Changes in ROS/RNS Levels in Endothelial Cells in Experimental Bacteremia.

A high-precision system was developed for the quantification of biological analytes in single cells (reactive oxygen species (ROS) and reactive nitrogen species (RNS)) based on the electrochemical amperometric method. The efficacy of this system was evaluated using an experimental bacteremia model. Endothelial cells exhibited increased ROS/RNS production when stimulated by Staphylococcus aureus. However, they remained inactive when exposed to either unprimed or primed neutrophils that had been pre-incubated with bacteria. It is noteworthy that the sequential stimulation of endothelial cells with bacteria followed by neutrophils resulted in a significant increase in the ROS/RNS level, which demonstrated a correlation with the number of neutrophils in contact with the endothelial cells. These results highlight the potential of our system to quantitatively assess ROS/RNS dynamics in complex biological systems. They also offer insights into the interplay between various cellular components in experimental bacteremia.

Effect of LDL Extracted from Human Plasma on Membrane Stiffness in Living Endothelial Cells and Macrophages via Scanning Ion Conductance Microscopy.

Mechanical properties of living cells play a crucial role in a wide range of biological functions and pathologies, including atherosclerosis. We used low-stress Scanning Ion-Conductance Microscopy (SICM) correlated with confocal imaging and demonstrated the topographical changes and mechanical properties alterations in EA.hy926 and THP-1 exposed to LDL extracted from CVD patients' blood samples. We show that the cells stiffened in the presence of LDL, which also triggered caveolae formation. Endothelial cells accumulated less cholesterol in the form of lipid droplets in comparison to THP-1 cells based on fluorescence intensity data and biochemical analysis; however, the effect on Young's modulus is higher. The cell stiffness is closely connected to the distribution of lipid droplets along the z-axis. In conclusion, we show that the sensitivity of endothelial cells to LDL is higher compared to that of THP-1, triggering changes in the cytoskeleton and membrane stiffness which may result in the increased permeability of the intima layer due to loss of intercellular connections and adhesion.

Single-Cell Analysis with Silver-Coated Pipette by Combined SERS and SICM.

The study of individual cell processes that occur both on their surface and inside is highly interesting for the development of new medical drugs, cytology and cell technologies. This work presents an original technique for fabricating the silver-coated pipette and its use for the cell analysis by combination with surface-enhanced Raman spectroscopy (SERS) and scanning ion-conducting microscopy (SICM). Unlike the majority of other designs, the pipette opening in our case remains uncovered, which is important for SICM. SERS-active Ag nanoparticles on the pipette surface are formed by vacuum-thermal evaporation followed by annealing. An array of nanoparticles had a diameter on the order of 36 nm and spacing of 12 nm. A two-particle model based on Laplace equations is used to calculate a theoretical enhancement factor (EF). The surface morphology of the samples is investigated by scanning electron microscopy while SICM is used to reveal the surface topography, to evaluate Young's modulus of living cells and to control an injection of the SERS-active pipettes into them. A Raman microscope-spectrometer was used to collect characteristic SERS spectra of cells and cell components. Local Raman spectra were obtained from the cytoplasm and nucleus of the same HEK-293 cancer cell. The EF of the SERS-active pipette was 7 × 105. As a result, we demonstrate utilizing the silver-coated pipette for both the SICM study and the molecular composition analysis of cytoplasm and the nucleus of living cells by SERS. The probe localization in cells is successfully achieved.

Scanning Ion-Conductance Microscopy for Studying Mechanical Properties of Neuronal Cells during Local Delivery of Glutamate.

Mechanical properties of neuronal cells have a key role for growth, generation of traction forces, adhesion, migration, etc. Mechanical properties are regulated by chemical signaling, neurotransmitters, and neuronal ion exchange. Disturbance of chemical signaling is accompanied by several diseases such as ischemia, trauma, and neurodegenerative diseases. It is known that the disturbance of chemical signaling, like that caused by glutamate excitotoxicity, leads to the structural reorganization of the cytoskeleton of neuronal cells and the deviation of native mechanical properties. Thus, to investigate the mechanical properties of living neuronal cells in the presence of glutamate, it is crucial to use noncontact and low-stress methods, which are the advantages of scanning ion-conductance microscopy (SICM). Moreover, a nanopipette may be used for the local delivery of small molecules as well as for a probe. In this work, SICM was used as an advanced technique for the simultaneous local delivery of glutamate and investigation of living neuronal cell morphology and mechanical behavior caused by an excitotoxic effect of glutamate.

Scanning Ion-Conductance Microscopy for Studying ß-Amyloid Aggregate Formation on Living Cell Surfaces.

ß-Amyloid aggregation on living cell surfaces is described as responsible for the neurotoxicity associated with different neurodegenerative diseases. It is suggested that the aggregation of ß-amyloid (Aß) peptide on neuronal cell surface leads to various deviations of its vital function due to myriad pathways defined by internalization of calcium ions, apoptosis promotion, reduction of membrane potential, synaptic activity loss, etc. These are associated with structural reorganizations and pathologies of the cell cytoskeleton mainly involving actin filaments and microtubules and consequently alterations of cell mechanical properties. The effect of amyloid oligomers on cells' Young's modulus has been observed in a variety of studies. However, the precise connection between the formation of amyloid aggregates on cell membranes and their effects on the local mechanical properties of living cells is still unresolved. In this work, we have used correlative scanning ion-conductance microscopy (SICM) to study cell topography, Young's modulus mapping, and confocal imaging of Aß aggregate formation on living cell surfaces. However, it is well-known that the cytoskeleton state is highly connected to the intracellular level of reactive oxygen species (ROS). The effect of Aß leads to the induction of oxidative stress, actin polymerization, and stress fiber formation. We measured the reactive oxygen species levels inside single cells using platinum nanoelectrodes to demonstrate the connection of ROS and Young's modulus of cells. SICM can be successfully applied to studying the cytotoxicity mechanisms of Aß aggregates on living cell surfaces.

Pitstop-2 and its novel derivative RVD-127 disrupt global cell dynamics and nuclear pores integrity by direct interaction with small GTPases.

Clathrin-mediated endocytosis (CME) is an essential cell physiological process of broad biomedical relevance. Since the recent introduction of Pitstop-2 as a potent CME inhibitor, we and others have reported on substantial clathrin-independent inhibitory effects. Herein, we developed and experimentally validated a novel fluorescent derivative of Pitstop-2, termed RVD-127, to clarify Pitstop-2 diverse effects. Using RVD-127, we were able to trace additional protein targets of Pitstop-2. Besides inhibiting CME, Pitstop-2 and RVD-127 proved to directly and reversibly bind to at least two members of the small GTPase superfamily Ran and Rac1 with particularly high efficacy. Binding locks the GTPases in a guanosine diphosphate (GDP)-like conformation disabling their interaction with their downstream effectors. Consequently, overall cell motility, mechanics and nucleocytoplasmic transport integrity are rapidly disrupted at inhibitor concentrations well below those required to significantly reduce CME. We conclude that Pitstop-2 is a highly potent, reversible inhibitor of small GTPases. The inhibition of these molecular switches of diverse crucial signaling pathways, including nucleocytoplasmic transport and overall cell dynamics and motility, clarifies the diversity of Pitstop-2 activities. Moreover, considering the fundamental importance and broad implications of small GTPases in physiology, pathophysiology and drug development, Pitstop-2 and RVD-127 open up novel avenues.

ROS Production by a Single Neutrophil Cell and Neutrophil Population upon Bacterial Stimulation.

The reactive oxygen species (ROS) production by a single neutrophil after stimulation with S. aureus and E. coli was estimated by an electrochemical amperometric method with a high time resolution. This showed significant variability in the response of a single neutrophil to bacterial stimulation, from a "silent cell" to a pronounced response manifested by a series of chronoamperometric spikes. The amount of ROS produced by a single neutrophil under the influence of S. aureus was 5.5-fold greater than that produced under the influence of E. coli. The response of a neutrophil granulocyte population to bacterial stimulation was analyzed using luminol-dependent biochemiluminescence (BCL). The stimulation of neutrophils with S. aureus, as compared to stimulation with E. coli, caused a total response in terms of ROS production that was seven-fold greater in terms of the integral value of the light sum and 13-fold greater in terms of the maximum peak value. The method of ROS detection at the level of a single cell indicated the functional heterogeneity of the neutrophil population, but the specificity of the cellular response to different pathogens was the same at the cellular and population levels.

Scanning ion-conductance microscopy technique for studying the topography and mechanical properties of Candida parapsilosis yeast microorganisms.

Super-resolution microscopy is widely used in the development of novel antimicrobial testing in vitro. In the presented work, a scanning protocol was developed by the method of scanning ion-conducting microscopy (SICM), which makes it possible to study microorganisms without rigid fixation and in saline, obtaining an index map of nanosized structures. The effect of azole and echinocandins drugs on the morphology and mechanical properties of Candida parapsilosis yeast was studied. The findings are consistent with previously proposed drug mechanisms and reports that have examined antifungal agents using AFM, SEM, and TEM. We have shown that the SICM method is capable of scanning and detecting the nanomechanical properties of yeast non-invasively.

Sensing Cells-Peptide Hydrogel Interaction In Situ via Scanning Ion Conductance Microscopy.

Peptide-based hydrogels were shown to serve as good matrices for 3D cell culture and to be applied in the field of regenerative medicine. The study of the cell-matrix interaction is important for the understanding of cell attachment, proliferation, and migration, as well as for the improvement of the matrix. Here, we used scanning ion conductance microscopy (SICM) to study the growth of cells on self-assembled peptide-based hydrogels. The hydrogel surface topography, which changes during its formation in an aqueous solution, were studied at nanoscale resolution and compared with fluorescence lifetime imaging microscopy (FLIM). Moreover, SICM demonstrated the ability to map living cells inside the hydrogel. A zwitterionic label-free pH nanoprobe with a sensitivity > 0.01 units was applied for the investigation of pH mapping in the hydrogel to estimate the hydrogel applicability for cell growth. The SICM technique that was applied here to evaluate the cell growth on the peptide-based hydrogel can be used as a tool to study functional living cells.

PSMA-targeted small-molecule docetaxel conjugate: Synthesis and preclinical evaluation.

Prostate cancer is one of the most commonly diagnosed men's cancers and remains one of the leading causes of cancer death. The development of approaches to the treatment of this oncological disease is an ongoing process. In this work, we have carried out the selection of ligands for the creation of conjugates based on the drug docetaxel and synthesized a series of three docetaxel conjugates. In vitro cytotoxicity of these molecules was evaluated using the MTT assay. Based on the assay results, we selected the conjugate which showed cytotoxic potential close to unmodified docetaxel. At the same time, the molar solubility of the resulting compound increased up to 20 times in comparison with the drug itself. In vivo evaluation on 22Rv1 (PSMA+) xenograft model demonstrated a good potency of the synthesized conjugate to inhibit tumor growth: the inhibition turned out to be more than 80% at a dose of 30 mg/kg. Pharmacokinetic parameters of conjugate distribution were analyzed. Also, it was found that PSMA-targeted docetaxel conjugate is less toxic than docetaxel itself, the decrease of molar acute toxicity in comparison with free docetaxel was up to 20%. Obtained conjugate PSMA-DOC is a good candidate for further expanded preclinical trials because of high antitumor activity, fewer side toxic effects and better solubility.

Synthesis, Characterization, and Preclinical Evaluation of a Small-Molecule Prostate-Specific Membrane Antigen-Targeted Monomethyl Auristatin E Conjugate.

Prostate cancer is the second most common type of cancer among men. Its main method of treatment is chemotherapy, which has a wide range of side effects. One of the solutions to this challenge is targeted delivery to prostate cancer cells. Here we synthesized a novel small-molecule PSMA-targeted conjugate based on the monomethyl auristatin E. Its structure and conformational properties were investigated by NMR spectroscopy. Cytotoxicity, intracellular reactive oxygen species induction, and stability under liver microsomes and P450-cytochrome species were investigated for this conjugate. The conjugate demonstrated 77-85% tumor growth inhibition levels on 22Rv1 (PSMA (+)) xenografts, compared with a 37% inhibition level on PC-3 (PSMA (-)) xenografts, in a single dose of 0.3 mg/kg and a sufficiently high therapeutic index of 21. Acute, chronic, and subchronic toxicities and pharmacokinetics have shown that the synthesized conjugate is a promising potential agent for the chemotherapy of prostate cancer.

Mapping mechanical properties of living cells at nanoscale using intrinsic nanopipette-sample force interactions.

Mechanical properties of living cells determined by cytoskeletal elements play a crucial role in a wide range of biological functions. However, low-stress mapping of mechanical properties with nanoscale resolution but with a minimal effect on the fragile structure of cells remains difficult. Scanning Ion-Conductance Microscopy (SICM) for quantitative nanomechanical mapping (QNM) is based on intrinsic force interactions between nanopipettes and samples and has been previously suggested as a promising alternative to conventional techniques. In this work, we have provided an alternative estimation of intrinsic force and stress and demonstrated the possibility to perform qualitative and quantitative analysis of cell nanomechanical properties of a variety of living cells. Force estimation on decane droplets with well-known elastic properties, similar to living cells, revealed that the forces applied using a nanopipette are much smaller than in the case using atomic force microscopy. We have shown that we can perform nanoscale topography and QNM using a scanning procedure with no detectable effect on live cells, allowing long-term QNM as well as detection of nanomechanical properties under drug-induced alterations of actin filaments and microtubulin.