Label-free mapping of cetuximab in multi-layered tumor oral mucosa models by atomic force-microscopy-based infrared spectroscopy.

Sensitive mapping of drugs and drug delivery systems is pivotal for the understanding and improvement of treatment options. Since labeling alters the physicochemical and potentially the pharmacological properties of the molecule of interest, its label-free detection by photothermal expansion is investigated. We report on a proof-of-concept study to map the cetuximab distribution by atomic-force microscopy-based infrared spectroscopy (AFM-IR). The monoclonal antibody cetuximab was applied to a human tumor oral mucosa model, consisting of a tumor epithelium on a lamina propria equivalent. Hyperspectral imaging in the wavenumber regime between 903 cm-1 and 1312 cm-1 and a probing distance between the data points down to 10 × 10 nm are used for determining the local drug distribution. The local distinction of cetuximab from the tissue background is gained by linear combination modeling making use of reference spectra of the drug and untreated models. The results from this approach are compared to principal component analyses, yielding comparable results. Even single molecule detection appears feasible. The results indicate that cetuximab penetrates the cytosol of tumor cells but does not bind to structures in the cell membrane. In conclusion, AFM-IR mapping of cetuximab proved to sensitively determine drug concentrations at an unprecedented spatial resolution without the need for drug labeling.

Visualization of Nanocarriers and Drugs in Cells and Tissue.

In this chapter, the visualization of nanocarriers and drugs in cells and tissue is reviewed. This topic is tightly connected to modern drug delivery, which relies on nanoscopic drug formulation approaches and the ability to probe nanoparticulate systems selectively in cells and tissue using advanced spectroscopic and microscopic techniques. We first give an overview of the breadth of this research field. Then, we mainly focus on topical drug delivery to the skin and discuss selected visualization techniques from spectromicroscopy, such as scanning transmission X-ray microscopy and fluorescence lifetime imaging. These techniques rely on the sensitive and quantitative detection of the topically applied drug delivery systems and active substances, either by exploiting their molecular properties or by introducing environmentally sensitive probes that facilitate their detection.

X-Ray absorption spectroscopy of H3O.

We report the X-ray absorption of isolated H3O+ cations at the O 1s edge. The molecular ions were prepared in a flowing afterglow ion source which was designed for the production of small water clusters, protonated water clusters, and hydrated ions. Isolated H2O+ cations have been analyzed for comparison. The spectra show significant differences in resonance energies and widths compared to neutral H2O with resonances shifting to higher energies by as much as 10 eV and resonance widths increasing by as much as a factor of 5. The experimental results are supported by time-dependent density functional theory calculations performed for both molecular cations, showing a good agreement with the experimental data. The spectra reported here could enable the identification of the individual molecules in charged small water clusters or liquid water using X-ray absorption spectroscopy.

Compressed FTIR spectroscopy using low-rank matrix reconstruction.

Fourier transform infrared (FTIR) spectroscopy is a powerful technique in analytical chemistry. Typically, spatially distributed spectra of the substance of interest are conducted simultaneously using FTIR spectrometers equipped with array detectors. Scanning-based methods such as near-field FTIR spectroscopy, on the other hand, are a promising alternative providing higher spatial resolution. However, serial recording severely limits their application due to the long acquisition times involved and the resulting stability issues. We demonstrate that it is possible to significantly reduce the measurement time of scanning methods by applying the mathematical technique of low-rank matrix reconstruction. Data from a previous pilot study of Leishmania strains are analyzed by randomly selecting 5% of the interferometer samples. The results obtained for bioanalytical fingerprinting using the proposed approach are shown to be essentially the same as those obtained from the full set of data. This finding can significantly foster the practical applicability of high-resolution serial scanning techniques in analytical chemistry and is also expected to improve other applications of FTIR spectroscopy and spectromicroscopy.

Data-based modeling of drug penetration relates human skin barrier function to the interplay of diffusivity and free-energy profiles.

Based on experimental concentration depth profiles of the antiinflammatory drug dexamethasone in human skin, we model the time-dependent drug penetration by the 1D general diffusion equation that accounts for spatial variations in the diffusivity and free energy. For this, we numerically invert the diffusion equation and thereby obtain the diffusivity and the free-energy profiles of the drug as a function of skin depth without further model assumptions. As the only input, drug concentration profiles derived from X-ray microscopy at three consecutive times are used. For dexamethasone, skin barrier function is shown to rely on the combination of a substantially reduced drug diffusivity in the stratum corneum (the outermost epidermal layer), dominant at short times, and a pronounced free-energy barrier at the transition from the epidermis to the dermis underneath, which determines the drug distribution in the long-time limit. Our modeling approach, which is generally applicable to all kinds of barriers and diffusors, allows us to disentangle diffusivity from free-energetic effects. Thereby we can predict short-time drug penetration, where experimental measurements are not feasible, as well as long-time permeation, where ex vivo samples deteriorate, and thus span the entire timescales of biological barrier functioning.

Modeling of Drug Diffusion Based on Concentration Profiles in Healthy and Damaged Human Skin.

Based on experimental drug concentration profiles in healthy as well as tape-stripped ex vivo human skin, we model the penetration of the antiinflammatory drug dexamethasone into the skin layers by the one-dimensional generalized diffusion equation. We estimate the position-dependent free-energy and diffusivity profiles by solving the conjugated minimization problem, in which the only inputs are concentration profiles of dexamethasone in skin at three consecutive penetration times. The resulting free-energy profiles for damaged and healthy skin show only minor differences. In contrast, the drug diffusivity in the first 10 µm of the upper skin layer of damaged skin is 200-fold increased compared to healthy skin, which reflects the corrupted barrier function of tape-stripped skin. For the case of healthy skin, we examine the robustness of our method by analyzing the behavior of the extracted skin parameters when the number of input and output parameters are reduced. We also discuss techniques for the regularization of our parameter extraction method.

Qualifying X-ray and Stimulated Raman Spectromicroscopy for Mapping Cutaneous Drug Penetration.

Research on topical drug delivery relies on reconstructed human skin (RHS) in addition to ex vivo human and animal skin, each with specific physiological features. Here, we compared the penetration of dexamethasone from an ethanolic hydroxyethyl cellulose gel into ex vivo human skin, murine skin, and RHS. For comprehensive insights into skin morphology and penetration enhancing mechanisms, scanning transmission X-ray microscopy (STXM), liquid chromatography tandem-mass spectrometry (LC-MS/MS), and stimulated Raman spectromicroscopy (SRS) were combined. STXM offers high spatial resolution with label-free drug detection and is therefore sensitive to tissue damage. Despite differences in sample preparation and data analysis, the amounts of dexamethasone in RHS, detected and quantified by STXM and LC-MS/MS, were very similar and increased during the first 100 min of exposure. SRS revealed interactions between the gel and the stratum corneum or, more specifically, its protein and lipid structures. Similar to both types of ex vivo skin, higher protein-to-lipid ratios within the stratum corneum of RHS indicated reduced lipid amounts after 30 min of ethanol exposure. Extended ethanol exposure led to a continued reduction of lipids in the ex vivo matrixes, while protein integrity appeared to be compromised in RHS, which led to declining protein signals. In conclusion, LC-MS/MS proved the predictive capability of STXM for label-free drug detection. Combining STXM with SRS precisely dissected the penetration enhancing effects of ethanol. Further studies on topical drug delivery should consider the potential of these complementary techniques.

Adsorption of Mono- and Divalent 4-(Dimethylamino)pyridines on Gold Surfaces: Studies by Surface-Enhanced Raman Scattering and Density Functional Theory.

The adsorption thermodynamics of 4-(dimethylamino)pyridine (DMAP) and its five divalent derivatives di-DMAP- n (2 ≤ n ≤ 6) with gradually increasing methylene-spacer lengths n binding to planar gold surfaces has been studied by surface-enhanced Raman spectroscopy (SERS) and density functional theory (DFT). SERS intensities of the totally symmetrical breathing mode of the pyridine ring at approximately 1007 cm-1 are used to monitor the surface coverage of the DMAP and di-DMAP- n ligands on gold surfaces at different concentrations. The equilibrium constant as a measure of the binding affinity is obtained from these measurements by using a modified Langmuir isotherm. Due to multivalent binding to the gold substrate, a characteristic enhancement of the binding affinity of di-DMAP- n compared to the monovalent DMAP is observed for all divalent species. First principles calculations of the di-DMAP- n ligands on an ideal Au(111) surface model as well as step terrace models have been performed to understand the adsorption structures and the multivalent binding enhancements. Furthermore, Raman spectra of the adsorbed molecules have been studied by first principles calculations to correlate the binding affinities to experimentally determined adsorption constants. The joint experimental and theoretical investigation of an oscillatory behavior of the binding affinity as a function of the methylene-spacer length in mono- and divalent 4-(dimethylamino)pyridines reveals that the molecular architecture plays an important role for the structure-function interplay of multivalently bound adsorbates.

Laminar flow in microfluidics investigated by spatially-resolved soft X-ray absorption and infrared spectroscopy.

The application of soft X-ray absorption spectroscopy (XAS) to liquid cells based on microfluidics for chemical state analysis of light elements is much more difficult than hard X-ray absorption since soft X-rays cannot deeply penetrate a microfluidic cell. In this study, we have newly developed a microfluidic cell for spatially resolved XAS, where a 100 nm thick Si3N4 membrane is used for the measurement window to transmit soft X-rays for keeping the microfluidic flow at a width and depth of 50 µm. The π* peak of pyridine near the N K-edge XAS shows characteristic energy shifts near the liquid-liquid interface in a laminar flow of pyridine and water. The distributions of the molar fractions of pyridine and water near the liquid-liquid interface have been determined from the energy shifts of the π* peak probed at different geometric positions, where pyridine is mixed in the water part of the laminar flow and vice versa. The spatial distribution of both species has also been studied by infrared microscopy, using the same microfluidic setup. The present work clearly shows that these spectroscopic techniques are easily applicable to chemical and biological reactions prepared by microfluidics.

Shape-Dependent Dissolution and Cellular Uptake of Silver Nanoparticles.

The cellular uptake and dissolution of trigonal silver nanoprisms (edge length 42 ± 15 nm, thickness 8 ± 1 nm) and mostly spherical silver nanoparticles (diameter 70 ± 25 nm) in human mesenchymal stem cells (hMSC's) and human keratinocytes (HaCaT cells) were investigated. Both particles are stabilized by polyvinylpyrrolidone (PVP), with the prisms additionally stabilized by citrate. The nanoprisms dissolved slightly in pure water but strongly in isotonic saline or at pH 4, corresponding to the lowest limit for the pH during cellular uptake. The tips of the prisms became rounded within minutes due to their high surface energy. Afterward, the dissolution process slowed down due to the presence of both PVP stabilizing Ag{100} sites and citrate blocking Ag{111} sites. On the contrary, nanospheres, solely stabilized by PVP, dissolved within 24 h. These results correlate with the finding that particles in both cell types have lost >90% of their volume within 24 h. hMSC's took up significantly more Ag from nanoprisms than from nanospheres, whereas HaCaT cells showed no preference for one particle shape. This can be rationalized by the large cellular interaction area of the plateletlike nanoprisms and the bending stiffness of the cell membranes. hMSC's have a highly flexible cell membrane, resulting in an increased uptake of plateletlike particles. HaCaT cells have a membrane with a 3 orders of magnitude higher Young's modulus than for hMSC. Hence, the energy gain due to the larger interaction area of the nanoprisms is compensated for by the higher energy needed for cell membrane deformation compared to that for spheres, leading to no shape preference.

Laser Scanning Microscopic Investigations of the Decontamination of Soot Nanoparticles from the Skin.

BACKGROUND/AIMS: Airborne pollutants, such as nano-sized soot particles, are increasingly being released into the environment as a result of growing population densities and industrialization. They can absorb organic and metal compounds with potential biological activity, such as polycyclic aromatic hydrocarbons and airborne pollen allergens. Local and systemic toxicities may be induced in the skin if the particulates release their harmful components upon dermal contact. METHODS: In the present study, skin pretreatments with serum and/or shield as barrier formulations prior to exposure and washing with a cleanser subsequent to exposure were evaluated as a protection and decontamination strategy using laser scanning microscopy. RESULTS: The results indicate that while the application of serum and a cleanser was insufficient for decontamination, the pretreatment with shield prior to nanoparticle exposure followed by washing led to the removal of a considerable amount of the carbon black particles. The combined application of serum and shield before the administration of carbon black particles and subsequent washing led to their elimination from the skin samples. CONCLUSION: The application of barrier-enhancing formulations in combination with a cleanser may reduce the penetration of harmful airborne particulates by preventing their adhesion to the skin and facilitating their removal by subsequent washing with the cleanser.

Lysosomal Dysfunction Caused by Cellular Accumulation of Silica Nanoparticles.

Nanoparticles (NPs) are widely used as components of drugs or cosmetics and hold great promise for biomedicine, yet their effects on cell physiology remain poorly understood. Here we demonstrate that clathrin-independent dynamin 2-mediated caveolar uptake of surface-functionalized silica nanoparticles (SiNPs) impairs cell viability due to lysosomal dysfunction. We show that internalized SiNPs accumulate in lysosomes resulting in inhibition of autophagy-mediated protein turnover and impaired degradation of internalized epidermal growth factor, whereas endosomal recycling proceeds unperturbed. This phenotype is caused by perturbed delivery of cargo via autophagosomes and late endosomes to SiNP-filled cathepsin B/L-containing lysosomes rather than elevated lysosomal pH or altered mTOR activity. Given the importance of autophagy and lysosomal protein degradation for cellular proteostasis and clearance of aggregated proteins, these results raise the question of beneficial use of NPs in biomedicine and beyond.

Enhancing the sensitivity of nano-FTIR spectroscopy.

Synchrotron radiation-based nano-FTIR spectroscopy utilizes the highly brilliant and ultra-broadband infrared (IR) radiation provided by electron storage rings for the infrared spectroscopic characterization of samples at the nanoscale. In order to exploit the full potential of this approach we investigated the influence of the properties of the radiation source, such as the electron bunch shape and spectral bandwidth of the emitted radiation, on near-field infrared spectra of silicon-carbide (SiC). The adapted configuration of the storage ring optics enables a modification of the transverse electron bunch profile allowing an increase of the measured near-field signal amplitude. Additionally, the decay of the signal amplitude due to the decreasing storage ring current is also eliminated. Further options for improving the sensitivity of nano-FTIR spectroscopy, which can also be applied to other broadband radiation sources, are the adaption of the spectral bandwidth to the wavelength range of interest or the use of polarization optics. The sensitivity enhancement emerging from these options is verified by comparing near-field spectra collected from crystalline SiC samples. The improvement in sensitivity by combining these approaches is demonstrated by acquiring nano-FTIR spectra from thin organic films, which show weak resonances in the IR-regime.

Charge Effects on the Efflorescence in Single Levitated Droplets.

The influence of electrical excess charges on the crystallization from supersaturated aqueous sodium chloride solutions is reported. This is accomplished by efflorescence studies on single levitated microdroplets using optical and electrodynamic levitation. Specifically, a strong increase in efflorescence humidity is observed as a function of the droplet's negative excess charge, ranging up to -2.1 pC, with a distinct threshold behavior, increasing the relative efflorescence humidity, at which spontaneous nucleation occurs, from 44% for the neutral microparticle to 60%. These findings are interpreted by using molecular dynamics simulations for determining plausible structural patterns located near the particle surface that could serve as suitable precursors for the formation of critical clusters overcoming the nucleation barrier. These results, facilitating heterogeneous nucleation in the case of negatively charged microparticles, are compared to recent work on charge-induced nucleation of neat supercooled water, where a distinctly different nucleation behavior as a function of droplet charge has been observed.

Nanoscale plasmonic phenomena in CVD-grown MoS(2) monolayer revealed by ultra-broadband synchrotron radiation based nano-FTIR spectroscopy and near-field microscopy.

Nanoscale plasmonic phenomena observed in single and bi-layers of molybdenum disulfide (MoS(2)) on silicon dioxide (SiO(2)) are reported. A scattering type scanning near-field optical microscope (s-SNOM) with a broadband synchrotron radiation (SR) infrared source was used. We also present complementary optical mapping using tunable CO(2)-laser radiation. Specifically, there is a correlation of the topography of well-defined MoS(2) islands grown by chemical vapor deposition, as determined by atomic force microscopy, with the infrared (IR) signature of MoS(2). The influence of MoS(2) islands on the SiO(2) phonon resonance is discussed. The results reveal the plasmonic character of the MoS(2) structures and their interaction with the SiO(2) phonons leading to an enhancement of the hybridized surface plasmon-phonon mode. A theoretical analysis shows that, in the case of monolayer islands, the coupling of the MoS(2) optical plasmon mode to the SiO(2) surface phonons does not affect the infrared spectrum significantly. For two-layer MoS(2), the coupling of the extra inter-plane acoustic plasmon mode with the SiO(2) surface transverse phonon leads to a remarkable increase of the surface phonon peak at 794 cm(-1). This is in agreement with the experimental data. These results show the capability of the s-SNOM technique to study local multiple excitations in complex non-homogeneous structures.

Nanoscale plasmonic phenomena in CVD-grown MoS2 monolayer revealed by ultra-broadband synchrotron radiation based nano-FTIR spectroscopy and near-field microscopy: publisher's note.

This publisher's note amends the Acknowledgments of a recent publication [Opt. Express24, 1154 (2016)10.1364/OE.24.001154].

Role of the polymer matrix on the photoluminescence of embedded CdSe quantum dots.

The photoluminescence (PL) of CdSe quantum dots (QDs) that form stable nanocomposites with polymer liquid crystals (LCs) as smectic C hydrogen-bonded homopolymers from a family of poly[4-(n-acryloyloxyalkyloxy)benzoic acids] is reported. The matrix that results from the combination of these units with methoxyphenyl benzoate and cholesterol-containing units has a cholesteric structure. The exciton PL band of QDs in the smectic matrix is redshifted with respect to QDs in solution, whereas a blueshift is observed with the cholesteric matrix. The PL lifetimes and quantum yield in cholesteric nanocomposites are higher than those in smectic ones. This is interpreted in terms of a higher order of the smectic matrix in comparison to the cholesteric one. CdSe QDs in the ordered smectic matrix demonstrate a splitting of the exciton PL band and an enhancement of the photoinduced differential transmission. These results reveal the effects of the structure of polymer LC matrices on the optical properties of embedded QDs, which offer new possibilities for photonic applications of QD-LC polymer nanocomposites.

Characterization of semiconductor materials using synchrotron radiation-based near-field infrared microscopy and nano-FTIR spectroscopy.

We describe the application of scattering-type near-field optical microscopy to characterize various semiconducting materials using the electron storage ring Metrology Light Source (MLS) as a broadband synchrotron radiation source. For verifying high-resolution imaging and nano-FTIR spectroscopy we performed scans across nanoscale Si-based surface structures. The obtained results demonstrate that a spatial resolution below 40 nm can be achieved, despite the use of a radiation source with an extremely broad emission spectrum. This approach allows not only for the collection of optical information but also enables the acquisition of near-field spectral data in the mid-infrared range. The high sensitivity for spectroscopic material discrimination using synchrotron radiation is presented by recording near-field spectra from thin films composed of different materials used in semiconductor technology, such as SiO2, SiC, SixNy, and TiO2.

Skin barrier disruptions in tape stripped and allergic dermatitis models have no effect on dermal penetration and systemic distribution of AHAPS-functionalized silica nanoparticles.

The skin is a potential site of entry for nanoparticles (NP) but the role of disease-associated barrier disturbances on the path and extent of skin penetration of NP remains to be characterized. Silica nanoparticles (SiO2-NP) possess promising potential for various medical applications. Here, effects of different skin barrier disruptions on the penetration of N-(6-aminohexyl)-aminopropyltrimethoxysilane (AHAPS) functionalized SiO2-NP were studied. AHAPS-SiO2-NP (55±6 nm diameter) were topically applied on intact, tape stripped or on inflamed skin of SKH1 mice with induced allergic contact dermatitis for one or five consecutive days, respectively. Penetration of AHAPS-SiO2-NP through the skin was not observed regardless of the kind of barrier disruption. However, only after subcutaneous injection, AHAPS-SiO2-NP were incorporated by macrophages and transported to the regional lymph node only. Adverse effects on cells or tissues were not observed. In conclusion, AHAPS-SiO2-NP seem to not cross the normal or perturbed mouse skin. From the clinical editor: Skin is a potential site of entry for nanoparticles; however, it is poorly understood how skin diseases may alter this process. In tape-stripped skin and allergic contact dermatitis models the delivery properties of AHAPS-SiO2 nanoparticles remained unchanged, and in neither case were these NP-s able to penetrate the skin. No adverse effects were noted on the skin in these models and control mice.

Near-field imaging and nano-Fourier-transform infrared spectroscopy using broadband synchrotron radiation.

We demonstrate scanning near-field optical microscopy with a spatial resolution below 100 nm by using low intensity broadband synchrotron radiation in the IR regime. The use of such a broadband radiation source opens up the possibility to perform nano-Fourier-transform infrared spectroscopy over a wide spectral range.