Biomedical subjects
Publications and source records attributed to Bradford G Orr.
The formation of mixed monolayers of hydridospherosilsesquioxane clusters (H(8)Si(8)O(12)) and alkylsilanes (H(2n+1)C(n)SiH(3)) on Au has been investigated using X-ray photoelectron and reflection-absorption infrared spectroscopies and scanning tunneling microscopy. All of the techniques indicate the displacement of the majority of the siloxane clusters from the surface in favor of the alkylsilane.
Polycationic polymers are used extensively in biology to disrupt cell membranes and thus enhance the transport of materials into the cell. The highly polydisperse nature of many of these materials makes obtaining a mechanistic understanding of the disruption processes difficult. To design an effective mechanistic study, a monodisperse class of polycationic polymers, poly(amidoamine) (PAMAM) dendrimers, has been studied in the context of supported dimyristoylphosphatidylcholine (DMPC) lipid bilayers using atomic force microscopy (AFM). Aqueous solutions of amine-terminated generation 7 (G7) PAMAM dendrimers caused the formation of 15-40-nm-diameter holes in lipid bilayers. This effect was significantly reduced for smaller G5 dendrimers. For G3, no hole formation was observed. In addition to dendrimer size, surface chemistry had a strong influence on dendrimer-lipid bilayer interactions. In particular, acetamide-terminated G5 did not cause hole formation in bilayers. In all instances, the edges of bilayer defects proved to be points of highest dendrimer activity. A proposed mechanism for the removal of lipids by dendrimers involves the formation of dendrimer-filled lipid vesicles. By considering the thermodynamics, interaction free energy, and geometry of these self-assembled vesicles, a model that explains the influence of polymer particle size and surface chemistry on the interactions with lipid membranes was developed. These results are of general significance for understanding the physical and chemical properties of polycationic polymer interactions with membranes that lead to the transport of materials across cell membranes.
Apoptosis is defined by a distinct set of morphological changes observed during cell death including loss of focal adhesions, the formation of cell membrane buds or blebs, and a decrease in total cell volume. Recent studies suggest that these dramatic morphological changes, particularly apoptotic volume decrease (AVD), are an early prerequisite to apoptosis and precede key biochemical time-points. Here we use atomic force microscopy to observe early stage AVD of KB cells undergoing staurosporine-induced apoptosis. After a 3-h exposure to 1 microM staurosporine, a 32% decrease in total cell height and a 50% loss of total cell volume is observed accompanied by only a 15% change in cell diameter. The observed AVD precedes key biochemical hallmarks of apoptosis such as loss of mitochondrial membrane potential, phosphatidyl serine translocation, nuclear fragmentation, and measurable caspase-3 activity. This suggests that morphological volume changes occur very early in the induction of apoptosis.
The interaction of an antimicrobial peptide, MSI-78, with phospholipid bilayers has been investigated using atomic force microscopy, circular dichroism, and nuclear magnetic resonance (NMR). Binding of amphipathic peptide helices with their helical axis parallel to the membrane surface leads to membrane thinning. Atomic force microscopy of supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers in the presence of MSI-78 provides images of the membrane thinning process at a high spatial resolution. This data reveals that the membrane thickness is not reduced uniformly over the entire bilayer area. Instead, peptide binding leads to the formation of distinct domains where the bilayer thickness is reduced by 1.1 +/- 0.2 nm. The data is interpreted using a previously published geometric model for the structure of the peptide-lipid domains. In this model, the peptides reside at the hydrophilic-hydrophobic boundary in the lipid headgroup region, which leads to an increased distance between lipid headgroups. This picture is consistent with concentration-dependent 31P and 2H NMR spectra of MSI-78 in mechanically aligned DMPC bilayers. Furthermore, 2H NMR experiments on DMPC-d54 multilamellar vesicles indicate that the acyl chains of DMPC are highly disordered in the presence of the peptide as is to be expected for the proposed structure of the peptide-lipid assembly.
Polycationic polymers are known to disrupt lipid bilayers. In this letter, we report the dependence of this disruption on the lipid structural phase. DMPC bilayers are exposed to two polycationic polymeric nanoparticles, PAMAM dendrimers and MSI-78. We find that regions of the bilayer that are in the gel phase are unaffected by the presence of polymers, whereas the liquid phase is disrupted.
Chemisorbed monolayers of octylgermane (C8H17GeH3) on gold have been formed by vapor deposition in ultrahigh vacuum. The monolayer has been characterized by X-ray photoelectron and reflection absorption infrared spectroscopies (XPS and RAIRS) and scanning tunneling microscopy (STM). XPS data indicate the monolayer can be oxidized by exposure to ozone. STM images exhibit a complex pattern which can be modeled as strain-mediated spinodal decomposition.
The oxidation of alkylsilane monolayers on Au has been studied by X-ray photoelectron spectroscopy, reflection-absorption infrared spectroscopy, contact-angle measurements, and scanning tunneling microscopy. Exposure of the monolayers at 298 K to pure O(2) or H(2)O (>5 x 10(-5) Torr and >150 000 L) does not cause oxidation. Ambient atmosphere only causes oxidation if direct sight lines are maintained to the sample. Ozone exposure results in rapid monolayer oxidation. Oxidation initially occurs only at the Si atom, resulting in formation of a cross-linked siloxane monolayer that retains alkyl surface termination. Prolonged ozone exposures result in the oxidation and subsequent loss of the alkyl chain.
Although it is known that noncollagenous proteins of dental origin bind to the hydroxyapatite crystal surfaces, no measure of their binding strength has been calculated. This experiment used -COOH-capped generation 7 PAMAM dendrimers as nanoprobes of the biological hydroxyapatite nanorod surfaces. Dendrimer distribution was characterized using AFM. The results showed dendrimers to be spaced at intervals along the c-axis of the crystals. From these observations and assuming a fully ionized -COOH dendrimer, a mathematical model of the binding capacity of the crystal surface with the dendrimer was developed. The Monte Carlo method was used to simulate the binding process between the dendrimer and crystal surface, and the binding strength of the -COOH groups to the surface was calculated to be 90 +/- 20 kJ/mol. These results support the CFM studies which have described alternating bands of charge domains on the crystal surface and that the binding strength will be dependent on both the intensity of the charge on the protein and the crystal surface.
Scanning tunneling microscopy data of a mixed monolayer comprised of a 40:60 ratio of H8Si8O12 and C6H13-H7Si8O12 clusters on gold are presented. The images display a composite monolayer surface with well-defined domain regions of the individual components. Holes present at face-centered cubic (fcc) sites of the starting Au/H7Si8O12 adsorbate layer indicate the location of active sites for impinging C6H13-H7Si8O12 clusters. Adsorption of a C6H13-H7Si8O12 cluster likely yields a mobile hydrogen atom available to recombine with and desorb an adjacent H8Si8O12 cluster. Hydrogen atom diffusion along substrate [121] directions is the proposed pattern formation mechanism of the mixed monolayer. Imaging of the spherosiloxane cluster domains identifies a novel terrace-based active site located in the fcc regions of the Au(111) 23 x square root3 surface reconstruction.
Atomic force microscopy (AFM) is employed to observe the effect of poly(amidoamine) (PAMAM) dendrimers on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers. Aqueous solutions of generation 7 PAMAM dendrimers cause the formation of holes 15-40 nm in diameter in previously intact bilayers. This effect is observed for two different branch end-groups--amine and carboxyl. In contrast, carboxyl-terminated core-shell tectodendrimer clusters do not create holes in the lipid membrane but instead show a strong affinity to adsorb to the edges of existing bilayer defects. A possible mechanism for the formation of holes in the lipid bilayer is proposed. The dendrimers remove lipid molecules from the substrate and form aggregates consisting of a dendrimer surrounded by lipid molecules. Dynamic light scattering (DLS) measurements as well as 31P NMR data support this explanation. The fact that tectodendrimers behave differently suggests that their cluster-like architecture plays an important role in their interaction with the lipid bilayer.
Studies of electrically induced morphological changes in neurons have either been limited by the resolution of light microscopy or the cell fixation required for electron microscopy. Atomic force microscopy (AFM), however, mechanically maps cell topography, offering exquisite resolution of evolving processes in three dimensions. In this paper, we present a microelectrode array (MEA) based platform for the real-time detection of subtle, electrically induced variations in neuronal morphology, with AFM. This platform required the customized design and production of a silicon-based MEA, integration with a commercial AFM, and the development of biological techniques for culture of neuroblastoma (SH-SY5Y) cells onto the device. Biphasic pulse trains (1 Hz) of electric current were delivered to a microelectrode interfaced with a neuroblastoma cell, and the AFM continuously recorded a cross-sectional height profile. Proof-of-principle experiments demonstrate that electric stimulation may induce fluctuations ranging in the 100-300-nm range, 75-fold greater than the systemic resolution, but smaller than the resolution of light microscopy modalities. In addition, the real-time capabilities of AFM captured a collapse (30%-40%) of a neurite cross section, seconds after electric stimulation. Ultimately, this platform can be used to nanocharacterize cell responses to electric stimulation and other biochemical cues, for use in neuronal patterning and regeneration studies.
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Scanning tunneling microscopy (STM), in conjunction with X-ray photoemission (XPS) and reflection-absorption infrared (RAIRS) spectroscopy, has been used to investigate the reaction of octahydridosilsesquioxane clusters (H(8)Si(8)O(12)) on the Si(100)-2x1 and Si(111)-7x7 surfaces. The clusters exhibit a markedly different reactivity upon exposure to the two clean silicon surfaces. STM data is presented that, in conjunction with XPS and RAIRS data, provides numerous constraints upon possible geometries for the chemisorbed clusters. The sum of the data is consistent with a dissociative reaction mechanism on Si(100)-2x1, resulting in cluster attachment to the surface via a single vertex. Conversely, data of Si(111)-7x7 subject to a saturation exposure of H(8)Si(8)O(12) is presented that is highly suggestive of cluster decomposition on the surface.
We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine- nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine-terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide-terminated PAMAM dendrimers.
Interactions of polycationic polymers with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and live cell membranes (KB and Rat2) have been investigated using atomic force microscopy (AFM), cytosolic enzyme assays, confocal laser scanning microscopy (CLSM), and a fluorescence-activated cell sorter (FACS). Polycationic polymers poly-L-lysine (PLL), polyethylenimine (PEI), and diethylaminoethyl-dextran (DEAE-DEX) and sphere-like poly(amidoamine) (PAMAM) dendrimers are employed because of their importance for gene and drug delivery. AFM studies indicate that all the polycationic polymers cause the formation and/or expansion of preexisting defects in supported DMPC bilayers in the concentration range of 1-3 microg/mL. By way of contrast, hydroxyl-containing neutral linear poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) do not induce hole formation or expand the size of preexisting defects in the same concentration range. All polymers tested are not toxic to KB or Rat2 cells up to a 12 microg/mL concentration (XTT assay). In the concentration range of 6-12 microg/mL, however, significant amounts of the cytosolic enzymes lactate dehydrogenase (LDH) and luciferase (LUC) are released. PEI, which possesses the greatest density of charged groups on its chain, shows the most dramatic increase in membrane permeability. In addition, treatment with polycationic polymers allows the small dye molecules propidium idodide (PI) and fluorescein (FITC) to diffuse in and out of the cells. CLSM images also show internalization of PLL labeled with FITC dye. In contrast, controls of membrane permeability using the neutral linear polymers PEG and PVA show dramatically less LDH and LUC leakage and no enhanced dye diffusion. Taken together, these data are consistent with the hypothesis that polycationic polymers induce the formation of transient, nanoscale holes in living cells and that these holes allow a greatly enhanced exchange of materials across the cell membrane.