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Nano-Motion Analysis for Rapid and Label Free Assessing of Cancer Cell Sensitivity to Chemotherapeutics
Optimization of chemotherapy is crucial for cancer patients. Timely and costly efficient treatments are emerging due to the increasing incidence of cancer worldwide. Here, we present a methodology of nano-motion analysis that could be developed to serve as a screening tool able to determine the best chemotherapy option for a particular patient within hours. Materials and Methods: Three different human cancer cell lines and their multidrug resistant (MDR) counterparts were analyzed with an atomic force microscope (AFM) using tipless cantilevers to adhere the cells and monitor their nano-motions. Results: The cells exposed to doxorubicin (DOX) differentially responded due to their sensitivity to this chemotherapeutic. The death of sensitive cells corresponding to the drop in signal variance occurred in less than 2 h after DOX application, while MDR cells continued to move, even showing an increase in signal variance. Conclusions: Nano-motion sensing can be developed as a screening tool that will allow simple, inexpensive and quick testing of different chemotherapeutics for each cancer patient. Further investigations on patient-derived tumor cells should confirm the method’s applicability.
A perspective view on the nanomotion detection of living organisms and its features
The insurgence of newly arising, rapidly developing health threats, such as drug-resistant bacteria and cancers, is one of the most urgent public-health issues of modern times. This menace calls for the development of sensitive and reliable diagnostic tools to monitor the response of single cells to chemical or pharmaceutical stimuli. Recently, it has been demonstrated that all living organisms oscillate at a nanometric scale and that these oscillations stop as soon as the organisms die. These nanometric scale oscillations can be detected by depositing living cells onto a micro-fabricated cantilever and by monitoring its displacements with an atomic force microscope-based electronics. Such devices, named nanomotion sensors, have been employed to determine the resistance profiles of life-threatening bacteria within minutes, to evaluate, among others, the effect of chemicals on yeast, neurons, and cancer cells. The data obtained so far demonstrate the advantages of nanomotion sensing devices in rapidly characterizing microorganism susceptibility to pharmaceutical agents. Here, we review the key aspects of this technique, presenting its major applications. and detailing its working protocols.
Nanomotion Detection Method for Testing Antibiotic Resistance and Susceptibility of Slow-Growing Bacteria
Infectious diseases are caused by pathogenic microorganisms and are often severe. Time to fully characterize an infectious agent after sampling and to find the right antibiotic and dose are important factors in the overall success of a patient's treatment. Previous results suggest that a nanomotion detection method could be a convenient tool for reducing antibiotic sensitivity characterization time to several hours. Here, the application of the method for slow-growing bacteria is demonstrated, taking Bordetella pertussis strains as a model. A low-cost nanomotion device is able to characterize B. pertussis sensitivity against specific antibiotics within several hours, instead of days, as it is still the case with conventional growth-based techniques. It can discriminate between resistant and susceptible B. pertussis strains, based on the changes of the sensor's signal before and after the antibiotic addition. Furthermore, minimum inhibitory and bactericidal concentrations of clinically applied antibiotics are compared using both techniques and the suggested similarity is discussed.
AFM contribution to unveil pro- and eukaryotic cell mechanical properties
Atomic force microscopy is nowadays a well-established technique that permits the investigation of numerous parameters of living matter. In particular, it allows the exploration of the mechanical properties of living organisms in almost physiological conditions. Here, we focus on the use of this technology to review recent contributions that relates the physiology and pathology of bacteria, yeast, plant and mammalian cells to their nano-mechanical properties.
Nanomechanical sensor applied to blood culture pellets: a fast approach to determine the antibiotic susceptibility against agents of bloodstream infections
The management of bloodstream infection, a life-threatening disease, largely relies on early detection of infecting microorganisms and accurate determination of their antibiotic susceptibility to reduce both mortality and morbidity. Recently we developed a new technique based on atomic force microscopy capable of detecting movements of biologic samples at the nanoscale. Such sensor is able to monitor the response of bacteria to antibiotic's pressure, allowing a fast and versatile susceptibility test. Furthermore, rapid preparation of a bacterial pellet from a positive blood culture can improve downstream characterization of the recovered pathogen as a result of the increased bacterial concentration obtained. Using artificially inoculated blood cultures, we combined these two innovative procedures and validated them in double-blind experiments to determine the susceptibility and resistance of Escherichia coli strains (ATCC 25933 as susceptible and a characterized clinical isolate as resistant strain) towards a selection of antibiotics commonly used in clinical settings. On the basis of the variance of the sensor movements, we were able to positively discriminate the resistant from the susceptible E. coli strains in 16 of 17 blindly investigated cases. Furthermore, we defined a variance change threshold of 60% that discriminates susceptible from resistant strains. By combining the nanomotion sensor with the rapid preparation method of blood culture pellets, we obtained an innovative, rapid and relatively accurate method for antibiotic susceptibility test directly from positive blood culture bottles, without the need for bacterial subculture.
Mitochondrial activity detected by cantilever based sensor
Our team recently demonstrated that cantilever based devices can detect signature of life in a chemistry independent manner. In this technique, the organism of interest is attached onto a classical AFM cantilever. If alive, it induces nanometre scale oscillations of the cantilever that disappear once the organism is killed. The technique was successfully used on bacteria, yeast, vegetal and mammalian cells. In this work we demonstrate that the method can also be applied to sub-cellular organelles, such as mitochondria. Mitochondria are involved in cellular energy production and are present in most eukaryotic cells. Nowadays, it is believed that mitochondria were originally prokaryotes that colonized eukaryotic cells and that live in an endosymbiotic way ever since. Here we present that mitochondria are also animated by nanometre scale oscillations that depend on their metabolic state and that stop once they are inhibited. This observation opens novel avenues to investigate the numerous mitochondria-related diseases in humans.
Fixation and Permeabilization Approaches for Scanning Electrochemical Microscopy of Living Cells
Scanning electrochemical microscopy (SECM) has been widely used for the electrochemical imaging of dynamic topographical and metabolic changes in alive adherent mammalian cells. However, extracting intracellular information by SECM is challenging, since it requires redox species to travel in and out the lipid cell membrane. Herein, we present cell fixation and permeabilization approaches as an alternative tool for visualizing cell properties by SECM. With this aim, adherent cells were analyzed in the SECM feedback mode in three different conditions: (i) alive; (ii) fixed, and (iii) fixed and permeabilized. The fixation was carried out with formaldehyde and does not damage lipid membranes. Therefore, this strategy can be used for the SECM investigation of cell topography or the passive transport of the redox mediator into the cells. Additional permeabilization of the cell membrane after fixation enables the analysis of the intracellular content through the coupling of SECM with immunoassay strategies for the detection of specific biomarkers. The latter was successfully applied as an easy and fast screening approach to detect the expression of the melanoma-associated marker tyrosinase in adherent melanoma cell lines corresponding to different cancer progression stages using the SECM substrate generation–tip collection mode. The present approach is simple, fast, and reliable and can open new ways to analyze cell cultures with electrochemically based scanning probe techniques.
Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy
Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.
Détecter la vie grâce à la microscopie à force atomique
L’existence de créatures extraterrestres est une question qui a toujours fasciné l’humanité. Des efforts considérables sont investis de nos jours dans la recherche de la vie, qu’elle soit dans notre système solaire ou dans notre galaxie. La recherche « directe » d’organismes vivants dans notre système solaire a débuté sur Mars en 1975 avec les sondes Viking et se poursuit actuellement sur la planète rouge avec le robot Curiosity. D’après plusieurs études récentes, de nombreux corps célestes du système solaire, comme Titan, Encelade (lunes de Saturne), Europe ou Callysto (lunes de Jupiter) pourraient abriter des organismes vivants, et ces lunes seront très certainement la cible de prochaines missions spatiales.
Detecting nanoscale vibrations as signature of life
The quest to find life in extreme and extraterrestrial environments is exciting and touches many research fields. One of the common signatures of life is movement: Even small microorganisms vibrate in response to their metabolic activity. Thus, we have devised a nanomotion detector to study these fluctuations and to associate them to the metabolic activity of the specimens. This technique does not measure the chemical response of life, which would require prior knowledge of the metabolic pathways involved. Instead, it monitors the physical manifestation of any kind of metabolic activity the microorganisms might have. Here, we show how this nanomotion detector can study any living system, paving the way to a complementary approach to the study of life in extreme environments.
Development of lyophilized spherical particles of poly(epsilon-caprolactone) and examination of their morphology, cytocompatibility and influence on the formation of reactive oxygen species
A common limitation of using polymeric micro and nanoparticles in long-term conservation is due to their poor physical and chemical stability. Freeze-drying is one of the most convenient methods that enable further reconstitution of micro and nanoparticles for therapeutical use. Nevertheless, this process generates various stresses during freezing and desiccation steps. This paper underlines the combined outcomes of freeze drying method and physicochemical solvent/non-solvent approach to design biocompatible poly(epsilon-caprolactone) (PCL) nanospheres and evaluate influence of different cryoprotectants (glucose, saccharose, polyvinyl alcohol or polyglutamic acid) on the outcome of freeze-dried PCL particles. Samples were characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and dynamic light scattering method (DLS). In vitro studies used, include MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), testing cytotoxicity as the quality of being toxic to cells, and DCFH-DA assay (2’,7’-dichlordihydrofluorescein-diacetate), testing the possible increase in ROS levels. It was found that cryoprotection with 1% glucose solution is an optimal for obtaining uniform, spherical but also biocompatible PCL nanoparticles for biomedical purposes.
The Cyclic Cystine Ladder of Theta-Defensins as a Stable, Bifunctional Scaffold: A Proof-of-Concept Study Using the Integrin-Binding RGD Motif
Peptides have the specificity and size required to target the protein–protein interactions involved in many diseases. Some cyclic peptides have been utilised as scaffolds for peptide drugs because of their stability; however, other cyclic peptide scaffolds remain to be explored. θ-Defensins are cyclic peptides from mammals; they are characterised by a cyclic cystine ladder motif and have low haemolytic and cytotoxic activity. Here we demonstrate the potential of the cyclic cystine ladder as a scaffold for peptide drug design by introducing the integrin-binding Arg-Gly-Asp (RGD) motif into the θ-defensin RTD-1. The most active analogue had an IC50 of 18 nM for the αvβ3 integrin as well as high serum stability, thus demonstrating that a desired bioactivity can be imparted to the cyclic cystine ladder. This study highlights how θ-defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a β-strand or turn conformation. Furthermore, the symmetry of the cyclic cystine ladder presents the opportunity to design peptides with dual bioactive epitopes to increase activity and specificity.