D. V. Bagrov

Mechanical properties of fibroblasts depend on level of cancer transformation.

Recently, it was revealed that tumor cells are significantly softer than normal cells. Although this phenomenon is well known, it is connected with many questions which are still unanswered. Among these questions are the molecular mechanisms which cause the change in stiffness and the correlation between cell mechanical properties and their metastatic potential. We studied mechanical properties of cells with different levels of cancer transformation. Transformed cells in three systems with different transformation types (monooncogenic N-RAS, viral and cells of tumor origin) were characterized according to their morphology, actin cytoskeleton and focal adhesion organization. Transformation led to reduction of cell spreading and thus decreasing the cell area, disorganization of actin cytoskeleton, lack of actin stress fibers and decline in the number and size of focal adhesions. These alterations manifested in a varying degree depending on type of transformation. Force spectroscopy by atomic force microscopy with spherical probes was carried out to measure the Youngs modulus of cells. In all cases the Youngs moduli were fitted well by log-normal distribution. All the transformed cell lines were found to be 40-80% softer than the corresponding normal ones. For the cell system with a low level of transformation the difference in stiffness was less pronounced than for the two other systems. This suggests that cell mechanical properties change upon transformation, and acquisition of invasive capabilities is accompanied by significant softening.

Hydrolytic Degradation of Poly(3-hydroxybutyrate), Polylactide and their Derivatives: Kinetics, Crystallinity, and Surface Morphology

Hydrolytic degradations of biodegradable poly(3-hydroxybutyrate) (PHB), polylactide (PLA) and their derivatives were explored by kinetic and structure methods at 37 and 70°C in phosphate buffer. It was revealed the kinetic profiles for copolymer PHBV (20% of 3-hydroxyvalerate) and the blend PHB-PLA (1:1 wt. ratio). The intensity of biopolymer hydrolysis depending on temperature is characterized by total weight loss and the viscosity-averaged molecular weight decrement (ΔMW) as well as by WAXS and AMF techniques. Characterization of PHB and PHBV includes both ΔMW and crystallinity evolution (x-ray diffraction) as well as the AFM analysis of PHB film surfaces before and after aggressive medium exposition. The degradation is enhanced in the series PHBV < PHB < PHB-PLA blend < PLA. The impact of MW on the biopolymer hydrolysis is shown.

The effects of confluency on cell mechanical properties

Mechanical properties of cells depend on various external and internal factors, like substrate stiffness and surface modifications, cell ageing and disease state. Some other currently unknown factors may exist. In this study we used force spectroscopy by AFM, confocal microscopy and flow cytometry to investigate the difference between single non-confluent and confluent (in monolayer) Vero cells. In all cases the stiffness values were fitted by log-normal rather than normal distribution. Log-normal distribution was also found for an amount of cortical actin in cells by flow cytometry. Cells in the monolayer were characterized by a significantly lower (1.4-1.7 times) Youngs modulus and amount of cortical actin than in either of the single non-confluent cells or cells migrating in the experimental wound. Youngs modulus as a function of indentation speed followed a weak power law for all the studied cell states, while the value of the exponent was higher for cells growing in monolayer. These results show that intercellular contacts and cell motile state significantly influence the cell mechanical properties.

Application of the Johnson-Kendall-Roberts model in AFM-based mechanical measurements on cells and gel.

The force-distance curves (FCs) obtained by the atomic force microscope (AFM) with colloid probes contain information about both the viscoelastic properties and adhesion of a sample. Here, we processed both the approach and retraction parts of FCs obtained on polyacrylamide gels (in water or PBS) and Vero cells (in a culture medium). The Johnson-Kendall-Roberts model was applied to the retraction curves to account for the adhesion. The effects of loading rate, holding time and indentation depth on adhesion force and Youngs modulus, calculated from approach and retraction curves, were studied. It was shown that both bulk and local interfacial viscoelasticity can affect the observed approach-retraction hysteresis and measured parameters. The addition of 1% bovine serum albumin (BSA) decreased adhesion of the probe to the PAA gel surface, so interfacial viscoelasticity effects were diminished. On the contrary, the adhesiveness of Vero cells increased after BSA addition, indicating the complex nature of the cell-probe interaction.

Atomic force microscopic study of the structure of high-density polyethylene deformed in liquid medium by crazing mechanism

A procedure has been developed for the direct atomic force microscopic (AFM) examination of the native structure of high‐density polyethylene (HDPE) deformed in an adsorption‐active liquid medium (AALM) by the crazing mechanism. The AFM investigation has been carried out in the presence of a liquid medium under conditions preventing deformed films from shrinkage. Deformation of HDPE in AALM has been shown to proceed through the delocalized crazing mechanism and result in the development of a fibrillar‐porous structure. The structural parameters of the crazed polymer have been determined. The obtained AFM images demonstrate a nanosized nonuniformity of the deformation and enable one to observe the structural rearrangements that take place in the deformed polymer after removal of the liquid medium and stress relaxation. A structural similarity has been revealed between HDPE deformed in the AALM and hard elastic polymers.

Atomic force microscopy of animal cells: Advances and prospects

We review the advances of the method of atomic force microscopy (AFM) for investigating the animal cells and analyze its development, paying much attention to studies of living cells. We consider the specific features and tasks of AFM, and a number of special AFM-based techniques. We discuss the choice of probe geometry for studies of animal cells, determination of cell adhesion on substrate, mapping of the cell surface using chemically modified cantilevers, and analysis of the distribution of molecular components inside the cell with the use of micro- and nanosurgical approaches, as well as combining AFM with optical and laser scanning confocal microscopy, and the possible applications of AFM in biotechnology and medicine.

Application of vasoactive and matrix-modifying drugs can improve polyplex delivery to tumors upon intravenous administration

Low efficacy of cationic polymer-based formulations (polyplexes) for systemic gene delivery to tumors remains the crucial concern for their clinical translation. Here we show that modulating the physiological state of a tumor using clinically approved pharmaceuticals can improve delivery of intravenously injected polyplexes to murine melanoma tumors with different characteristics. Direct comparison of drugs with different mechanisms of action has shown that application of nitroglycerin or losartan improved extravasation and tumor uptake of polyplex nanoparticles, whereas angiotensin II had almost no effect on polyplex accumulation and microdistribution in the tumor tissue. Application of nitroglycerin and losartan caused from 2- to 6-fold enhanced efficacy of polyplex-mediated gene delivery depending on the tumor model. The results obtained on polyplex behavior in tumor tissues depending on physiological state of the tumor can be relevant to optimize delivery of polyplexes and other nanomedicines with similar physicochemical properties.

Features of the delocalized crazing of high-density polyethylene in poly(ethylene oxide) solutions

It is shown that the deformation of high-density polyethylene films in poly(ethylene oxide) solutions occurs via the delocalized-crazing mechanism and leads to the formation of polymer blends. With an increase in the draw ratio, the concentration of poly(ethylene oxide) in the blend increases and significantly exceeds the theoretical values calculated from the polyethylene porosity and the poly(ethylene oxide) concentration in the solution. It is suggested that the high concentration of poly(ethylene oxide) in the blends is due to its adsorption on a highly developed surface of polyethylene deformed via the crazing mechanism. The composition of the resulting blends is independent of the strain rate.

Morphology and aggregation of RADA-16-I peptide studied by AFM, NMR and molecular dynamics simulations

RADA‐16‐I is a self‐assembling peptide which forms biocompatible fibrils and hydrogels. We used molecular dynamics simulations, atomic‐force microscopy, NMR spectroscopy, and thioflavin T binding assay to examine size, structure, and morphology of RADA‐16‐I aggregates. We used the native form of RADA‐16‐I (H‐(ArgAlaAspAla)4‐OH) rather than the acetylated one commonly used in the previous studies. At neutral pH, RADA‐16‐I is mainly in the fibrillar form, the fibrils consist of an even number of stacked β‐sheets. At acidic pH, RADA‐16‐I fibrils disassemble into monomers, which form an amorphous monolayer on graphite and monolayer lamellae on mica. RADA‐16‐I fibrils were compared with the fibrils of a similar peptide RLDL‐16‐I. Thickness of β‐sheets measured by AFM was in excellent agreement with the molecular dynamics simulations. A pair of RLDL‐16‐I β‐sheets was thicker (2.3 ± 0.4 nm) than a pair of RADA‐16‐I β‐sheets (1.9 ± 0.1 nm) due to the volume difference between alanine and leucine residues.

Atomic force microscopy of living and fixed Xenopus laevis embryos

Xenopus laevis embryos are a rather simple and at the same time a very interesting animal model, which is widely used for research in developmental biology. Intensive coordinated cell movements take place during the multi-cellular organism development. Little is known of the cellular, molecular and biomechanical mechanisms of these movements. The conceptual framework for analysis of cell interactions within integrated populations is poorly developed. We have used atomic force microscopy (AFM) to observe the surface of fixed X. laevis embryos at different stages of their development. We have developed a new sample preparation protocol for these observations. The obtained images were compared with scanning electronic microscopy (SEM) data. Cell rearrangement during morphogenesis in vivo was also visualized by AFM. In the current paper we discuss facilities and challenges of using this technique for further embryo researching.