Glycan distribution and density in native skin's stratum corneum.

BACKGROUND: The glycosylation of proteins on the surface of corneocytes is believed to play an important role in cellular adhesion in the stratum corneum (SC) of human skin. Mapping with accuracy the localization of glycans on the surface of corneocytes through traditional methods of immunohistochemistry and electron microscopy remains a challenging task as both approaches lack enough resolution or need to be performed in high vacuum conditions. MATERIALS AND METHODS: We used an advanced mode of atomic force microscope (AFM), with simultaneous topography and recognition imaging to investigate the distribution of glycans on native (no chemical preparation) stripped samples of human SC. The AFM cantilever tips were functionalized with anti-heparan sulfate antibody and the lectin wheat germ agglutinin (WGA) which binds specifically to N-acetyl glucosamine and sialic acid. RESULTS: From the recognition imaging, we observed the presence of the sulfated glycosaminoglycan, heparan sulfate, and the glycans recognized by WGA on the surface of SC corneocytes in their native state. These glycans were found associated with bead-like domains which represent corneodesmosomes in the SC layers. Glycan density was calculated to be ~1200 molecules/µm2 in lower layers of SC compared to an important decrease, (~106 molecules/µm2 ) closer to the surface due probably to corneodesmosome degradation. CONCLUSION: Glycan spatial distribution and degradation is first observed on the surface of SC in native conditions and at high resolution. The method used can be extended to precisely localize the presence of other macromolecules on the surface of skin or other tissues where the maintenance of its native state is required.

A single-molecule approach to explore binding, uptake and transport of cancer cell targeting nanotubes.

In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic 'roadmap' that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity.

AFM imaging of functionalized carbon nanotubes on biological membranes.

Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

AFM imaging of functionalized double-walled carbon nanotubes.

We present a comparative study of several non-covalent approaches to disperse, debundle and non-covalently functionalize double-walled carbon nanotubes (DWNTs). We investigated the ability of bovine serum albumin (BSA), phospholipids grafted onto amine-terminated polyethylene glycol (PL-PEG(2000)-NH(2)), as well as a combination thereof, to coat purified DWNTs. Topographical imaging with the atomic force microscope (AFM) was used to assess the coating of individual DWNTs and the degree of debundling and dispersion. Topographical images showed that functionalized DWNTs are better separated and less aggregated than pristine DWNTs and that the different coating methods differ in their abilities to successfully debundle and disperse DWNTs. Height profiles indicated an increase in the diameter of DWNTs depending on the functionalization method and revealed adsorption of single molecules onto the nanotubes. Biofunctionalization of the DWNT surface was achieved by coating DWNTs with biotinylated BSA, providing for biospecific binding of streptavidin in a simple incubation step. Finally, biotin-BSA-functionalized DWNTs were immobilized on an avidin layer via the specific avidin-biotin interaction.