Site-site interactions within high-loading PAMAM dendrimer resin beads

Combinatorial chemistry and solid-phase synthesis have revolutionised the process of drug discovery in the last decade. Ever since the concept of split and mix synthesis was introduced in 1988, and in particular the concept of one bead one compound, this approach has been associated with the possibility of generating thousands or millions of compounds in only a relatively small number of synthetic steps. The demand for high loading resins has therefore increased over the last few years. Our research has focussed on bead-loading enhancement via a dendrimerisation process. Dendrimer resin beads have shown compatibility for many chemical conditions, however high functional group density could produce undesirable site-site interactions.     

Formation and diffusivity characterization of supported lipid bilayers with complex lipid compositions

The moving edge of a hydrodynamically manipulated supported lipid bilayer (SLB) can be used to catalyze SLB formation of adsorbed lipid vesicles that do not undergo spontaneous SLB formation upon adsorption on SiO(2). By removing the lipid reservoir of an initially formed SLB, we show how a hydrodynamically moved SLB patch composed of POPC can be used to form isolated SLBs with compositions that to at least 95% represent that of the adsorbed lipid vesicles. The concept is used to investigate the diffusivity of lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine-DHPE) in SLBs made from complex lipid compositions, revealing a decrease in diffusivity by a factor of 2 when the cholesterol content was increased from 0% to 50%. We also demonstrate how the concept can be used to induce stationary domains in SLBs containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol (39:21:40 mol %, respectively). Because the method serves as a means to form SLBs with lipid compositions that hamper SLB formation via spontaneous rupture of adsorbed lipid vesicles, it opens up the possibility for new biophysical investigations of SLBs with more nativelike compositions.     

PAMAM (poly-amido amine) dendrimer supported copper nanoparticles for chemoselective nitro reduction

Present paper reports the synthesis of biocompatible poly (amidoamine) (PAMAM) dendrimer by Michael addition (up to Generation 2) which has been used as a matrix for preparing copper nanoparticles using green solvent. X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy – dispersive X-ray spectroscopy (EDX) were used to determine the surface morphology and elemental composition of the nanoparticle. The results confirmed the FCC structure of the prepared dendrimer encapsulated Cu nanoparticles with an average crystallite size of 4 ​nm. Purely aqueous-phase chemoselective reduction of nitro group in various aromatic nitro substrates such as p-nitrophenol, p-nitrobenzaldehyde and 2, 4 dinitrophenol have been employed in the presence of eco-friendly dendrimer encapsulated Cu nanoparticles using hydrazine hydrate as reducing agent. The nanocatalyst showed high reusability up to seven runs exhibiting no significant loss in its catalytic activity. As the synthesised material is found to be biocompatible, this catalytic strategy can be used to catalyse similar biochemical reactions.     

Molecular interactions between lipid bilayers and a water-soluble polymer

Molecular interactions between lipid bilayers (liposomes) and chondroitin sulfate C (CS), a water soluble polymer, have been investigated in terms of zeta-potential, particle size, microscopic-viscosity, microscopic-polarity of liposomes and permeability of calcein. Microscopic morphology is dramatically changed by the addition of CS to the positively charged liposomes (Pos.L), while it is not changed by the addition to uncharged liposomes (Unc.L) or negatively charged liposomes (Neg.L). The absolute value of the particle size of Pos.L increases with the addition of CS, while the zeta- potential of Pos.L decreases. Permeability of Pos.L decreases with an increase in the concentration of CS. Phase transition temperature of Pos.L is changed after the addition of CS. These values, however, are not changed for the other liposomes by the addition of CS. The results of gel filtration chromatography show that CS is absorbed on the Pos.L surface. Microscopic viscosity is also increased by the addition of CS to Pos.L due to the adsorption of CS.     

Creating fluid and air-stable solid supported lipid bilayers

Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.     

Interfacial interactions of hydrophobic peptides with lipid bilayers

Four hydrophobic laminin-related peptides and their corresponding parent peptides were synthesized to use them to target liposomes to tumoral cells. The peptide sequence was YIGSR((NH(2))), and hydrophobic residues linked to the alpha-amino terminal end were decanoyl, myristoyl, stearoyl, and cholesteryl-succinoyl. Before use in biological systems, a physicochemical study was carried out in order to determine their interaction with DPPC bilayers that could compromise both the toxicity and the stability of liposomal preparations. The experiments were based on DSC, fluorescence polarization, outer-membrane destabilization, and vesicle leakage. These peptides showed in general a low interaction with the vesicles, promoting in all cases the rigidification of bilayers. This lack of strong disturbances in the ordered state of phospholipid molecules seems more likely due to the similarity of peptide acyl chains with those of lipids than to the absence of interactions. The bulkiness of cholesteryl derivative as well as its tendency toward aggregation resulted in weak interaction levels except in thermograms. The binding of peptides to the surface of liposomes loaded with doxorubicin resulted in preparations with good entrapment yields and small size, required for long circulating vesicles (especially for the myristoyl derivative). The alternative method based on the reaction of parent peptide to the surface of liposomes through an amide linkage was slightly more efficient when the peptide was linked to the carboxy-terminal end of the DSPE-PEG-COOH-containing liposomes. Nevertheless, the final decision must be made with the simplicity of the procedure and reduction in losses during all the steps of the processes taken into consideration.     

Fluid supported lipid bilayers containing monosialoganglioside GM1: a QCM-D and FRAP study

In an effort to use model fluid membranes for immunological studies, we compared the formation of planar phospholipid bilayers supported on silicon dioxide surfaces with and without incorporation of glycolipids as the antigen for in situ antibody binding. Dynamic light scattering measurements did not differentiate the hydrodynamic volumes of extruded small unilamellar vesicles (E-SUVs) containing physiologically relevant concentrations (0.5-5 mol%) of monosialoganglioside GM1 (GM1) from exclusive egg yolk L-alpha-phosphatidylcholine (egg PC) E-SUVs. However, quantifiable differences in deposition mass and dissipative energy loss emerged in the transformation of 5 mol% GM1/95 mol% egg PC E-SUVs to planar supported lipid bilayers (PSLBs) by vesicle fusion on thermally evaporated SiO2, as monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Compared to the 100 mol% egg PC bilayers on the same surface, E-SUVs containing 5 mol% GM1 reached a approximately 12% higher mass and a lower dissipative energy loss during bilayer transformation. PSLBs with 5 mol% GM1 are approximately 18% heavier than 100 mol% egg PC and approximately 11% smaller in projected area per lipid, indicating an increased rigidity and a tighter packing. Subsequent binding of polyclonal immunoglobulin G anti-GM1 to the PSLBs was performed in situ and showed specificity. The anti-GM1 to GM1 ratios at equilibrium were roughly proportional to the concentrations of anti-GM1 administered in the solution. Fluorescence recovery after photobleaching was utilized to verify the retained, albeit reduced lateral fluidity of the supported membranes. Five moles percentage of GM1 membranes (GM1 to PC ratio approximately 1:19) decorated with 1 mol% N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine (Texas Red DHPE) exhibited an approximately 16% lower diffusion coefficient of 1.32+/-0.06 microm2/s, compared to 1.58+/-0.04 microm2/s for egg PC membranes without GM1 (p<0.01). The changes in vesicle properties and membrane lateral fluidity are attributed to the interactions of GM1 with itself and GM1 with other membrane lipids. This system allows for molecules of interest such as GM1 to exist on a more biologically relevant surface than those used in conventional methods such as ELISA. Our analysis of rabbit serum antibodies binding to GM1 demonstrates this platform can be used to test for the presence of anti-lipid antibodies in serum.     

Heterogeneous molecular distribution in supported multicomponent lipid bilayers

Membrane domains contribute important structural and functional attributes to biological membranes. We describe the heterogeneous nanoscale distribution of lipid molecules within microscale membrane domains in multicomponent lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). The lipids were labeled with the fluorescent lipid analogues Bodipy-PC and DiI-C20:0 to identify the distribution of individual membrane components. We used a near-field scanning optical microscope (NSOM) at room temperature to identify the nanoscale structures in the membrane. Simultaneous multicolor NSOM imaging at the emission maxima of the fluorescent analogues revealed a patchy distribution of Bodipy-PC and DiI-C20:0 indicative of phase separations in the bilayer. In a cholesterol-free system (DPPC/DLPC = 1:1), NSOM images proved that the two phosphatidylcholine molecules can coexist in domains at the micrometer level but form nanoscopic patches within the domains; DPPC occurs at the edge of the domains, whereas DLPC is present throughout the domains. In the presence of cholesterol (DPPC/DLPC = 7:3, chol = 18.9%), the two lipid molecules were more miscible but incomplete phase separations also occurred. The average domain sizes were 140-200 nm, well below the resolution capabilities of diffraction-limited light microscopy techniques; the domains were unresolvable by confocal microscopy. Our high-resolution NSOM studies of membrane domain behavior provide a better understanding of complex membrane phase phenomena in multicomponent biological membranes.     

Disruption of supported lipid bilayers by semihydrophobic nanoparticles

Understanding the interaction between functional nanoparticles and cell membranes is critical to use nanomaterials for broad biomedical applications with minimal cytotoxicity. In this work, we have investigated the effect of adsorbed semihydrophobic nanoparticles (NPs) on the dynamics and morphology of model cell membranes. We have systematically varied the degree of surface hydrophobicity of carboxyl end-functionalized polystyrene NPs of varied size in buffer solutions with varied ionic strength. It is observed that semihydrophobic NPs can readily adsorb on neutral SLBs and drag lipids from SLBs to NP surfaces. Above a critical NP concentration, the disruption of SLBs is observed, accompanied with the formation and rapid growth of lipid-poor regions on NP-adsorbed SLBs. In the study of the effect of solution ionic strength on NP surface hydrophobic degree and the growth of lipid-poor regions, we have concluded that the hydrophobic interaction enhanced by screened electrostatic interaction underlies the envelopment of NPs by lipids that are attracted from SLBs to the surface of NPs or their aggregates. Hence, the formation and growth of lipid-poor regions, or vaguely referred as "pores" or "holes" in the literature, can be controlled by NP concentration, size, and surface hydrophobicity, which is critical to design functional nanomaterials for effective nanomedicine while minimizing possible cytotoxicity.     

Dynamic dendrimer at electrified interface: potential dependent adsorption-desorption and reorientation of a 4-pyridyl-modified PAMAM dendrimer

Potential-dependent dynamic adsorption-desorption and reorientation of a 4-pyridyl modified PAMAM G2 dendrimer at a Au(111) electrode has been demonstrated.     

Parallel-oriented fibrogenesis of a beta-sheet forming peptide on supported lipid bilayers

Peptide self-assembly on substrates is currently an intensively studied topic that provides a promising strategy for fabrication of soft materials and is also important for revealing the surface chemistry of amyloidogenic proteins that aggregate on cell membranes. We investigated the fibrogenesis of a beta-sheet forming peptide Abeta(26-35) on supported lipid bilayers (SLBs) by in situ atomic force microscopy (AFM), circular dichroism (CD), and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The results show that the Abeta(26-35) nanofilaments' growth is oriented to a specific direction and formed a highly ordered, large-scale, parallel-oriented surface pattern on membranes. The parallel-oriented fibrogenesis of Abeta(26-35) was able to occur on different lipid membranes rather than on solid substrates. It implies that the parallel-oriented fibrogenesis was associated with the distinct properties of lipid membranes, such as the fluid nature of lipid molecules on membranes. The membrane fluidity may allow the peptide assemblies to float at the water-membrane interface and easily orient to an energetically favorable state. These results provide an insight into the surface chemistry of peptide self-assembly on lipid membranes and highlight a possible way to fabricate supramolecular architectures on the surface of soft materials.     

Arrays of mobile tethered vesicles on supported lipid bilayers

We report a new system of laterally mobile, arrayed vesicles that are encoded with DNA to control tethering to fluid-supported phospholipid bilayers. The motion of individual fluorescently labeled vesicles, specifically bound, are easily visualized by fluorescence video microscopy and observed to collide reversibly on the surface. This system is an ideal model for studying interactions involving membranes, in particular integral membrane proteins.     

Detection of supported lipid bilayers using their electric charge

We describe an electronic detection method for charged lipid bilayers supported on a Si 3N 4/SiO 2/Si substrate. The flat-band voltage was used to monitor the charge of the bilayers. We show that the flat-band voltage varies with lipid adsorption depending on the polarity and mole ratio of the charged lipids, the salt concentration, and the surface coverage. Cationic and anionic bilayers produced a decrease and an increase in the flat-band voltage, respectively. The voltage change increased as the percentage of charged lipid components was elevated in the planar bilayers with full surface coverage. In addition, the voltage variation increased when the salt concentration was decreased or when the surface coverage of planar bilayer patches was increased. These results demonstrate that charged bilayers can be detected from the field effect that they exert on a solid support.     

Asymmetric distribution of anionic phospholipids in supported lipid bilayers

Lipid bilayers with a controlled content of anionic lipids are a prerequisite for the quantitative study of hydrophobic-electrostatic interactions of proteins with lipid bilayers. Here, the asymmetric distribution of zwitterionic and anionic lipids in supported lipid bilayers is studied by neutron reflectometry. We prepare POPC/POPS (3:1) unilamellar vesicles in a high-salt-concentration buffer. Initially, no fusion of the vesicles to a SiO(2) surface is observed over hours and days. Once the isotonic buffer is exchanged with hypotonic buffer, vesicle fusion and bilayer formation occur by osmotic shock. Neutron reflectivity on the bilayers formed this way reveals the presence of anionic lipids (d(31)-POPS) in the outer bilayer leaflet only, and no POPS is observed in the leaflet facing the SiO(2) substrate. We argue that this asymmetric distribution of POPS is induced by the electrostatic repulsion of the phosphatidylserines from the negatively charged hydroxy surface groups of the silicon block. Such bilayers with controlled and high contents of anionic lipids in the outer leaflet are versatile platforms for studying anionic lipid protein interactions that are key elements in signal transduction pathways in the cytoplasmic leaflet of eukaryotic cells.     

Photoelectric measurements of self-assembled and supported planar lipid bilayers: a new technique for studying apoptosis

A new method based on photoelectrochemistry for analyzing apoptosis of bilayer lipid membranes (s-BLMs) containing MCF-7 nuclei is reported. The s-BLM cell responded to white light (200–800 nm). During the apoptosis induced by Taxol, the photoelectric current of the cell decreased, suggesting degradation of the nuclear DNA. Electron transfer along the DNA double helix and along the nuclear skeleton is assumed in the interpretation. This novel photoelectric analytical method may provide a rapid and sensitive technique to evaluate apoptosis.     

Copper-free click conjugation of methotrexate to a PAMAM dendrimer platform

The synthesis of a generation 5 (G5) poly(amidoamine) (PAMAM) dendrimer platform having cyclooctyne ligands that were subsequently be used for a copper-free Huisgen 1,3-dipolar cycloaddition (click reaction) with azido modified methotrexate is described. The G5 PAMAM dendrimer was first partially (70%) acetylated and then coupled with 20 cyclooctyne ligands through amide bonds. The remaining primary amine groups on the dendrimer surface were neutralized by acetylation. The platform was then ‘clicked’ with different numbers (5, 10, and 17) of γ-azido functionalized methotrexate. The copper-free click reactions were stoichiometric with excellent yields.     

Spectral investigation of coordination of cuprum cations and protons at PAMAM dendrimer peripherally modified with 1,8-naphthalimide units

The investigations aim at revealing the ability of a 1,8-napthalimide-modified poly(amidoamine) dendrimer from second generation to respond to the presence of cuprum cations and protons in the environment. It has been established that a single Cu(2+) cation present in the dendrimer molecule is capable of quenching more than 78% of its fluorescence what is an indication of high sensitiveness. An enhancement of the fluorescence emission of the dendrimer has been observed in acidic medium. It has been established that the processes of coordinating the ions in different sites of the dendrimer are reversible.     

Patterned supported lipid bilayers and monolayers on poly(dimethylsiloxane)

A simple and practical method for patterning supported lipid bilayers on poly(dimethylsiloxane) is presented. By using electron microscopy grids to laterally control the extent of plasma oxidation, the substrate is partitioned into regions of different hydrophilicities. Addition of vesicles then results in the spontaneous formation of lipid bilayers and monolayers side-by-side on the surface, separated by regions that contain no lipid and/or a region with adhering vesicles. By using millimeter-sized plastic masks we are able to control the formation of these lipid structures on macroscopic patches by simply varying the plasma-cleaning time. For the first time, we are able to influence, in a controlled fashion, the chemical composition of a substrate in such a way that it supports fluid lipid monolayers, rejects lipid adhesion, adsorbs intact lipid vesicles, or supports fluid bilayers.