Manfait, O. Piot, Shedding light on the laser wavelength effect in Raman analysis of skin epidermises, Analyst 137 (2012) 4241–4246.

L. Franzen, M. Windbergs / Advanced Drug Delivery Reviews 89 (2015) 91–104 [43] N. Everall, Depth profiling with confocal Raman microscopy, part II, Spectroscopy 19 (2004) 16–27. [44] N. Everall, Depth profining with confocal Raman microscopy, part I, Spectroscopy 19 (2004) 22–27. [45] N.J. Everall, Confocal Raman microscopy: common errors and artefacts, Analyst 135 (2010) 2512–2522. [46] R. Mendelsohn, C.H. Xiao, C.R. Flach, C. Marcott, Uncertainties in depth determination and comparison of multivariate with univariate analysis in confocal Raman studies of a laminated polymer and skin, Appl. Spectrosc. 58 (2004) 382–389. [47] N.J. Everall, Confocal Raman microscopy: why the depth resolution and spatial accuracy can be much worse than you think, Appl. Spectrosc. 54 (2000) 1515–1520. [48] K.J. Baldwin, D.N. Batchelder, Confocal Raman microspectroscopy through a planar interface, Appl. Spectrosc. 55 (2001) 517–524. [49] C. Sourisseau, J.L. Bruneel, J.C. Lassegues, In-depth analyses by confocal Raman microspectrometry: experimental features and modeling of the refraction effects, J. Raman Spectrosc. 33 (2002) 815–828. [50] O. Piot, A. Tfayli, M. Manfait, Confocal Raman microspectroscopy on excised human skin: uncertainties in depth profiling and mathematical correction applied to dermatological drug permeation, J. Biophotonics 1 (2008) 140–153. [51] L. Franzen, J. Anderski, M. Windbergs, Quantitative detection of caffeine in human skin by confocal Raman spectroscopy - A systematic in vitro validation study, Eur. J. Pharm. Biopharm. (2015) http://dx.doi.org/10.1016/j.ejpb.2015.03. 026 (Submitted). [52] L. Franzen, D. Selzer, J.W. Fluhr, U.F. Schaefer, M. Windbergs, Towards drug quantification in human skin with confocal Raman microscopy, Eur. J. Pharm. Biopharm. 84 (2013) 437–444. [53] A.C. Williams, H.G.M. Edwards, B.W. Barry, Fourier-transform Raman-spectroscopy — a novel application for examining human stratum–corneum, Int. J. Pharm. 81 (1992) R11–R14. [54] B.W. Barry, H.G.M. Edwards, A.C. Williams, Fourier-transform Raman and infrared vibrational study of human skin — assignment of spectral bands, J. Raman Spectrosc. 23 (1992) 641–645. [55] A.C. Williams, H.G.M. Edwards, B.W. Barry, The ‘Iceman’: molecular structure of 5200-year-old skin characterised by Raman spectroscopy and electron microscopy, Biochim. Biophys. Acta Protein Struct. Mol. 1246 (1995) 98–105. [56] A.C. Williams, H.G.M. Edwards, B.W. Barry, Raman-spectra of human keratotic biopolymers — skin, callus, hair and nail, J. Raman Spectrosc. 25 (1994) 95–98. [57] S.M. Ali, F. Bonnier, A. Tfayli, H. Lambkin, K. Flynn, V. McDonagh, C. Healy, T. Clive Lee, F.M. Lyng, H.J. Byrne, Raman spectroscopic analysis of human skin tissue sections ex-vivo: evaluation of the effects of tissue processing and dewaxing, J. Biomed. Opt. 18 (2013) 61202. [58] R. Vyumvuhore, A. Tfayli, H. Duplan, A. Delalleau, M. Manfait, A. Baillet-Guffroy, Effects of atmospheric relative humidity on stratum corneum structure at the molecular level: ex vivo Raman spectroscopy analysis, Analyst 138 (2013) 4103–4111. [59] M. Gasior-Glogowska, M. Komorowska, J. Hanuza, M. Maczka, A. Zajac, M. Ptak, R. Bedzinski, M. Kobielarz, K. Maksymowicz, P. Kuropka, S. Szotek, FT-Raman spectroscopic study of human skin subjected to uniaxial stress, J. Mech. Behav. Biomed. Mater. 18 (2013) 240–252. [60] R. Vyumvuhore, A. Tfayli, H. Duplan, A. Delalleau, M. Manfait, A. Baillet-Guffroy, Raman spectroscopy: a tool for biomechanical characterization of stratum corneum, J. Raman Spectrosc. 44 (2013) 1077–1083. [61] A.N.C. Anigbogu, A.C. Williams, B.W. Barry, H.G.M. Edwards, Fourier-transform Raman-spectroscopy of interactions between the penetration enhancer dimethylsulfoxide and human stratum–corneum, Int. J. Pharm. 125 (1995) 265–282. [62] C. Adlhart, W. Baschong, Surface distribution and depths profiling of particulate organic UV absorbers by Raman imaging and tape stripping, Int. J. Cosmet. Sci. 33 (2011) 527–534. [63] A. Tfayli, O. Piot, F. Pitre, M. Manfait, Follow-up of drug permeation through excised human skin with confocal Raman microspectroscopy, Eur. Biophys. J. Biophy. 36 (2007) 1049–1058. [64] S. Tfaili, C. Gobinet, G. Josse, J.F. Angiboust, A. Baillet, M. Manfait, O. Piot, Vibrational spectroscopies for the analysis of cutaneous permeation: experimental limiting factors identified in the case of caffeine penetration, Anal. Bioanal. Chem. 405 (2013) 1325–1332. [65] M. Ashtikar, C. Matthaus, M. Schmitt, C. Krafft, A. Fahr, J. Popp, Non-invasive depth profile imaging of the stratum corneum using confocal Raman microscopy: first insights into the method, Eur J Pharm Sci. [66] L. Franzen, M. Windbergs, Accessing Raman spectral variability in human stratum corneum for quantitative in vitro depth profiling, J. Raman Spectrosc. 45 (2014) 82–88. [67] Q. Tu, C. Chang, Diagnostic applications of Raman spectroscopy, Nanomed. Nanotechnol. 8 (2012) 545–558. [68] M. Gniadecka, P. Philipsen, L. Hansen, J. Hercogova, K. Rossen, H. Thomsen, H. Wulf, Malignant melanoma diagnosis by Raman spectroscopy and artificial neural network, J. Invest. Dermatol. 113 (1999) 464. [69] M. Gniadecka, P.A. Philipsen, S. Sigurdsson, S. Wessel, O.F. Nielsen, D.H. Christensen, J. Hercogova, K. Rossen, H.K. Thomsen, R. Gniadecki, L.K. Hansen, H.C. Wulf, Melanoma diagnosis by Raman spectroscopy and neural networks: structure alterations in proteins and lipids in intact cancer tissue, J. Invest. Dermatol. 122 (2004) 443–449. [70] A. Nijssen, K. Maquelin, L.F. Santos, P.J. Caspers, T.C.B. Schut, J.C.D. Hollander, M.H.A. Neumann, G.J. Puppels, Discriminating basal cell carcinoma from perilesional skin using high wave-number Raman spectroscopy, J. Biomed. Opt. 12 (2007). [71] A. Nijssen, T.C.B. Schut, F. Heule, P.J. Caspers, D.P. Hayes, M.H.A. Neumann, G.J. Puppels, Discriminating basal cell carcinoma from its surrounding tissue by Raman spectroscopy, J. Invest. Dermatol. 119 (2002) 64–69.

103

[72] M.A. Short, H. Lui, D. McLean, H.S. Zeng, A. Alajlan, X.K. Chen, Changes in nuclei and peritumoral collagen within nodular basal cell carcinomas via confocal microRaman spectroscopy, J. Biomed. Opt. 11 (2006). [73] C.A. Lieber, S.K. Majumder, D. Billheimer, D.L. Ellis, A. Mahadevan-Jansen, Raman microspectroscopy for skin cancer detection in vitro, J. Biomed. Opt. 13 (2008). [74] M. Larraona-Puy, A. Ghita, A. Zoladek, W. Perkins, S. Varma, I.H. Leach, A.A. Koloydenko, H. Williams, I. Notingher, Discrimination between basal cell carcinoma and hair follicles in skin tissue sections by Raman micro-spectroscopy, J. Mol. Struct. 993 (2011) 57–61. [75] L. Silveira, F.L. Silveira, B. Bodanese, R.A. Za Ngaro, M.T. Pacheco, Discriminating model for diagnosis of basal cell carcinoma and melanoma in vitro based on the Raman spectra of selected biochemicals, J. Biomed. Opt. 17 (2012) 077003. [76] B.W. Barry, A.C. Williams, H.G.M. Edwards, Fourier transform Raman and IR spectra of snake skin, Spectrochim. Acta A: Mol. Spectrosc. 49 (1993) 801–807. [77] A.C. Williams, B.W. Barry, H.G. Edwards, Comparison of Fourier transform Raman spectra of mammalian and reptilian skin, Analyst 119 (1994) 563–566. [78] H.G.M. Edwards, D.W. Farwell, A.C. Williams, B.W. Barry, Raman spectroscopic studies of the skins of the Sahara sand viper, the carpet python and the American black rat snake, Spectrochim. Acta A 49 (1993) 913–919. [79] H.G.M. Edwards, D.E. Hunt, M.G. Sibley, FT-Raman spectroscopic study of keratotic materials: horn, hoof and tortoiseshell, Spectrochim. Acta A 54 (1998) 745–757. [80] R.L. Bronaugh, R.F. Stewart, E.R. Congdon, Methods for in vitro percutaneous absorption studies. II. Animal models for human skin, Toxicol. Appl. Pharmacol. 62 (1982) 481–488. [81] U. Jacobi, M. Kaiser, R. Toll, S. Mangelsdorf, H. Audring, N. Otberg, W. Sterry, J. Lademann, Porcine ear skin: an in vitro model for human skin, Skin Res. Technol. 13 (2007) 19–24. [82] S. Tfaili, C. Gobinet, G. Josse, J.F. Angiboust, M. Manfait, O. Piot, Confocal Raman microspectroscopy for skin characterization: a comparative study between human skin and pig skin, Analyst 137 (2012) 3673–3682. [83] J. Wu, T.G. Polefka, Confocal Raman microspectroscopy of stratum corneum: a preclinical validation study, Int. J. Cosmet. Sci. 30 (2008) 47–56. [84] M. Forster, M.A. Bolzinger, M.R. Rovere, O. Damour, G. Montagnac, S. Briancon, Confocal Raman microspectroscopy for evaluating the stratum corneum removal by 3 standard methods, Skin Pharmacol. Physiol. 24 (2011) 103–112. [85] L. Franzen, C. Mathes, S. Hansen, M. Windbergs, Advanced chemical imaging and comparison of human and porcine hair follicles for drug delivery by confocal Raman microscopy, J. Biomed. Opt. 18 (2013) 61210. [86] L.B. Lyndgaard, K.M. Sorensen, F. van den Berg, S.B. Engelsen, Depth profiling of porcine adipose tissue by Raman spectroscopy, J. Raman Spectrosc. 43 (2012) 482–489. [87] C.H. Xiao, D.J. Moore, C.R. Flach, R. Mendelsohn, Permeation of dimyristoylphosphatidylcholine into skin — structural and spatial information from IR and Raman microscopic imaging, Vib. Spectrosc. 38 (2005) 151–158. [88] C.H. Xiao, D.J. Moore, M.E. Rerek, C.R. Flach, R. Mendelsohn, Feasibility of tracking phospholipid permeation into skin using infrared and Raman microscopic imaging, J. Invest. Dermatol. 124 (2005) 622–632. [89] G. Mao, C.R. Flach, R. Mendelsohn, R.M. Walters, Imaging the distribution of sodium dodecyl sulfate in skin by confocal raman and infrared microspectroscopy, Pharm. Res. 29 (2012) 2189–2201. [90] M. Forster, M.A. Bolzinger, D. Ach, G. Montagnac, S. Briancon, Ingredients tracking of cosmetic formulations in the skin: a confocal Raman microscopy investigation, Pharm. Res. 28 (2011) 858–872. [91] G. Zhang, C.R. Flach, R. Mendelsohn, Tracking the dephosphorylation of resveratrol triphosphate in skin by confocal Raman microscopy, J. Control. Release 123 (2007) 141–147. [92] G.J. Zhang, D.J. Moore, K.B. Sloan, C.R. Flach, R. Mendelsohn, Imaging the prodrugto-drug transformation of a 5-fluorouracil derivative in skin by confocal Raman microscopy, J. Invest. Dermatol. 127 (2007) 1205–1209. [93] A. Tfayli, O. Piot, F. Draux, F. Pitre, M. Manfait, Molecular characterization of reconstructed skin model by Raman microspectroscopy: comparison with excised human skin, Biopolymers 87 (2007) 261–274. [94] S.M. Ali, F. Bonnier, K. Ptasinski, H. Lambkin, K. Flynn, F.M. Lyng, H.J. Byrne, Raman spectroscopic mapping for the analysis of solar radiation induced skin damage, Analyst 138 (2013) 3946–3956. [95] M. Pudlas, S. Koch, C. Bolwien, S. Thude, N. Jenne, T. Hirth, H. Walles, K. SchenkeLayland, Raman spectroscopy: a noninvasive analysis tool for the discrimination of human skin cells, Tissue Eng. Part C Methods 17 (2011) 1027–1040. [96] G. Bernard, M. Auger, J. Soucy, R. Pouliot, Physical characterization of the stratum corneum of an in vitro psoriatic skin model by ATR–FTIR and Raman spectroscopies, Biochim. Biophys. Acta Gen. Subj. 1770 (2007) 1317–1323. [97] G. Perna, M. Lastella, M. Lasalvia, E. Mezzenga, V. Capozzi, Raman spectroscopy and atomic force microscopy study of cellular damage in human keratinocytes treated with HgCl2, J. Mol. Struct. 834 (2007) 182–187. [98] G. Perna, M. Lasalvia, P. D'Antonio, N. L'Abbate, V. Capozzi, Identification of chemical modification in single human keratinocyte cells exposed to low doses of chlorpyriphos by Raman micro-spectroscopy, J. Raman Spectrosc. 42 (2011) 603–611. [99] P. Donfack, M. Rehders, K. Brix, P. Boukamp, A. Materny, Micro Raman spectroscopy for monitoring alterations between human skin keratinocytes HaCaT and their tumorigenic derivatives A5RT3-toward a Raman characterization of a skin carcinoma model, J. Raman Spectrosc. 41 (2010) 16–26. [100] A. Percot, M. Lafleur, Direct observation of domains in model stratum corneum lipid mixtures by Raman microspectroscopy, Biophys. J. 81 (2001) 2144–2153. [101] M. Ochalek, S. Heissler, J. Wohlrab, R.H.H. Neubert, Characterization of lipid model membranes designed for studying impact of ceramide species on drug diffusion and penetration, Eur. J. Pharm. Biopharm. 81 (2012) 113–120.

104


[102] B. Gotter, W. Faubel, R.H.H. Neubert, FTIR microscopy and confocal Raman microscopy for studying lateral drug diffusion from a semisolid formulation, Eur. J. Pharm. Biopharm. 74 (2010) 14–20. [103] A. Tfayli, E. Guillard, M. Manfait, A. Baillet-Guffroy, Molecular interactions of penetration enhancers within ceramides organization: a Raman spectroscopy approach, Analyst 137 (2012) 5002–5010. [104] M.G. Shim, B.C. Wilson, Development of an in vivo Raman spectroscopic system for diagnostic applications, J. Raman Spectrosc. 28 (1997) 131–142. [105] M.G. Shim, B.C. Wilson, E. Marple, M. Wach, Study of fiber-optic probes for in vivo medical Raman spectroscopy, Appl. Spectrosc. 53 (1999) 619–627. [106] P.J. Caspers, G.W. Lucassen, R. Wolthuis, H.A. Bruining, G.J. Puppels, In vitro and in vivo Raman spectroscopy of human skin, Biospectroscopy 4 (1998) S31–S39. [107] P.J. Caspers, G.W. Lucassen, E.A. Carter, H.A. Bruining, G.J. Puppels, In vivo confocal Raman microspectroscopy of the skin: noninvasive determination of molecular concentration profiles, J. Invest. Dermatol. 116 (2001) 434–442. [108] P.J. Caspers, G.W. Lucassen, G.J. Puppels, Combined in vivo confocal Raman spectroscopy and confocal microscopy of human skin, Biophys. J. 85 (2003) 572–580. [109] L. Chrit, C. Hadjur, S. Morel, G. Sockalingum, G. Lebourdon, F. Leroy, M. Manfait, In vivo chemical investigation of human skin using a confocal Raman fiber optic microprobe, J. Biomed. Opt. 10 (2005). [110] L. Knudsen, C.K. Johansson, P.A. Philipsen, M. Gniadecka, H.C. Wulf, Natural variations and reproducibility of in vivo near-infrared Fourier transform Raman spectroscopy of normal human skin, J. Raman Spectrosc. 33 (2002) 574–579. [111] Z.W. Huang, H. Lui, X.K. Chen, A. Alajlan, D.I. McLean, H.S. Zeng, Raman spectroscopy of in vivo cutaneous melanin, J. Biomed. Opt. 9 (2004) 1198–1205. [112] A. Tfayli, E. Guillard, M. Manfait, A. Baillet-Guffroy, Raman spectroscopy: feasibility of in vivo survey of stratum corneum lipids, effect of natural aging, Eur. J. Dermatol. 22 (2012) 36–41. [113] F.J. Gonzalez, C. Castillo-Martinez, M. Martinez-Escaname, M.G. Ramirez-Elias, F.I. Gaitan-Gaona, C. Oros-Ovalle, B. Moncada, Noninvasive estimation of chronological and photoinduced skin damage using Raman spectroscopy and principal component analysis, Skin Res. Technol. 18 (2012) 442–446. [114] P.J. Caspers, G.W. Lucassen, H.A. Bruining, G.J. Puppels, Automated depth-scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin, J. Raman Spectrosc. 31 (2000) 813–818. [115] M. Egawa, H. Tagami, Comparison of the depth profiles of water and water-binding substances in the stratum corneum determined in vivo by Raman spectroscopy between the cheek and volar forearm skin: effects of age, seasonal changes and artificial forced hydration, Br. J. Dermatol. 158 (2008) 251–260. [116] M. Egawa, T. Kajikawa, Changes in the depth profile of water in the stratum corneum treated with water, Skin Res. Technol. 15 (2009) 242–249. [117] N. Nakagawa, M. Matsumoto, S. Sakai, In vivo measurement of the water content in the dermis by confocal Raman spectroscopy, Skin Res. Technol. 16 (2010) 137–141. [118] M. Boncheva, J. de Sterke, P.J. Caspers, G.J. Puppels, Depth profiling of stratum corneum hydration in vivo: a comparison between conductance and confocal Raman spectroscopic measurements, Exp. Dermatol. 18 (2009) 870–876. [119] L. Chrit, P. Bastien, G.D. Sockalingum, D. Batisse, F. Leroy, M. Manfait, C. Hadjur, An in vivo randomized study of human skin moisturization by a new confocal Raman fiber-optic microprobe: assessment of a glycerol-based hydration cream, Skin Pharmacol. Physiol. 19 (2006) 207–215. [120] L. Chrit, P. Bastien, B. Biatry, J.T. Simonnet, A. Potter, A.M. Minondo, F. Flament, R. Bazin, G.D. Sockalingum, F. Leroy, M. Manfait, C. Hadjur, In vitro and in vivo confocal Raman study of human skin hydration: assessment of a new moisturizing agent, pMPC, Biopolymers 85 (2007) 359–369. [121] M. Egawa, T. Hirao, M. Takahashi, In vivo estimation of stratum corneum thickness from water concentration profiles obtained with Raman spectroscopy, Acta Derm. Venereol. 87 (2007) 4–8. [122] S. Bielfeldt, V. Schoder, U. Ely, A. van der Pol, J. de Sterke, K.-P. Wilhelm, Assessment of human stratum corneum thickness and its barrier properties by in-vivo confocal Raman spectroscopy, IFSCC Magazine2009. (01/2009). [123] A. Böhling, S. Bielfeldt, A. Himmelmann, M. Keskin, K.P. Wilhelm, Comparison of the stratum corneum thickness measured in vivo with confocal Raman spectroscopy and confocal reflectance microscopy, Skin Res. Technol. 20 (2014) 50–57. [124] M.E. Darvin, J.W. Fluhr, P. Caspers, A. van der Pool, H. Richter, A. Patzelt, W. Sterry, J. Lademann, In vivo distribution of carotenoids in different anatomical locations of human skin: comparative assessment with two different Raman spectroscopy methods, Exp. Dermatol. 18 (2009) 1060–1063. [125] J. Lademann, P.J. Caspers, A. van der Pol, H. Richter, A. Patzelt, L. Zastrow, M. Darvin, W. Sterry, J.W. Fluhr, In vivo Raman spectroscopy detects increased epidermal

[126]

[127]

[128]

[129]

[130]

[131]

[132] [133]

[134]

[135] [136]

[137]

[138]

[139]

[140]

[141]

[142]

[143]

[144] [145]

antioxidative potential with topically applied carotenoids, Laser Phys. Lett. 6 (2009) 76–79. S.F. Haag, B. Taskoparan, M.E. Darvin, N. Groth, J. Lademann, W. Sterry, M.C. Meinke, Determination of the antioxidative capacity of the skin in vivo using resonance Raman and electron paramagnetic resonance spectroscopy, Exp. Dermatol. 20 (2011) 483–487. S. Scarmo, B. Cartmel, H. Lin, D.J. Leffell, I.V. Ermakov, W. Gellermann, P.S. Bernstein, S.T. Mayne, Single v. multiple measures of skin carotenoids by resonance Raman spectroscopy as a biomarker of usual carotenoid status, Br. J. Nutr. 110 (2013) 911–917. J.W. Fluhr, S. Sassning, O. Lademann, M.E. Darvin, S. Schanzer, A. Kramer, H. Richter, W. Sterry, J. Lademann, In vivo skin treatment with tissue-tolerable plasma influences skin physiology and antioxidant profile in human stratum corneum, Exp. Dermatol. 21 (2012) 130–134. P.J. Caspers, A.C. Williams, E.A. Carter, H.G.M. Edwards, B.W. Barry, H.A. Bruining, G.J. Puppels, Monitoring the penetration enhancer dimethyl sulfoxide in human stratum corneum in vivo by confocal Raman spectroscopy, Pharm. Res. 19 (2002) 1577–1580. M. Melot, P.D.A. Pudney, A.M. Williamson, P.J. Caspers, A. Van Der Pol, G.J. Puppels, Studying the effectiveness of penetration enhancers to deliver retinol through the stratum corneum by in vivo confocal Raman spectroscopy, J. Control. Release 138 (2009) 32–39. R. Mateus, H. Abdalghafor, G. Oliveira, J. Hadgraft, M.E. Lane, A new paradigm in dermatopharmacokinetics — confocal Raman spectroscopy, Int. J. Pharm. 444 (2013) 106–108. D. Mohammed, P.J. Matts, J. Hadgraft, M.E. Lane, In vitro–in vivo correlation in skin permeation, Pharm. Res. (2013) 1–7. M. Egawa, H. Iwaki, In vivo evaluation of the protective capacity of sunscreen by monitoring urocanic acid isomer in the stratum corneum using Raman spectroscopy, Skin Res. Technol. 14 (2008) 410–417. M. Egawa, J. Nomura, H. Iwaki, The evaluation of the amount of cis- and transurocanic acid in the stratum corneum by Raman spectroscopy, Photochem. Photobiol. Sci. 9 (2010) 730–733. P.J. Caspers, A. van der Pol, J. de Sterke, Penetration of sunscreen agents monitored in vivo by confocal Raman spectroscopy, J. Invest. Dermatol. 126 (2006) 73. H.C. Broding, A. van der Pol, J. de Sterke, C. Monse, M. Fartasch, T. Bruning, In vivo monitoring of epidermal absorption of hazardous substances by confocal Raman micro-spectroscopy, J. Dtsch. Dermatol. Ges. 9 (2011) 618–626. M. Egawa, N. Kunizawa, T. Hirao, T. Yamamoto, K. Sakamoto, T. Terui, H. Tagami, In vivo characterization of the structure and components of lesional psoriatic skin from the observation with Raman spectroscopy and optical coherence tomography: a pilot study, J. Dermatol. Sci. 57 (2010) 66–69. T.C. Bakker Schut, R. Wolthuis, P.J. Caspers, G.J. Puppels, Real-time tissue characterization on the basis of in vivo Raman spectra, J. Raman Spectrosc. 33 (2002) 580–585. J. Alda, C. Castillo-Martinez, R. Valdes-Rodriguez, D. Hernandez-Blanco, B. Moncada, F.J. Gonzalez, Use of Raman spectroscopy in the analysis of nickel allergy, J. Biomed. Opt. 18 (2013) 61206. G.M. O'Regan, P.M.J.H. Kemperman, A. Sandilands, H. Chen, L.E. Campbell, K. Kroboth, R. Watson, M. Rowland, G.J. Puppels, W.H.I. McLean, P.J. Caspers, A.D. Irvine, Raman profiles of the stratum corneum define 3 filaggrin genotype-determined atopic dermatitis endophenotypes, J Allergy Clin Immun 126 (2010) 574–580 e571. A.C. Baclig, T.C.B. Schut, G.M. O'Regan, A.D. Irvine, W.H.I. McLean, G.J. Puppels, P.J. Caspers, Possibilities for human skin characterization based on strongly reduced Raman spectroscopic information, J. Raman Spectrosc. 44 (2013) 340–345. V. Wascotte, P. Caspers, J. de Sterke, M. Jadoul, R.H. Guy, V. Preat, Assessment of the “Skin reservoir” of urea by confocal Raman microspectroscopy and reverse iontophoresis in vivo, Pharm. Res. 24 (2007) 1897–1901. K.U. Schallreuter, J. Moore, S. Behrens-Williams, A. Panske, M. Harari, H. Rokos, J.M. Wood, In vitro and in vivo identification of ‘pseudocatalase’ activity in Dead Sea water using Fourier transform Raman spectroscopy, J. Raman Spectrosc. 33 (2002) 586–592. H. Lui, J.H. Zhao, D. McLean, H.S. Zeng, Real-time Raman spectroscopy for in vivo skin cancer diagnosis, Cancer Res. 72 (2012) 2491–2500. P.A. Philipsen, L. Knudsen, M. Gniadecka, M.H. Ravnbak, H.C. Wulf, Diagnosis of malignant melanoma and basal cell carcinoma by in vivo NIR–FT Raman spectroscopy is independent of skin pigmentation, Photochem. Photobiol Sci. 12 (2013) 770–776.

Diffusion profile of macromolecules within and between human skin layers for (trans)dermal drug delivery.

Dyspigmentation, skin physiology, and a novel approach to skin lightening.

Effect of skin metabolism on dermal delivery of testosterone: qualitative assessment using a new short-term skin model.

Drug delivery nanoparticles in skin cancers.

Acellular Dermal Matrix to Treat Full Thickness Skin Defects: Follow-Up Subjective and Objective Skin Quality Assessments.

Using skin for drug delivery and diagnosis in the critically ill.

Physiology of skin.

Efficient dermal delivery of retinyl palmitate: Progressive polarimetry and Raman spectroscopy to evaluate the structure and efficacy.

Evaluation of dermal irritation and skin sensitization due to vitacoxib.

Skin Matters: A Review of Topical Treatments for Chronic Pain. Part One: Skin Physiology and Delivery Systems.

Inflammatory Bowel Disease and Skin Cancer: An Assessment of Patient Risk Factors, Knowledge, and Skin Practices.

Newborn infant skin: physiology, development, and care.

Confocal Raman microscopic investigation of the effectiveness of penetration enhancers for procaine delivery to the skin.

Liposomes for (trans)dermal drug delivery: the skin-PVPA as a novel in vitro stratum corneum model in formulation development.

Raman Plus X: Biomedical Applications of Multimodal Raman Spectroscopy.

Advances in drug delivery systems (DDSs) to release growth factors for wound healing and skin regeneration.

Skin physiology in pruritus of advanced ageing.

Advances in the in Vivo Raman Spectroscopy of Malignant Skin Tumors Using Portable Instrumentation.

Skin delivery of kojic acid-loaded nanotechnology-based drug delivery systems for the treatment of skin aging.

Melanosomes in dermal Schwann cells of human and rodent skin.

Cefazolin potency against methicillin-resistant Staphylococcus aureus: a microbiologic assessment in support of a novel drug delivery system for skin and skin structure infections.

Human age and skin physiology shape diversity and abundance of Archaea on skin.

Erratum: Cefazolin potency against methicillin-resistant Staphylococcus aureus: a microbiologic assessment in support of a novel drug delivery system for skin and skin structure infections [Erratum].

Raman spectroscopy: in vivo quick response code of skin physiological status.