Electrophoretic mobility and molecular distribution studies of poly(amidoamine) dendrimers of defined charges

Generation 5 ethylenediamine (EDA)-cored poly(amidoamine) (PAMAM) dendrimers (E5, E denotes the EDA core and 5 the generation number) with different degrees of acetylation and carboxylation were synthesized and used as a model system to investigate the effect of charge and the influence of dendrimer surface modifications on electrophoretic mobility (EM) and molecular distribution. The surface-modified dendrimers were characterized by size-exclusion chromatography, 1H NMR, MALDI-TOF-MS, PAGE, and CE. The focus of our study was to determine how EM changes as a function of particle charge and molecular mass, and how the molecular distribution changes due to surface modifications. We demonstrate that partially modified dendrimers have much broader migration peaks than those of fully surface functionalized or unmodified E5 dendrimers due to variations in the substitution of individual dendrimer surfaces. EM decreased nonlinearly with increases in surface acetylation for both PAMAM acetamides and PAMAM succinamic acids, indicating a complex migration activity in CE separations that is not solely due to charge/mass ratio changes. These studies provide new insights into dendrimer properties under an electric field, as well as into the characterization of dendrimer-based materials being developed for medical applications.     

Surface-immobilized PAMAM-dendrimers modified with cationic or anionic terminal functions: physicochemical surface properties and conformational changes after application of liquid interface stress

Functionalization of surfaces with highly branched dendrimer molecules has gained attractiveness for various applications because the number of functional groups exceeds those of surfaces functionalized with self-assembled monolayers. So far, little is known about the physicochemical properties of dendrimer functionalized surfaces, especially if the flexibility of dendrimer structure remains after covalent immobilization. Therefore, the purpose of this study was to covalently immobilize polyamidoamine (PAMAM) dendrimer molecules exhibiting terminal amine and carboxyl groups to silicon model surfaces and to explore their properties and structure at the solid-air and solid-liquid interface. Our results show that the surface free energy is higher for PAMAM coatings than for analogously terminated SAMs and also higher for carboxyl than amine functionalized coatings. Furthermore, several findings suggest that conformational freedom of the dendrimers was preserved after surface immobilization. Wet compared to dry PAMAMNH(2) surfaces show reduced hydrophilicity and increased contact angle hysteresis, whereas PAMAMCOOH surfaces become more hydrophilic and showed decreased hysteresis. Streaming current measurements showed an unexpected behavior for PAMAMCOOH surfaces in that they reveal a net positive surface charge over a wide pH range in spite of the carboxylated periphery. All of these results indicate a certain degree of masking, burrowing, back-folding and unfolding of functional groups upon environmental changes.     

Interaction forces between poly(amidoamine) (PAMAM) dendrimers adsorbed on gold surfaces

Interaction forces between two gold surfaces with adsorbed poly(amidoamine) (PAMAM) dendrimers (generations G3.0 and G5.0) have been investigated using colloidal probe atomic force microscopy (AFM). In the absence of dendrimers or at their low concentrations, an attractive force derived from the van der Waals interaction was observed. On the other hand, this attractive interaction changed to repulsion with increasing dendrimer concentration. The origin of the repulsion can be attributed to either an electric double layer interaction or a steric effect of the adsorbed dendrimers, depending on the concentration of dendrimer. The steric hindrance was also influenced by the generation of the dendrimer; the force-detectable distance in the presence of PAMAM G5.0 dendrimer was slightly longer than that in the presence of G3.0 dendrimer. In order to estimate the occupied area of each dendrimer adsorbed on gold, quartz crystal microbalance (QCM) measurement was also carried out.     

Conformation and distribution of groups on the surface of amphiphilic polyether-co-polyester dendrimers: effect of molecular architecture

Amphiphilic polyester-co-polyether (PEPE) dendrimers synthesized from poly(ethylene glycol) (PEG) were examined to understand the influence of alterations in the architecture of dendrimers on their conformation at interfaces and distribution of various groups on their surface. Effect of changes in the number of branching points, type of terminal functional groups and generation of dendrimer was primarily evaluated. Dendrimers were deposited on mica by spin coating at 0.1 mg/mL. Tapping mode atomic force microscopy (AFM) was employed for the visualization of dendrimer topographies while, X-ray photoelectron spectroscopy (XPS), AFM phase and force imaging were used as the tools for characterization of their surfaces. Individual dendrimer molecules could be imaged by AFM, which showed that they are round or oval in topography. Dendrimers were also flattened on mica but the extent of flattening differed with the chemical structure; for instance, third generation dendrimers were more flattened than second generation dendrimers whereas, dendrimers with higher number of branches had greater height above the mica surface. Hydrophilic and hydrophobic groups present towards the aerial interface existed in distinct zones rather than being distributed randomly, except in dendrimer with higher number of branches. The percentage of various hydrophobic groups on the surface of dendrimer was enhanced by increase in the number of branches but, was lowered by the presence of hydroxyl groups as the pendant terminal groups. Furthermore, the core of dendrimers was not always located towards the centre, its position was found to be altered by the number of branching points, type of terminal functional groups and the generation of dendrimer.     

Divergent growth of coordination dendrimers on surfaces

Divergent growth of surface-initiated dendritic nanostructures on gold surfaces in a highly controlled, stepwise manner is demonstrated, using metal-organic coordination as the binding motif. The repeat unit for dendrimer growth was a branched, C3-symmetrical ligand building block bearing three bis-hydroxamate groups. The surface initiation sites for dendrimer growth were obtained by the formation of a mixed monolayer comprising isolated bis-hydroxamate disulfide anchor ligands and octanethiol (OT) at very low anchor/OT ratios. Following functionalization of the surface with spaced anchors, alternate immersion in solutions of Zr4+ ions and the branched ligand afforded surface-confined dendrimers of increasing generation, where the number of generations is conveniently controlled by the number of coordination binding sequences. The heights of different generation dendrimers are in excellent agreement with values predicted by molecular models, as well as with thicknesses of branched multilayers prepared by the same procedure on full anchor monolayers. At higher generation numbers, gradual dendrimer overlap and coalescence are observed, eventually resulting in a continuous overlayer and a transition from 3D to 1D growth. A mechanism for the development of dendritic coordination nanostructures on surfaces is discussed.     

Unraveling dendrimer translocation across cell membrane mimics

Poly(amidoamine) (PAMAM) dendrimers are promising candidates in several applications within the medical field. However, it is still to date not fully understood whether they are able to passively translocate across lipid bilayers. Recently, we used fluorescence microscopy to show that PAMAM dendrimers induced changes in the permeability of lipid membranes but the dendrimers themselves could not translocate to be released into the vesicle lumen. Because of the lack of resolution, these experiments could not assess whether the dendrimers were able to translocate but remained attached to the membrane. Using quartz crystal microbalance with dissipation monitoring and neutron reflectivity, a structural investigation was performed to determine how dendrimers interact with zwitterionic and negatively charged lipid bilayers. We hereby show that dendrimers adsorb on top of lipid bilayers without significant dendrimer translocation, regardless of the lipid membrane surface charge. Thus, most likely dendrimers are actively transported through cell membranes by protein-mediated endocytosis in agreement with previous cell studies. Finally, the higher activity of PAMAM dendrimers for phosphoglycerol-containing membranes is in line with their high antimicrobial activity against Gram-negative bacteria.     

Specific binding structures of dendrimers on lipid bilayer membranes

Dissipative particle dynamics simulations are used to study the specific binding structures of polyamidoamine (PAMAM) dendrimers on amphiphilic membranes and the permeation mechanisms. Mutually consistent coarse-grained (CG) models both for PAMAM dendrimers and for dimyristoylphosphatidylcholine (DMPC) lipid molecules are constructed. The PAMAM CG model describes correctly the conformational behavior of the dendrimers, and the DMPC CG model can properly give the surface tension of the amphiphilic membrane. A series of systematic simulations is performed to investigate the binding structures of the dendrimers on membranes with varied length of the hydrophobic tails of amphiphiles. The permeability of dendrimers across membranes is enhanced upon increasing the dendrimer size (generation). The length of the hydrophobic tails of amphiphiles in turn affects the dendrimer conformation, as well as the binding structure of the dendrimer-membrane complexes. The negative curvature of the membrane formed in the dendrimer-membrane complexes is related to dendrimer concentration. Higher dendrimer concentration together with increased dendrimer generation is observed to enhance the permeability of dendrimers across the amphiphilic membranes.     

Detecting the adsorption of dye molecules in homogeneous poly(propylene imine) dendrimer monolayers by surface plasmon resonance sensor

In this work, we studied guest-host interactions between various dye molecules and the fifth-generation poly(propylene imine) (PPI-5) dendrimers in aqueous solutions using a surface plasmon resonance (SPR) sensor. The effect of the properties of guest and host molecules (e.g., charge and shape) and media (e.g., pH and ion strength) on affinity between guest and host molecules was investigated. Based on an immobilized homogeneous monolayer of PPI-5 dendrimer tethered to carboxyl-terminal self-assembled monolayers, the adsorption behavior of a group of dye molecules in PPI-5 was obtained. Results show that the strong affinity of PPI-5 to Rose Bengal and erythrosine B is attributed to the good match in charge and shape between the cavities of the dendrimer and the dye molecules. Maximum adsorption around a pH value of 7 was observed. The kinetic behaviors of different dye molecules in dendrimers were also studied. A fundamental understanding of guest-host interactions in dendrimers will guide the design of new-generation sensors and drug delivery carriers.     

Adsorption of poly(amido amine) (PAMAM) dendrimers on silica: importance of electrostatic three-body attraction

Adsorption of poly(amido amine) (PAMAM) dendrimers to silicon oxide surfaces was studied as a function of pH, ionic strength, and dendrimer generation. By combining optical reflectometry and atomic force microscopy (AFM), the adsorbed layers can be fully characterized and an unequivocal determination of the adsorbed mass becomes possible. For early stages, the adsorption process is transport limited and of first order with respect to the dendrimer solution concentration. For later stages, the surface saturates and the adsorbed dendrimers form loose but correlated liquidlike surface structures. This correlation is evidenced by a peak in the pair correlation function determined by AFM. The maximum adsorbed amount increases with increasing ionic strength and pH. The increase with the ionic strength is explained by the random sequential adsorption (RSA) model and electrostatic repulsion between the dendrimers. The adsorbing dendrimers interact by the repulsive screened Coulomb potential, whose range decreases with increasing ionic strength and thus leads to increasing adsorbed densities. The pH increase is interpreted as an effect of the substrate and is quantitatively explained by the extended three-body RSA model. This model stipulates the importance of a three-body interaction acting between two adsorbing dendrimers and the charged substrate. The presence of the charged substrate weakens the repulsion between the adsorbing dendrimers and thus leads to higher surface densities. This effect can be interpreted as an additional attractive three-body interaction, which acts in addition to the usual two-body repulsion and originates from the additional screening of the Coulomb repulsion by the counterions accumulating in the diffuse layer.     

Importance of size-to-charge ratio in construction of stable and uniform nanoscale RNA/dendrimer complexes

Formation of RNA/dendrimer complexes between various RNA molecules and PAMAM dendrimers was studied using atomic force microscopy. Our results demonstrate that effective construction of stable nanoscale and uniform RNA/dendrimer complexes depends critically on the size of the RNA molecule, the dendrimer generation and the charge ratio between the dendrimer and the RNA. Larger RNA molecules, higher generations of dendrimers and larger dendrimer-to-RNA charge ratios lead to the formation of stable, uniform nanoscale RNA/dendrimer complexes. These findings provide new insights in developing dendrimer systems for RNA delivery.     

Adsorption of charged dendrimers: a Brownian dynamics study

The adsorption of isolated charged dendrimers onto oppositely charged flat surfaces is studied in this work using Brownian dynamics simulations. The dendrimer is modeled as a freely jointed bead-rod chain in which excluded-volume interactions are modeled by a repulsive Lennard-Jones potential and bead-bead and bead-surface electrostatic interactions are described by screened Coulombic potentials. Adsorption behavior is studied as a function of inverse screening length, dendrimer generation, and dendrimer charge distribution. Adsorbed dendrimers adopt a disclike conformation in which they flatten in the direction normal to the surface and expand in the direction parallel to the surface. As the inverse screening length increases, the dendrimer expands in the normal direction and contracts in the parallel direction, adopting a conformation that is more stretched in the normal direction. When the inverse screening length becomes sufficiently large, the dendrimer desorbs and adopts a spherelike conformation. Bead density profiles show that adsorbed dendrimers form a two-layer structure, with one layer corresponding to adsorbed beads and a second, less dense layer corresponding to beads one rod length away from the surface. They also reveal how the distribution of monomers within the dendrimer and near the surface can be tailored by changing various problem parameters. The results presented here are expected to be helpful in providing qualitative guidance for dendrimer design in various applications.     

Electrostatic layer-by-layer deposition of photoactive dendrimers with triviologen-like cores on their surfaces. Synthesis and electrochemical and photocurrent generation measurements

The stepwise assembly of Fréchet-type dendrimers with naphthalene peripheral groups and positively charged viologen-like cores on quartz and ITO surfaces utilizing the layer-by-layer approach was investigated. We were able to deposit only the (+6) charged dendrimers series on ITO. The number of assembled dendrimers was found to increase as we go to higher-generation dendrimers. This dendrimer generation effect was evident from the UV-vis and electrochemical measurements of the assembled dendrimers. The half-wave potentials (E1/2) of the dendrimers shift to less negative values as the dendrimer generation increases in acetonitrile and to more negative values when assembled on ITO. Anodic photocurrent generation was seen upon light irradiation of the second- and third-generation dendrimers, NB1V3+6 and NB2V3+6, assembled on ITO but not for the zero-generation one, NV3+6. This observation was attributed to a fast charge recombination process in NV3+6 when compared to that of NB1V3+6 and NB2V3+6 dendrimers.     

Quantitative FT-IRRAS spectroscopic studies of the interaction of avidin with biotin on functionalized quartz surfaces

The interaction of avidin with biotin was studied on functionalized quartz surfaces terminated with 3-aminopropyltrimethoxysilane (3-APTMS), 2,2'-(ethylenedioxy)bis(ethylenediamine) (DADOO), and fourth-generation amine-terminated polyamidoamine (G4-NH2 PAMAM) dendrimers with the use of Fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS). In particular, the molecular recognition ability of these surfaces was quantified through FT-IRRAS in combination with the use of an alkyne dicobalt hexacarbonyl probe coupled with avidin. The degree of nonspecific adsorption of avidin was determined by exposure of the amine-terminated and/or biotinylated surfaces to solutions of biotin-saturated avidin. The results indicate that the biotinylated 3-APTMS layer exhibits a very low specific binding capacity for avidin (on the order of 0.15 pmol of avidin/cm2) and substantial nonspecific adsorption. Both the binding capacity and the specificity were greatly improved when the 3-APTMS layer on quartz was modified through serial chemisorption of glutaraldehyde (GA), DADOO, and/or G4-NH2 PAMAM dendrimer layers. Among these layers, the biotinylated G4-NH2 PAMAM dendrimer layer exhibited the highest capacity for avidin binding (2.02 pmol of avidin/cm2) with a specificity of approximately 90%. This effect can be attributed to the efficient packing/ordering of the binding dendrimer layer, leading to a more dense and better organized layer of biotin headgroups on the subsequent biotinylated surface.     

Enhancement of composite‐metal interfacial adhesion strength by dendrimer

The interface between carbon fiber reinforced polymer composites and metal plays a critical role in determining the strength of epoxy/metal laminated composites. We propose to introduce one dendrimer layer into the epoxy/metal interface, aiming to enhance the interfacial adhesion strength so that the interface could more effectively transfer the load from epoxy to metal. In this paper, the preparation and adsorption of dendrimer layer onto the alumina surface were systematically investigated. The results show that a highly stable and nanopatterned dendrimers layer was dip‐coated onto alumina substrates by adsorbing poly (amidoamine) dendrimers. It was confirmed that the dendrimers were adsorbed onto the alumina via acid‐base chemical interactions. The adsorption depends on the reaction time. The adhesion property between dendrimers and alumina was examined by sonication method. Copyright © 2010 John Wiley & Sons, Ltd.     

Charging and aggregation of latex particles by oppositely charged dendrimers

Poly(amidoamine) (PAMAM) dendrimers were shown to adsorb strongly on negatively charged latex particles, and their effect on the particle charge and aggregation behavior was investigated by light scattering and electrophoretic mobility measurements. Time-resolved simultaneous static and dynamic light scattering was used to measure absolute aggregation rate constants. With increasing dendrimer dose, the overall charge could be tuned from negative to positive values through the isoelectric point (IEP). The aggregation is fast near the IEP and slows down further away. With decreasing ionic strength, the region of fast aggregation narrows and the dependence of the aggregation rate on the dendrimer dose is more pronounced. Surface charge heterogeneities become important for higher dendrimer generations. They widen the fast aggregation region, reduce the dependence of the aggregation rate on the dendrimer dose, and lead to an acceleration of the rate in the fast aggregation regime near the IEP. The ratio of the dendrimer charge and the particle charge exceeds the stoichiometric ratio of unity substantially and further increases with increasing generation. The tentative interpretation of such superstoichiometric charge neutralization involves coadsorption of anions and the finite thickness of the adsorbed dendrimer layer.     

Photocurrent generation in layer-by-layer assembled dendrimers with ruthenium tris-bipyridine peripheral groups and a viologen-like core

The photophysical and photoelectrochemical properties of first- and second-generation dendrimers with ruthenium tris-bipyridine peripheral groups and a tri-viologen like core (Ru3V3 and Ru6V3) were investigated in solution and when embedded within assembled films. The stepwise assembly of these dendrimers on quartz and ITO surfaces utilizing the layer-by-layer approach was investigated. The amount of the assembled dendrimers was found to increase on going to the higher generation dendrimer. This dendrimer generation effect was evident from the UV-vis, atomic force microscopy, and electrochemical measurements of the dendrimers in either solution phase or when embedded in films. The anodic and cathodic photocurrent generation was seen upon visible light irradiation, with higher photocurrents for Ru6V3 than Ru3V3. This observation was attributed to better light-harvesting properties, thicker films, and slower charge recombination processes in Ru6V3 when compared to Ru3V3.     

FT-IRRAS spectroscopic studies of the interaction of avidin with biotinylated dendrimer surfaces

The interaction of avidin with biotin on a functional Au surface containing fourth generation amine-terminated polyamidoamine (G4-NH₂ PAMAM) dendrimers was investigated through the use of Fourier transform infrared reflection–adsorption spectroscopy (FT-IRRAS). The first step in the fabrication of the functional surfaces used was the construction of an aldehyde-terminated self-assembled monolayer (SAM) through the treatment of Au-coated glass slides with ethanol solutions of self-synthesized 2-hydroxypentamethylene sulfide (HPMS). The as-formed aldehyde-terminated monolayer was subsequently immersed in methanol solutions of G4-NH₂ PAMAM dendrimer to obtain well-organized primary amine-terminated surfaces. Biotinylation of the amine-terminated layers thus obtained was accomplished by use of the N-succinimidyl ester of biotin. Each step of the synthetic process, as well as the performance of final surface for protein recognition was monitored by FT-IRRAS. In particular, the molecular recognition ability was examined and quantified by use of an alkyne dicobalt hexacarbonyl probe coupled with avidin. Non-specific adsorption of avidin was determined by exposure of the amine-terminated and/or biotinylated surfaces to solutions of biotin-saturated avidin. The results indicate that the biotinylated G4-NH₂ PAMAM dendrimer layers formed according to this procedure have a high capacity for binding avidin with relatively high specificity. The performance of these layers (i.e. both binding capacity and specificity) improve substantially when 6-mercapto-1-hexanol (MH) is present as a co-adsorbent during the formation of the initial aldehyde-terminated layers. This effect can be attributed to the dilution of the initial aldehyde-terminated SAM, leading to a more favorable spatial arrangement of the subsequent biotinylated surfaces.     

Fluorescence quenching mechanism of a polyphenylene polyelectrolyte with other macromolecules: cytochrome c and dendrimers

This study investigates the fluorescence quenching of a polyphenyl based polyelectrolyte by positively charged macromolecules (proteins and dendrimers). This work shows that the fluorescence quenching of the dendrimer materials does not involve energy transfer or electron transfer but is correlated to the overall charge on the dendrimer and its size. The quenching is hypothesized to result from conformational changes that occur upon binding the polyelectrolyte to the protein or dendrimer. This mechanism is qualitatively different from that invoked for small-molecule analytes.     

Mechanism of charging and supercharging molecules in electrospray ionization

The origin of the extent of charging and the mechanism by which multiply charged ions are formed in electrospray ionization have been hotly debated for over a decade. Many factors can affect the number of charges on an analyte ion. Here, we investigate the extent of charging of poly(propyleneimine) dendrimers (generations 3.0 and 5.0), cytochrome c, poly(ethylene glycol)s, and 1,n-diaminoalkanes formed from solutions of different composition. We demonstrate that in the absence of other factors, the surface tension of the electrospray droplet late in the desolvation process is a significant factor in determining the overall analyte charge. For poly(ethylene glycol)s, 1,n-diaminoalkanes, and poly(propyleneimine) dendrimers electrosprayed from single-component solutions, there is a clear relationship between the analyte charge and the solvent surface tension. Addition of m-nitrobenzyl alcohol (m-NBA) into electrospray solutions increases the charging when the original solution has a lower surface tension than m-NBA, but the degree of charging decreases when this compound is added to water, which has a higher surface tension. Similarly, the charging of cytochrome c ions formed from acidified denaturing solutions generally increases with increasing surface tension of the least volatile solvent. For the dendrimers investigated, there is a strong correlation between the average charge state of the dendrimer and the Rayleigh limiting charge calculated for a droplet of the same size as the analyte molecule and with the surface tension of the electrospray solvent. A bimodal charge distribution is observed for larger dendrimers formed from water/m-NBA solutions, suggesting the presence of more than one conformation in solution. A similar correlation is found between the extent of charging for 1,n-diaminoalkanes and the calculated Rayleigh limiting charge. These results provide strong evidence that multiply charged organic ions are formed by the charged residue mechanism. A significantly smaller extent of charging for both dendrimers and 1,n-diaminoalkanes would be expected if the ion evaporation mechanism played a significant role.     

PAMAM dendrimer-induced aggregation of 5(6)-carboxyfluorescein

The binding of the fluorescent polyanionic probe 5(6)-carboxyfluorescein (CF) to various generations of dendrimers (G3-G7) was studied in buffered aqueous media by absorbance and fluorescence spectroscopy and by isothermal titration calorimetry (ITC). Absorbance, fluorescence, and fluorescence anisotropy data were collected concurrently by using a multiwell plate format. Because ITC does not depend on the presence of a chromophore/fluorophore for measurement, it allowed the exploration of concentration ratios otherwise unattainable in the spectroscopy experiments. Qualitative dendrimer generational trends were observed and found to be consistent with dendrimer size and charge. However, a number of significant anomalies were found in the spectroscopic titration profiles, which led us to propose a binding model comprising multiple, concurrent binding regimes. The predictive value of the model was ascertained by construction of a binding simulation, which was consistent with the experimental results. Finally, ITC results afforded insights into the fundamental thermodynamic properties of the binding process along with trends found across dendrimer generations. Thermodynamic data were found to be in accordance with the proposed model.