Tuning the Extent and Depth of Penetration of Flexible Liposomes in Human Skin.

In this work we made an attempt to assess the effect of drug-induced changes of flexibility on the penetration of deformable vesicles into the human skin. Eight cationic liposomes with different degrees of flexibility were obtained by entrapping unfractionated heparin, enoxaparin, and nadroparin. The deformability was studied by a novel, facile, and reliable extrusion assay appositely developed and validated by means of quantitative nanoscale mechanical AFM measurements of vesicle elastic modulus (log10(YM)). The proposed extrusion assay, determining the forces involved in vesicles deformation, resulted very sensitive to evidence of minimal changes in bilayer rigidity (σ) and vesicle deformation (K). The drug loading caused a reduction of liposome flexibility with respect to the reference plain liposomes and in accordance to the heparin type, drug to cationic lipid (DOTAP) ratio, and drug distribution within the vesicles. Interestingly, the σ and log10(YM) values perfectly correlated (R2 = 0.935), demonstrating the reliability of the deformability data obtained with both approaches. The combination of TEM and LC-MS/MS spectrometry allowed the pattern of the penetration of the entire vesicles into the skin to be followed. In all cases, intact liposomes in the epidermis layers were observed and a relationship between the depth of penetration and the liposome flexibility was found, supporting the hypothesis of the whole vesicle penetration mechanism. Moreover, the results of the extent (R24) of vesicle penetration in the human skin samples showed a direct relation to the flexibility values (σ1 = 0.65 ± 0.10 MPa → R24 = 3.33 ± 0.02 µg/mg; σ2 = 0.95 ± 0.04 MPa → R24 = 1.18 ± 0.26 µg/mg; σ3 = 1.89 ± 0.30 MPa → R24 = 0.53 ± 0.33 µg/mg).

Pulsed Force Kelvin Probe Force Microscopy.

Measurement of the contact potential difference (CPD) and work functions of materials are important in analyzing their electronic structures and surface residual charges. Kelvin probe force microscopy (KPFM), an imaging technique of atomic force microscopy, has been widely used for surface potential and work function mapping at the nanoscale. However, the conventional KPFM variants are often limited in their spatial resolution to 30-100 nm under ambient conditions. The continuingly decreasing size and increasing complexity of photoactive materials and semiconductor devices will present future challenges in uncovering their nanometer-scale electrical properties through KPFM. Here, we introduce a KPFM technique based on the pulsed force mode of atomic force microscopy. Our technique, named pulsed force Kelvin Probe Force Microscopy (PF-KPFM), is a single-pass technique that utilizes the intrinsic Fermi level alignment between the AFM tip and the conductive sample without the need for an external oscillating voltage. Induced cantilever oscillations generated by a spontaneous redistribution of electrons between tip and sample are extracted and used to obtain the cantilever oscillation amplitude and to derive the surface potential. The spatial resolution of PF-KPFM is shown to be <10 nm under ambient conditions. The high spatial resolution surface potential mapping enables in situ determination of ohmic and nonohmic contacts between metals and semiconductors, mapping boundaries of ferroelectric domains of BaTiO3, as well as characterization of protein aggregates. High spatial resolution measurements with PF-KPFM will facilitate further studies directed at uncovering electrical properties for emerging photoactive materials, biological samples, and semiconductor devices.

Rapid recognition and functional analysis of membrane proteins on human cancer cells using atomic force microscopy.

Understanding the physicochemical properties of cell surface signalling molecules is important for us to uncover the underlying mechanisms that guide the cellular behaviors. Atomic force microscopy (AFM) has become a powerful tool for detecting the molecular interactions on individual cells with nanometer resolution. In this paper, AFM peak force tapping (PFT) imaging mode was applied to rapidly locate and visually map the CD20 molecules on human lymphoma cells using biochemically sensitive tips. First, avidin-biotin system was used to test the effectiveness of using PFT imaging mode to probe the specific molecular interactions. The adhesion images obtained on avidin-coated mica using biotin-tethered tips obviously showed the recognition spots which corresponded to the avidins in the simultaneously obtained topography images. The experiments confirmed the specificity and reproducibility of the recognition results. Then, the established procedure was applied to visualize the nanoscale organization of CD20s on the surface of human lymphoma Raji cells using rituximab (a monoclonal anti-CD20 antibody)-tethered tips. The experiments showed that the recognition spots in the adhesion images corresponded to the specific CD20-rituximab interactions. The cluster sizes of CD20s on lymphoma Raji cells were quantitatively analyzed from the recognition images. Finally, under the guidance of fluorescence recognition, the established procedure was applied to cancer cells from a clinical lymphoma patient. The results showed that there were significant differences between the adhesion images obtained on cancer cells and on normal cells (red blood cell). The CD20 distributions on ten cancer cells from the patient were quantified according to the adhesion images. The experimental results demonstrate the capability of applying PFT imaging to rapidly investigate the nanoscale biophysical properties of native membrane proteins on the cell surface, which is of potential significance in developing novel biomarkers for cancer diagnosis and drug development.

Epoxy-acrylic core-shell particles by seeded emulsion polymerization.

We developed a novel method for synthesizing epoxy-acrylic hybrid latexes. We first prepared an aqueous dispersion of high molecular weight solid epoxy prepolymers using a mechanical dispersion process at elevated temperatures, and we subsequently used the epoxy dispersion as a seed in the emulsion polymerization of acrylic monomers comprising methyl methacrylate (MMA) and methacrylic acid (MAA). Advanced analytical techniques, such as scanning transmission X-ray microscopy (STXM) and peak force tapping atomic force microscopy (PFT-AFM), have elucidated a unique core-shell morphology of the epoxy-acrylic hybrid particles. Moreover, the formation of the core-shell morphology in the seeded emulsion polymerization process is primarily attributed to kinetic trapping of the acrylic phase at the exterior of the epoxy particles. By this new method, we are able to design the epoxy and acrylic polymers in two separate steps, and we can potentially synthesize epoxy-acrylic hybrid latexes with a broad range of compositions.

QUANTITATIVE NANOMECHANICAL MAPPING OF MARINE DIATOM IN SEAWATER USING PEAK FORCE TAPPING ATOMIC FORCE MICROSCOPY(1).

It is generally accepted that a diatom cell wall is characterized by a siliceous skeleton covered by an organic envelope essentially composed of polysaccharides and proteins. Understanding of how the organic component is associated with the silica structure provides an important insight into the biomineralization process and patterning on the cellular level. Using a novel atomic force microscopy (AFM) imaging technique (Peak Force Tapping), we characterized nanomechanical properties (elasticity and deformation) of a weakly silicified marine diatom Cylindrotheca closterium (Ehrenb.) Reimann et J. C. Lewin (strain CCNA1). The nanomechanical properties were measured over the entire cell surface in seawater at a resolution that was not achieved previously. The fibulae were the stiffest (200 MPa) and the least deformable (only 1 nm). Girdle band region appeared as a series of parallel stripes characterized by two sets of values of Young's modulus and deformation: one for silica stripes (43.7 Mpa, 3.7 nm) and the other between the stripes (21.3 MPa, 13.4 nm). The valve region was complex with average values of Young's modulus (29.8 MPa) and deformation (10.2 nm) with high standard deviations. After acid treatment, we identified 15 nm sized silica spheres in the valve region connecting raphe with the girdle bands. The silica spheres were neither fused together nor forming a nanopattern. A cell wall model is proposed with individual silica nanoparticles incorporated in an organic matrix. Such organization of girdle band and valve regions enables the high flexibility needed for movement and adaptation to different environments while maintaining the integrity of the cell.