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.

Mapping the intracellular distribution of carbon nanotubes after targeted delivery to carcinoma cells using confocal Raman imaging as a label-free technique.

The uptake of carbon nanotubes (CNTs) by mammalian cells and their distribution within cells is being widely studied in recent years due to their increasing use for biomedical purposes. The two main imaging techniques used are confocal fluorescence microscopy and transmission electron microscopy (TEM). The former, however, requires labeling of the CNTs with fluorescent dyes, while the latter is a work-intensive technique that is unsuitable for in situ bio-imaging. Raman spectroscopy, on the other hand, presents a direct, straightforward and label-free alternative. Confocal Raman microscopy can be used to image the CNTs inside cells, exploiting the strong Raman signal connected to different vibrational modes of the nanotubes. In addition, cellular components, such as the endoplasmic reticulum and the nucleus, can be mapped. We first validate our method by showing that only when using the CNTs' G band for intracellular mapping accurate results can be obtained, as mapping of the radial breathing mode (RBM) only shows a small fraction of CNTs. We then take a closer look at the exact localization of the nanotubes inside cells after folate receptor-mediated endocytosis and show that, after 8-10 h incubation, the majority of CNTs are localized around the nucleus. In summary, Raman imaging has enormous potential for imaging CNTs inside cells, which is yet to be fully realized.

Single-molecule recognition imaging microscopy.

Atomic force microscopy is a powerful and widely used imaging technique that can visualize single molecules and follow processes at the single-molecule level both in air and in solution. For maximum usefulness in biological applications, atomic force microscopy needs to be able to identify specific types of molecules in an image, much as fluorescent tags do for optical microscopy. The results presented here demonstrate that the highly specific antibody-antigen interaction can be used to generate single-molecule maps of specific types of molecules in a compositionally complex sample while simultaneously carrying out high-resolution topographic imaging. Because it can identify specific components, the technique can be used to map composition over an image and to detect compositional changes occurring during a process.

Reconstitution of membrane fusion sites. A total internal reflection fluorescence microscopy study of influenza hemagglutinin-mediated membrane fusion.

Influenza hemagglutinin (HA, strain A/PR/8/34) was purified and reconstituted into supported planar membranes in a two-step process: 1) HA was purified by C12E8 detergent solubilization followed by detergent removal with Biobeads; (2) the purified HA was then incorporated into "viroplanes," i.e. supported planar membranes which contained the viral membrane proteins. This step was accomplished by a spontaneous reaction of the HA-proteoliposomes with a phospholipid monolayer that was supported on a quartz microscope slide. The reconstitution of the HA into the planar membranes was followed by total internal reflection fluorescence microscopy (TIRFM) using fluorescein-labeled HA. By changing the solution concentration of HA, surface concentrations between 2.4 x 10(4) and 4.3 x 10(4) HA monomers/micron 2 were reached. Greater than 90% of all HA molecules were oriented with their ectodomain facing away from the substrate toward the large aqueous compartment of the measuring cell. Binding experiments with conformation-sensitive monoclonal antibodies against HA established that the reconstituted HA could undergo the low pH-induced conformational change in the supported bilayer. Binding of vesicles containing the fluorescent lipid analog N-(7-nitro-2,1,3-benzoxadiazol-4-yl)egg phosphatidylethanolamine was also measured by TIRFM. Vesicle binding was promoted when sialic acid-containing gangliosides or negatively charged lipids were included in these target membranes. Membrane fusion of the HA bound vesicles was monitored by measuring long range (over several micrometers) lateral diffusion coefficients of the lipids in the bound layer by fluorescence recovery after photobleaching. The vesicles did not fuse at pH 7.4, but efficient vesicle fusion occurred on the viroplanes after acidification of the environment with pH 5 buffer. This fusion reaction was only observed when the bound vesicles exceeded a critical threshold surface concentration. The successful reconstitution of membrane fusion sites in a planar supported membrane system opens new possibilities for studying fusion intermediates by localized spectroscopy and microscopy.