Force Spectroscopy on a Cell Drum: AFM Measurements on the Basolateral Side of Cells via Inverted Cell Cultures

The elasticity of a cell is one of the most critical measures of the difference between cancerous cells and healthy cells: cancer cells tend to be softer than healthy cells, and highly invasive cells tend to be more elastic than less aggressive cells. In this work, we present the novel “bottom-up” cell force spectroscopy method for the biophysical characterization of cancer cells, in which an atomic force microscopy (AFM) tip approach from the backside of a net-shaped culture substrate exposing the basolateral cell membrane drum, and compare it with the conventional “top-down” AFM measurements. We used two different human pancreatic carcinoma cell lines, PaTu8988S and PaTu8988T. Our bottom-up AFM tip approach provided a more statistically synchronized distribution of the measured elastic moduli of the cells, demonstrating its superior applicability for the clinical use of force spectroscopy, which is not attainable with the conventional top-down AFM approach

Selective Inactivation of Resistant Gram-Positive Pathogens with a Light-Driven Hybrid Nanomaterial

Herein, we present a straightforward strategy to disperse highly insoluble photosensitizers in aqueous environments, without major synthetic efforts and keeping their photosensitizing abilities unaffected. A layered nanoclay was employed to adsorb and to solubilize a highly efficient yet hydrophobic Si­(IV) phthalocyaninate in water. The aggregation of the photoactive dye was correlated with its photophysical properties, particularly with the ability to produce highly cytotoxic singlet oxygen. Moreover, the resulting hybrid nanomaterial is able to selectively photoinactivate Gram-positive pathogens, due to local interactions between the bacterial membranes and the negatively charged nanodiscs. Nanotoxicity assays confirmed its innocuousness toward eukaryotic cells, showing that it constitutes a new class of “phototriggered magic bullet” for the inactivation of pathogens in phototherapy, as well as in the development of coatings for self-disinfecting surfaces

<i>In vitro</i> Luciferase expression of HUVECs transduced by MNBs/Ad_luc complexes and evaluation of magnetically controlled gene delivery.

<p>Tranduction efficiency enhanced by MNBs. Transduction was carried out in 48-well plates. Ad (expressed as Ad amount in unit area (pfu/cm²)) and MNBs dose were varied. RLU were normalized to the protein content of cell lysates. Data are expressed as the mean ± SD. (<i>n</i> = 6, three independent experiments).</p

In <i>vitro</i> LacZ expression of HUVECs tranduced by MNBs/Ad_Laz complexes and evaluation of transduction specificity.

<p>(A) The transduced HUVECs formed a three-spot pattern determined by the locations of the magnets. (B–C) LacZ reporter gene expression in the confined area influenced by the magnetic field and in an area remote from the magnetic field. (D) The expression of reporter gene LacZ was detected and formed a clear border between the transduced and untransduced cells.</p

Intravenously MNBs/Ad_hVEGF injection restored cardiac function 4 weeks after MI assessed by catherization.

<p>Left ventricular function at both baseline and stress conditions revealed the increments of left ventricular dp/dt max and dp/dt min under baseline and dobutamine stress compared with MI-M⁻MNBs/Ad_hVEGF group. Meanwhile the ejection fraction (EF) was enhanced under the dobutamine stress compared with the MI-M⁻MNBs/Ad_hVEGF group. Data ware mean values± SEM. *<i>P</i><0.05MI-M⁺MNBs/Ad_hVEGF versus Sham, #<i>P</i><0.05 versus MI-saline, ζ<i>P</i><0.05versus MI-MNBs and τ<i>P</i><0.05 versus MI-M-AdVEGF. (MI-M⁺MNBs/Ad_hVEGF, n = 10; MI-M⁻MNBs/Ad_hVEGF, n = 10; MI-MNBs, n = 10; MI-Saline, n = 10 and sham-operated, n = 5).</p

Inflammation response after intravenously injection of MNBs/Ad_hVEGF complexes.

<p>FACs analyses of mononuclear cells from peripheral blood in different groups. CD8⁺T cells of peripheral blood significantly enhanced in MI-M⁺MNBs/Ad_hVEGF compared to sham and other MI treated groups Data ware mean values± SEM. *<i>P</i><0.05MI-M⁺MNBs/Ad_hVEGF versus Sham, #<i>P</i><0.05 versus MI-saline, ζ<i>P</i><0.05versus MI-MNBs and τ<i>P</i><0.05 versus MI-M-AdVEGF. (MI-M⁺ MNBs/Ad_hVEGF, n = 4; MI-M⁻MNBs/Ad_hVEGF, n = 4; MI-MNBs, n = 10; MI-Saline, n = 4 and sham-operated, n = 4).</p

Characterization of MNBs/Ad complexes.

<p>Ad concentration (expressed as Ad amount in unit area (pfu/cm²)) was 8×10⁷ pfu/cm² and MNBs dose were varied. MNBs/Ad complexes group was increased whereas the naked Ad group was reduced with the increase of the MNBs dose. The complexes group became close to constant 100% from the MNBs dose at 16 µl. Data are expressed as the mean ± SD. (<i>n</i> = 6, three independent experiments).</p

Real time PCR analysis for the distribution and expression of magnetically controlled gene delivery after systemic infusion of MNBs/Ad_hVEGF or MNBs/Ad_GFP complexes.

<p>Quantitative Real-time PCR analysis for hVEGF gene expression in hearts, livers, spleens, lungs and kidneys in MI-M⁺MNBs/Ad_hVEGF, MI-M⁻MNBs/Ad_hVEGF and MI-saline groups. *<i>P</i><0.05 versus heart in MI-M⁻MNBs/Ad_hVEGF, #<i>P</i><0.05 verus liver in MI-M⁻MNBs/Ad_hVEGF. (MI-M⁺MNBs/Ad_hVEGF, n = 4; MI-M⁻MNBs/Ad_hVEGF, n = 4; MI-Saline, n = 4).</p

Cytotoxicity of MNBs/Ad_luc complexes in HUVECs.

<p>Ad (expressed as Ad amount in unit area (pfu/cm²)) and MNBs dose were varied. Values were expressed as a percentage of viability of MNBs/Ad_luc treated cells relative to that of Ad_luc alone treated cells (control). Data are expressed as the mean ± SD. (<i>n</i> = 6, three independent experiments).</p

Experimental design.

<p>After LAD-ligation rats received the magnet fixed onto the area of blanched myocardium.</p