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.

Controlled Synthesis of Platinum and Silver Nanoparticles Using Multivalent Ligands.

Here, the controlled formation of platinum nanoparticles (PtNPs) and silver nanoparticles (AgNPs) using amine-functionalized multivalent ligands are reported. The effects of reaction temperature and ligand multivalency on the growth kinetics, size, and shape of PtNPs and AgNPs were systematically studied by performing a stepwise and a one-step process. PtNPs and AgNPs were prepared in the presence of amine ligands using platinum (II) acetylacetonate and silver (I) acetylacetonate, respectively. The effects of ligands and temperature on the formation of PtNPs were studied using a transmission electron microscope (TEM). For the characterization of AgNPs, additionally, ultraviolet-visible (UV-Vis) absorption was employed. The TEM measurements revealed that PtNPs prepared at different temperatures (160-200 °C, in a stepwise process) are monodispersed and of spherical shape regardless of the ligand multivalency or reaction temperature. In the preparation of PtNPs by the one-step process, ligands affect the shape of the PtNPs, which can be explained by the affinity of the ligands. The TEM and UV-Vis absorption studies on the formation of AgNPs with mono-, di-, and trivalent ligands showed narrower size distributions, while increasing the temperature from 80 °C to 120 °C and with a trivalent ligand in a one-step process.

Topical Delivery of Rapamycin by Means of Microenvironment-Sensitive Core-Multi-Shell Nanocarriers: Assessment of Anti-Inflammatory Activity in an ex vivo Skin/T Cell Co-Culture Model.

INTRODUCTION: Rapamycin (Rapa) is an immunosuppressive macrolide that inhibits the mechanistic target of rapamycin (mTOR) activity. Thanks to its anti-proliferative effects towards different cell types, including keratinocytes and T cells, Rapa shows promise in the treatment of skin diseases characterized by cell hyperproliferation. However, Rapa skin penetration is limited due to its lipophilic nature (log P = 4.3) and high molecular weight (MW = 914 g/mol). In previous studies, new microenvironment-sensitive core multishell (CMS) nanocarriers capable of sensing the redox state of inflamed skin were developed as more efficient and selective vehicles for macrolide delivery to inflamed skin. METHODS: In this study, we tested such redox-sensitive CMS nanocarriers using an inflammatory skin model based on human skin explants co-cultured with Jurkat T cells. Serine protease (SP) was applied on skin surface to induce skin barrier impairment and oxidative stress, whereas phytohaemagglutinin (PHA), IL-17A, and IL-22 were used to activate Jurkat cells. Activation markers, such as CD45 and CD69, phosphorylated ribosomal protein S6 (pRP-S6), and IL-2 release were monitored in activated T cells, whereas pro-inflammatory cytokines were measured in skin extracts and culture medium. RESULTS: We found that alteration of skin barrier proteins corneodesmosin (CDSN), occludin (Occl), and zonula occludens-1 (ZO-1) as well as oxidation-induced decrease of free thiol groups occurred upon SP-treatment. All Rapa formulations exerted inhibitory effects on T cells after penetration across ex vivo skin. No effects on skin inflammatory markers were detected. The superiority of the oxidative-sensitive CMS nanocarriers over the other formulations was observed with regard to drug delivery as well as downregulation of IL-2 release. CONCLUSION: Overall, our results demonstrate that nanocarriers addressing features of diseased skin are promising approaches to improve the topical delivery of macrolide drugs.

Improved Skin Permeability after Topical Treatment with Serine Protease: Probing the Penetration of Rapamycin by Scanning Transmission X-ray Microscopy.

Drug penetration in human skin ex vivo following a modification of skin barrier permeability is systematically investigated by scanning transmission X-ray microscopy. Element-selective excitation is used in the O 1s regime for probing quantitatively the penetration of topically applied rapamycin in different formulations with a spatial resolution reaching <75 nm. The data were analyzed by a comparison of two methods: (i) two-photon energies employing the Beer-Lambert law and (ii) a singular value decomposition approach making use of the full spectral information in each pixel of the X-ray micrographs. The latter approach yields local drug concentrations more reliably and sensitively probed than the former. The present results from both approaches indicate that rapamycin is not observed within the stratum corneum of nontreated skin ex vivo, providing evidence for the observation that this high-molecular-weight drug inefficiently penetrates intact skin. However, rapamycin is observed to penetrate more efficiently the stratum corneum when modifications of the skin barrier are induced by the topical pretreatment with the serine protease trypsin for variable time periods ranging from 2 to 16 h. After the longest exposure time to serine protease, the drug is even found in the viable epidermis. High-resolution micrographs indicate that the lipophilic drug preferably associates with corneocytes, while signals found in the intercellular lipid compartment were less pronounced. This result is discussed in comparison to previous work obtained from low-molecular-weight lipophilic drugs as well as polymer nanocarriers, which were found to penetrate the intact stratum corneum exclusively via the lipid layers between the corneocytes. Also, the role of the tight junction barrier in the stratum granulosum is briefly discussed with respect to modifications of the skin barrier induced by enhanced serine protease activity, a phenomenon of clinical relevance in a range of inflammatory skin disorders.

Oxidation-Sensitive Core-Multishell Nanocarriers for the Controlled Delivery of Hydrophobic Drugs.

A synthetic route for oxidation-sensitive core-multishell (osCMS) nanocarriers was established, and their drug loading and release properties were analyzed based on their structural variations. The nanocarriers showed a drug loading of 0.3-3 wt % for the anti-inflammatory drugs rapamycin and dexamethasone and the photosensitizer meso-tetra-hydroxyphenyl-porphyrin (mTHPP). Oxidative processes of the nanocarriers were probed in vitro by hydrogen peroxide, and the degradation products were identified by infrared spectroscopy supported by ab initio calculations, yielding mechanistic details on the chemical changes occurring in redox-sensitive nanocarriers. Oxidation-triggered drug release of the model drug Nile Red measured and assessed by time-dependent fluorescence spectroscopy showed a release of up to 80% within 24 h. The drug delivery capacity of the new osCMS nanocarriers was tested in ex vivo human skin with and without pretreatments to induce local oxidative stress. It was found that the delivery of mTHPP was selectively enhanced in skin under oxidative stress. The number and position of the thioether groups influenced the physicochemical as well as drug delivery properties of the carriers.

Sulfoxide-functionalized nanogels inspired by the skin penetration properties of DMSO.

Among polymeric nanocarriers, nanogels are especially promising non-irritating delivery vehicles to increase dermal bioavailability of therapeutics. However, accurately tailoring defined interactions with the amphiphilic skin barrier is still challenging. To address this limited specificity, we herein present a new strategy to combine biocompatible nanogels with the outstanding skin interaction properties of sulfoxide moieties. These chemical motifs are known from dimethyl sulfoxide (DMSO), a potent chemical penetration enhancer, which can often cause undesired skin damage upon long-term usage. By covalently functionalizing the nanogels' polymer network with such methyl sulfoxide side groups, tailor-made dermal delivery vehicles are developed to circumvent the skin disrupting properties of the small molecules. Key to an effective nanogel-skin interaction is assumed to be the specific nanogel amphiphilicity. This is examined by comparing the delivery efficiency of sulfoxide-based nanogels (NG-SOMe) with their corresponding thioether (NG-SMe) and sulfone-functionalized (NG-SO2Me) analogues. We demonstrate that the amphiphilic sulfoxide-based NG-SOMe nanogels are superior in their interaction with the likewise amphipathic stratum corneum (SC) showing an increased topical delivery efficacy of Nile red (NR) to the viable epidermis (VE) of excised human skin. In addition, toxicological studies on keratinocytes and fibroblasts show good biocompatibility while no perturbation of the complex protein and lipid distribution is observed via stimulated Raman microscopy. Thus, our NG-SOMe nanogels show high potential to effectively emulate the skin penetration enhancing properties of DMSO without its negative side effects.

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.

Polyglycerol-Based Thermoresponsive Nanocapsules Induce Skin Hydration and Serve as a Skin Penetration Enhancer.

The use of penetration enhancers (chemical or physical) has been proven to dramatically improve the penetration of therapeutics. Nevertheless, their use poses great risks, as they can lead to permanent damage of the skin, reduce its barrier efficiency, and result in the intrusion of harmful substances. Among the most used skin penetration enhancers, water is greatly accepted because skin quickly recovers from its exposure. Nanocapsules (NCs) represent a promising combination of the carrier system and penetration enhancer because their water-containing void combined with their polymer-based shell can be used to induce high local skin hydration, while simultaneously aiding the transport of drugs across the skin barrier. In this study, NCs were synthesized with a void core of 100 nm in diameter, a thermoresponsive shell based on different ratios of poly(N-isopropylacrylamide) and poly(N-isopropylmethacrylamide) as thermoresponsive polymers, and dendritic polyglycerol as a macromolecular cross-linker. These NCs can shrink or swell upon a thermal trigger, which was used to induce the release of the entrapped water in a controlled fashion. The interactions and effects of thermoresponsive NCs on the stratum corneum of excised human skin were investigated using fluorescence microscopy, high-resolution optical microscopy, and stimulated Raman spectromicroscopy. It could be observed that the thermoresponsive NCs increase the amount of deuterated water that penetrated into the viable epidermis. Moreover, NCs increased the skin penetration of a high-molecular weight dye (Atto Oxa12 NHS ester, MW = 835 g/mol) with respect to formulations in water or 30% DMSO, emphasizing the features of the NCs as a skin penetration enhancer.

Bio-Inspired Amphiphilic Block-Copolymers Based on Synthetic Glycopolymer and Poly(Amino Acid) as Potential Drug Delivery Systems.

In this work, a method to prepare hybrid amphiphilic block copolymers consisting of biocompatible synthetic glycopolymer with non-degradable backbone and biodegradable poly(amino acid) (PAA) was developed. The glycopolymer, poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG), was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Two methods for modifying the terminal dithiobenzoate-group of PMAG was investigated to obtain the macroinitiator bearing a primary aliphatic amino group, which is required for ring-opening polymerization of N-carboxyanhydrides of hydrophobic α-amino acids. The synthesized amphiphilic block copolymers were carefully analyzed using a set of different physico-chemical methods to establish their composition and molecular weight. The developed amphiphilic copolymers tended to self-assemble in nanoparticles of different morphology that depended on the nature of the hydrophobic amino acid present in the copolymer. The hydrodynamic diameter, morphology, and cytotoxicity of polymer particles based on PMAG-b-PAA were evaluated using dynamic light scattering (DLS) and transmission electron microscopy (TEM), as well as CellTiter-Blue (CTB) assay, respectively. The redox-responsive properties of nanoparticles were evaluated in the presence of glutathione taken at different concentrations. Moreover, the encapsulation of paclitaxel into PMAG-b-PAA particles and their cytotoxicity on human lung carcinoma cells (A549) and human breast adenocarcinoma cells (MCF-7) were studied.

Few-cycle laser driven reaction nanoscopy on aerosolized silica nanoparticles.

Nanoparticles offer unique properties as photocatalysts with large surface areas. Under irradiation with light, the associated near-fields can induce, enhance, and control molecular adsorbate reactions on the nanoscale. So far, however, there is no simple method available to spatially resolve the near-field induced reaction yield on the surface of nanoparticles. Here we close this gap by introducing reaction nanoscopy based on three-dimensional momentum-resolved photoionization. The technique is demonstrated for the spatially selective proton generation in few-cycle laser-induced dissociative ionization of ethanol and water on SiO2 nanoparticles, resolving a pronounced variation across the particle surface. The results are modeled and reproduced qualitatively by electrostatic and quasi-classical mean-field Mie Monte-Carlo (M3C) calculations. Reaction nanoscopy is suited for a wide range of isolated nanosystems and can provide spatially resolved ultrafast reaction dynamics on nanoparticles, clusters, and droplets.

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.

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.

pH-Sensitive Chitosan-Heparin Nanoparticles for Effective Delivery of Genetic Drugs into Epithelial Cells.

Chitosan has been extensively studied as a genetic drug delivery platform. However, its efficiency is limited by the strength of DNA and RNA binding. Expecting a reduced binding strength of cargo with chitosan, we proposed including heparin as a competing polyanion in the polyplexes. We developed chitosan-heparin nanoparticles by a one-step process for the local delivery of oligonucleotides. The size of the polyplexes was dependent on the mass ratio of polycation to polyanion. The mechanism of oligonucleotide release was pH-dependent and associated with polyplex swelling and collapse of the polysaccharide network. Inclusion of heparin enhanced the oligonucleotide release from the chitosan-based polyplexes. Furthermore, heparin reduced the toxicity of polyplexes in the cultured cells. The cell uptake of chitosan-heparin polyplexes was equal to that of chitosan polyplexes, but heparin increased the transfection efficiency of the polyplexes two-fold. The application of chitosan-heparin small interfering RNA (siRNA) targeted to vascular endothelial growth factor (VEGF) silencing of ARPE-19 cells was 25% higher. Overall, chitosan-heparin polyplexes showed a significant improvement of gene release inside the cells, transfection, and gene silencing efficiency in vitro, suggesting that this fundamental strategy can further improve the transfection efficiency with application of non-viral vectors.

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.

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.

Quasi-1D TiS3 Nanoribbons: Mechanical Exfoliation and Thickness-Dependent Raman Spectroscopy.

Quasi-one-dimensional (quasi-1D) materials enjoy growing interest due to their unusual physical properties and promise for miniature electronic devices. However, the mechanical exfoliation of quasi-1D materials into thin flakes and nanoribbons received considerably less attention from researchers than the exfoliation of conventional layered crystals. In this study, we investigated the micromechanical exfoliation of representative quasi-1D crystals, TiS3 whiskers, and demonstrate that they typically split into narrow nanoribbons with very smooth, straight edges and clear signatures of 1D TiS3 chains. Theoretical calculations show that the energies required for breaking weak interactions between the two-dimensional (2D) layers and between 1D chains within the layers are comparable and, in turn, are considerably lower than those required for breaking the covalent bonds within the chains. We also emulated macroscopic exfoliation experiments on the nanoscale by applying a local shear force to TiS3 crystals in different crystallographic directions using a tip of an atomic force microscopy (AFM) probe. In the AFM experiments, it was possible to slide the 2D TiS3 layers relative to each other as well as to remove selected 1D chains from the layers. We systematically studied the exfoliated TiS3 crystals by Raman spectroscopy and identified the Raman peaks whose spectral positions were most dependent on the crystals' thickness. These results could be used to distinguish between TiS3 crystals with thickness ranging from one to about seven monolayers. The conclusions established in this study for the exfoliated TiS3 crystals can be extended to a variety of transition metal trichalcogenide materials as well as other quasi-1D crystals. The possibility of exfoliation of TiS3 into narrow (few-nm wide) crystals with smooth edges could be important for the future realization of miniature device channels with reduced edge scattering of charge carriers.

Temperature-Dependent Structural Changes in Liquid Benzene.

Benzene is the simplest aromatic molecule with intermolecular π-π interactions. Because ordered liquids are key structures used to study chemical and biological phenomena in the liquid state, ordered structures of benzene confined in nanopores have been extensively studied, whereas those in the liquid state are still unknown. In this study, we address fundamental questions regarding whether ordered structures of benzene are formed in the liquid state by using carbon K-edge X-ray absorption spectroscopy (XAS) as a sensitive local probe. By comparing unexpected temperature behaviors of the π* peak in XAS spectra with model calculations, we have investigated temperature-dependent changes of ordered structures in liquid benzene caused by the increase in abundance of the parallel sandwich orientation relative to parallel displaced structures for the higher temperature. These results are confirmed by infrared spectroscopy with additional support of vibrational mode calculations.

Infrared Nanospectroscopy of Phospholipid and Surfactin Monolayer Domains.

A main challenge in understanding the structure of a cell membrane and its interactions with drugs is the ability to chemically study the different molecular species on the nanoscale. We have achieved this for a model system consisting of mixed monolayers (MLs) of the biologically relevant phospholipid 1,2-distearoyl-sn-glycero-phosphatidylcholine and the antibiotic surfactin. By employing nano-infrared (IR) microscopy and spectroscopy in combination with atomic force microscopy imaging, it was possible to identify and chemically detect domain formation of the two constituents as well as to obtain IR spectra of these species with a spatial resolution on the nanoscale. A novel method to enhance the near-field imaging contrast of organic MLs by plasmon interferometry is proposed and demonstrated. In this technique, the organic layer is deposited on gold and ML graphene substrates, the latter of which supports propagating surface plasmons. Plasmon reflections arising from changes in the dielectric environment provided by the organic layer lead to an additional contrast mechanism. Using this approach, the interfacial region between surfactin and the phospholipid has been mapped and a transition region is identified.