2,519 research outputs found

    Applications of AFM in pharmaceutical sciences

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    Atomic force microscopy (AFM) is a high-resolution imaging technique that uses a small probe (tip and cantilever) to provide topographical information on surfaces in air or in liquid media. By pushing the tip into the surface or by pulling it away, nanomechanical data such as compliance (stiffness, Young’s Modulus) or adhesion, respectively, may be obtained and can also be presented visually in the form of maps displayed alongside topography images. This chapter outlines the principles of operation of AFM, describing some of the important imaging modes and then focuses on the use of the technique for pharmaceutical research. Areas include tablet coating and dissolution, crystal growth and polymorphism, particles and fibres, nanomedicine, nanotoxicology, drug-protein and protein-protein interactions, live cells, bacterial biofilms and viruses. Specific examples include mapping of ligand-receptor binding on cell surfaces, studies of protein-protein interactions to provide kinetic information and the potential of AFM to be used as an early diagnostic tool for cancer and other diseases. Many of these reported investigations are from 2011-2014, both from the literature and a few selected studies from the authors’ laboratories

    Nanoscale imaging and force probing of biomolecular systems using atomic force microscopy: from single molecules to living cells

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    Detecting CD20-Rituximab interaction forces using AFM single-molecule force spectroscopy

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    The invention of atomic force microscopy (AFM) has provided new technology for measuring specific molecular interaction forces. Using AFM single-molecule force spectroscopy (SMFS) techniques, CD20-Rituximab rupture forces were measured on purified CD20 proteins, Raji cells, and lymphoma patient B cells. Rituximab molecules were linked onto AFM tips using AFM probe functionalization technology, and purified CD20 proteins were attached to mica using substrate functionalization technology. Raji cells (a lymphoma cell line) or lymphoma patient cells were immobilized on a glass substrate via electrostatic adsorption and chemical fixation. The topography of the purified CD20 proteins, Raji cells, and patient lymphoma cells was visualized using AFM imaging and the differences in the rupture forces were analyzed and measured. The results showed that the rupture forces between the CD20 proteins on Raji cells and Rituximab were markedly smaller than those for purified CD20 proteins and CD20 proteins on lymphoma patient B cells. These findings provide an effective experimental method for investigating the mechanisms underlying the variable efficacy of Rituximab. © 2011 Science China Press and Springer-Verlag Berlin Heidelberg.Link_to_subscribed_fulltex

    Application of atomic force microscopy in cancer research

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    Atomic force microscopy (AFM) allows for nanometer-scale investigation of cells and molecules. Recent advances have enabled its application in cancer research and diagnosis. The physicochemical properties of live cells undergo changes when their physiological conditions are altered. These physicochemical properties can therefore reflect complex physiological processes occurring in cells. When cells are in the process of carcinogenesis and stimulated by external stimuli, their morphology, elasticity, and adhesion properties may change. AFM can perform surface imaging and ultrastructural observation of live cells with atomic resolution under near-physiological conditions, collecting force spectroscopy information which allows for the study of the mechanical properties of cells. For this reason, AFM has potential to be used as a tool for high resolution research into the ultrastructure and mechanical properties of tumor cells. This review describes the working principle, working mode, and technical points of atomic force microscopy, and reviews the applications and prospects of atomic force microscopy in cancer research

    Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition and adhesion

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    Carbohydrates at the cell surface have been proposed as mediators in cell-cell recognition events involved in embryogenesis, metastasis, and other proliferation processes by calcium-dependent carbohydrate to carbohydrate interactions. They are the most prominently exposed structures on the surface of living cells, and with flexible chains and many binding sites are ideal to serve as the major players in initiating these cellular events. However, biological relevance of these type interactions is often questioned because of the very low affinity binding of single carbohydrate molecules and that they manifest themselves only through the contact of a large number of molecules tightly arranged in the membrane. Weak interactions are considerably more difficult to study and only a few biologically significant examples of direct carbohydrate-carbohydrate interactions have been reported, e.g. pioneering work showing glycosphingolipid self-interactions through multivalent interaction of Lewis X epitopes. However, there are no reports on the existence of specific proteoglycan self-interactions through carbohydrate-carbohydrate interactions in cellular recognition system, as it has been done with glycosphingolipids. Here, we used sponges, organisms on which the first proteoglycan-mediated cell-cell recognition in the animal kingdom was demonstrated, as a model system to study carbohydrate-mediated cellular recognition. We show that the interaction between single oligosaccharides from surface proteoglycans is relatively strong and comparable to protein-carbohydrate interactions, highly specific, and dependent on Ca2+-ions. 200 kDa glycans from the core protein of Microciona prolifera cell surface proteoglycans have been previously shown to mediate homotypic Microciona proteoglycan-proteoglycan interactions. Here, 200 kDa glycans from four different sponge species: Microciona prolifera, Halichondria panicea, Suberites fuscus and Cliona celata were purified and investigated for species-specific interactions. Selective recognition of glycans by live cells was studied to confirm the existence of glycan-glycan recognition system in biologically relevant situations. Mature sponge cells have the ability to reaggregate species-specifically and form homogenous aggregates on a shaker at the right shear forces in the presence of physiological 10 mM Ca2+. Live cells were allowed to aggregate with glycan-coated beads similar in size to small sponge cells in the presence of calcium. They specifically recognized beads coated with their own glycans and did not mix but separated from beads coated with glycans isolated from different species. The glycan-glycan recognition assay was developed to mimic species-specific cellcell recognition in sponges. 200 kDa glycans immobilized onto beads similar in size to small sponge cells assembled species-specifically in the presence of physiological calcium, at the same shear forces as in cell-cell aggregation. Glycans coated on beads aggregated with glycans from the same species coated on beads, and separated from glycans from other species. The glycan density necessary for specific live cellcell recognition in sponges is 828 molecules/ÎĽm2. In our studies, the glycan density necessary for specific glycan-coated bead was very similar: ~810 molecules/ÎĽm2. Mature live cells demonstrated specific recognition of 200 kDa glycans during selective-binding to glycans coated on surfaces in the presence of calcium. They strongly adhered to glycans from their own surface proteoglycans coated onto a solid polystyrene phase, while the binding to glycans from different proteoglycans was 3 - 5 times lower. Moreover, homotypic adhesion to glycan-coated plates enhanced sponge cell differentiation and formation of mineral skeleton (spicules). Larval cells, after settlement and spreading of larvae, can fuse species-specifically in nature. In our studies, live larval cells recognized and adhered specifically to glycans purified from adhesion proteoglycans from their "mother sponge". They showed almost no interaction with glycans from other species. As in cell-glycan adhesion assays, highly species-specific adhesion of 200 kDa glycans to glycan-coated surfaces could be observed in the presence of physiological calcium. Tested glycans bound strongly to glycans from the same species and showed up to a six fold reduction in binding to glycans from other species. Atomic force microscopy (AFM) was performed to measure for the first time adhesion forces between single glycan molecules obtained from different surface proteoglycans. Measurements revealed equally strong adhesion forces in the range of several hundred piconewtons (pN) between glycan molecules as between proteins and glycans measured in another recognition system. Moreover, statistically significant differences (p value < 0.01) were seen between homotypic (glycans from the same species) and heterotypic (glycans from different species) interactions. Moreover, the polyvalent character of binding characterized mainly interactions between glycans from the same species. This indicates that not only the higher adhesion force per binding site as such but also the higher amount of multiple interactions between glycans from the same species versus mixture of glycans from different species guaranteed the specificity of the glycan-mediated recognition. These findings confirm for the first time the existence of specific glycan-glycan recognition system between cell surface proteoglycans. We propose that these cell's outermost surface structures serve as important players in initiating the very first contacts between cells through highly species-specific and flexible carbohydratecarbohydrate interactions

    Carbohydrate-carbohydrate interaction provides adhesion force and specificity for cellular recognition and adhesion

    Get PDF
    Carbohydrates at the cell surface have been proposed as mediators in cell-cell recognition events involved in embryogenesis, metastasis, and other proliferation processes by calcium-dependent carbohydrate to carbohydrate interactions. They are the most prominently exposed structures on the surface of living cells, and with flexible chains and many binding sites are ideal to serve as the major players in initiating these cellular events. However, biological relevance of these type interactions is often questioned because of the very low affinity binding of single carbohydrate molecules and that they manifest themselves only through the contact of a large number of molecules tightly arranged in the membrane. Weak interactions are considerably more difficult to study and only a few biologically significant examples of direct carbohydrate-carbohydrate interactions have been reported, e.g. pioneering work showing glycosphingolipid self-interactions through multivalent interaction of Lewis X epitopes. However, there are no reports on the existence of specific proteoglycan self-interactions through carbohydrate-carbohydrate interactions in cellular recognition system, as it has been done with glycosphingolipids. Here, we used sponges, organisms on which the first proteoglycan-mediated cell-cell recognition in the animal kingdom was demonstrated, as a model system to study carbohydrate-mediated cellular recognition. We show that the interaction between single oligosaccharides from surface proteoglycans is relatively strong and comparable to protein-carbohydrate interactions, highly specific, and dependent on Ca2+-ions. 200 kDa glycans from the core protein of Microciona prolifera cell surface proteoglycans have been previously shown to mediate homotypic Microciona proteoglycan-proteoglycan interactions. Here, 200 kDa glycans from four different sponge species: Microciona prolifera, Halichondria panicea, Suberites fuscus and Cliona celata were purified and investigated for species-specific interactions. Selective recognition of glycans by live cells was studied to confirm the existence of glycan-glycan recognition system in biologically relevant situations. Mature sponge cells have the ability to reaggregate species-specifically and form homogenous aggregates on a shaker at the right shear forces in the presence of physiological 10 mM Ca2+. Live cells were allowed to aggregate with glycan-coated beads similar in size to small sponge cells in the presence of calcium. They specifically recognized beads coated with their own glycans and did not mix but separated from beads coated with glycans isolated from different species. The glycan-glycan recognition assay was developed to mimic species-specific cellcell recognition in sponges. 200 kDa glycans immobilized onto beads similar in size to small sponge cells assembled species-specifically in the presence of physiological calcium, at the same shear forces as in cell-cell aggregation. Glycans coated on beads aggregated with glycans from the same species coated on beads, and separated from glycans from other species. The glycan density necessary for specific live cellcell recognition in sponges is 828 molecules/ÎĽm2. In our studies, the glycan density necessary for specific glycan-coated bead was very similar: ~810 molecules/ÎĽm2. Mature live cells demonstrated specific recognition of 200 kDa glycans during selective-binding to glycans coated on surfaces in the presence of calcium. They strongly adhered to glycans from their own surface proteoglycans coated onto a solid polystyrene phase, while the binding to glycans from different proteoglycans was 3 - 5 times lower. Moreover, homotypic adhesion to glycan-coated plates enhanced sponge cell differentiation and formation of mineral skeleton (spicules). Larval cells, after settlement and spreading of larvae, can fuse species-specifically in nature. In our studies, live larval cells recognized and adhered specifically to glycans purified from adhesion proteoglycans from their "mother sponge". They showed almost no interaction with glycans from other species. As in cell-glycan adhesion assays, highly species-specific adhesion of 200 kDa glycans to glycan-coated surfaces could be observed in the presence of physiological calcium. Tested glycans bound strongly to glycans from the same species and showed up to a six fold reduction in binding to glycans from other species. Atomic force microscopy (AFM) was performed to measure for the first time adhesion forces between single glycan molecules obtained from different surface proteoglycans. Measurements revealed equally strong adhesion forces in the range of several hundred piconewtons (pN) between glycan molecules as between proteins and glycans measured in another recognition system. Moreover, statistically significant differences (p value < 0.01) were seen between homotypic (glycans from the same species) and heterotypic (glycans from different species) interactions. Moreover, the polyvalent character of binding characterized mainly interactions between glycans from the same species. This indicates that not only the higher adhesion force per binding site as such but also the higher amount of multiple interactions between glycans from the same species versus mixture of glycans from different species guaranteed the specificity of the glycan-mediated recognition. These findings confirm for the first time the existence of specific glycan-glycan recognition system between cell surface proteoglycans. We propose that these cell's outermost surface structures serve as important players in initiating the very first contacts between cells through highly species-specific and flexible carbohydratecarbohydrate interactions
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