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ABSTRACT: Cellulose nanocrystals (CNCs) were analyzed by asymmetric flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS) detection. Small fractions were collected from the output of the AF4 apparatus for investigation by transmission electron microscopy (TEM). The influence of CNC injection amount, the number of passes through a high-pressure homogenizer, and different CNC sources on the elution behavior and particle size distribution was investigated. The AF4-MALS results on crystal length were compared with those from TEM. Peak distortion and variation in elution profiles with the increase in sample load were observed. Good resolution was obtained when the injection mass varied from 20 to 40 μg, corresponding to injections of 4-8 μL at a starting concentration of ∼5 μg/μL; concentrations during the separation process and at the detector were significantly lower. As the number of homogenization treatments increased, the peak shape became narrower and more symmetrical. This indicates a narrowed crystal length distribution, but regardless of source or homogenization treatment, no CNC preparation was as uniform as tobacco mosaic virus, a well-known rigid rod model structure, whose length was found by AF4-MALS to be in agreement with literature values. CNCs derived from cotton contained longer crystals than those derived from microcrystalline cellulose, as shown by both AF4-MALS and TEM techniques. An advantage of AF4-MALS compared to TEM is the ability to sample large numbers of rodlike particles, which is challenging and time-consuming for TEM image analysis, especially without the presorting afforded by AF4. The good TMV results suggest a high degree of accuracy will pertain to the CNC size distribution measurements.
Biomacromolecules 07/2012; 13(9):2671-9. · 5.48 Impact Factor
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ABSTRACT: Etoposide (ETO), a widely used anti-cancer drug, is constrained by its low aqueous solubility and by side effects from both the drug and its solubilizing excipients. In this study, a recently discovered natural solubilizer rubusoside (RUB) was used to achieve the solubilization of ETO. Dynamic light scattering and freeze-fracture transmission electron microscopy studies showed that ETO and RUB formed ETO-RUB nanoparticles (∼6 nm in diameter). The powder of ETO-RUB nanoparticles was completely reconstitutable in water and remained stable in this solution at 25 and 37°C for at least 24h. Under other physiologic conditions, ETO solution was clear and free of precipitation at 25°C, but underwent various structural transformations. In PBS and simulated intestinal fluid, RUB-solubilized ETO underwent epimerization and equilibrated to cis-ETO. In simulated gastric fluid, RUB-solubilized ETO degraded to 4'-demethylepipodophyllotoxin-beta-d-glucoside and 4'-demethylepipodophyllotoxin. Higher temperatures favored epimerization or degradation. Furthermore, a side-by-side comparison with DMSO-solubilized ETO confirmed that the RUB-solubilized ETO showed no significant differences in cytotoxicity in colon, breast and prostate cancer cell lines. RUB effectively solubilized and stabilized etoposide, which sets the stage for further toxicology, bioavailability, and efficacy investigations.
International journal of pharmaceutics 06/2012; 434(1-2):453-9. · 2.96 Impact Factor
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ABSTRACT: Amine-functionalized colloidal silica finds use in a variety of applications and fundamental investigations. To explore convenient methods of synthesis and characterization of research-grade materials in relatively large quantities, nearly monodisperse colloidal silica particles were prepared by base-catalyzed hydrolysis of reagent-grade tetraethyl orthosilicate (TEOS) without the traditional time- and energy-consuming distillation step. Radius was varied reliably from 30 to 125 nm by changing the water/TEOS ratio. Asymmetric flow field flow fractionation (AF4) methods with online light scattering detection proved effective in assessing the uniformity of the various preparations. Even highly uniform commercial standards were resolved by AF4. The surface of the colloidal silica was decorated with amino groups using (3-aminopropyl) trimethoxysilane and spacer methyl groups from methyl-trimethoxysilane. The surface density of amino groups was quantified spectrophotometrically after reaction with ninhydrin; the nature of this analysis avoids interference from sample turbidity. As an alternative to the ninhydrin test, an empirical relationship between surface density of amino groups and zeta potential at low pH was found. The size of the colloidal silica was predictably decreased by etching with HF; this method will be effective for some preparations, despite a modest reduction in size uniformity.
Langmuir 03/2012; 28(13):5562-9. · 4.19 Impact Factor
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Pilong Li,
Sudeep Banjade,
Hui-Chun Cheng,
Soyeon Kim,
Baoyu Chen,
Liang Guo,
Marc Llaguno,
Javoris V Hollingsworth,
David S King,
Salman F Banani, Paul S Russo,
Qiu-Xing Jiang,
B Tracy Nixon,
Michael K Rosen
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ABSTRACT: Cells are organized on length scales ranging from ångström to micrometres. However, the mechanisms by which ångström-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.
Nature 03/2012; 483(7389):336-40. · 36.28 Impact Factor
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Michelle M Raiszadeh,
Mark M Ross, Paul S Russo,
Mary Ann Schaepper,
Weidong Zhou,
Jianghong Deng,
Daniel Ng,
April Dickson,
Cindy Dickson,
Monica Strom,
Carolina Osorio,
Thomas Soeprono,
Julia D Wulfkuhle,
Emanuel F Petricoin,
Lance A Liotta,
Wolff M Kirsch
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ABSTRACT: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC-MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC-MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC-MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers.
Journal of Proteome Research 02/2012; 11(4):2127-39. · 5.11 Impact Factor
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ABSTRACT: The aggregation of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in phosphate solutions was investigated as a function of pH, concentration, time, ionic strength, and solution preparation (either from dilution of a freshly prepared 2 mM stock or by direct preparation of μM solution concentrations) using a combination of complementary analytical techniques. UV-vis and fluorescence spectroscopy indicated the formation of staggered, side-by-side (J-type) assemblies. Their size and self-associative behavior were determined using analytical ultracentrifugation and small-angle X-ray scattering. Our results indicate that in neutral and basic solutions of H(2)TPPS(4-), porphyrin dimers and trimers are formed at micromolar concentrations and in the absence of NaCl to screen any ionic interactions. At these low concentrations and pH 4, the protonated H(4)TPPS(2-) species self-assembles, leading to the formation of particularly stable aggregates bearing 25 ± 3 macrocycles. At higher concentrations, these structures further organize or reorganize into tubular, rod-like shapes of various lengths, which were imaged by cryogenic and freeze-fracture transmission electron microscopy. Micron-scale fibrillar aggregates were obtained even at micromolar concentrations at pH 4 when prepared from dilution of a 2 mM stock solution, upon addition of NaCl, or both.
Biomacromolecules 01/2012; 13(1):60-72. · 5.48 Impact Factor
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ABSTRACT: Curcumin (CUR) is an active food compound, but its insolubility and instability in water contributes to low bioavailability. In this study, the solubility of CUR was enhanced by utilizing the solubilizing properties of rubusoside (RUB). The solubility of CUR in water increased linearly from 61 μg/mL to 2.318 mg/mL in the presence of RUB ranging from 1% to 10% (w/v). Dynamic light scattering and transmission electron microscopy studies found that CUR and RUB formed CUR-RUB nanoparticle (∼8 nm) complexes. The RUB-solubilized CUR was stable in physiological conditions and did not precipitate when diluted or degrade when spray-dried to a completely reconstitutable powder. Furthermore, cell viability assays demonstrated the efficacy of RUB-solubilized CUR against human colon, breast, and pancreatic cancer cell lines. The development of this new solubilized, stable, and biologically active CUR formulation lays the foundation for future bioavailability improvement.
Journal of Pharmaceutical Sciences 02/2011; 100(7):2778-89. · 3.06 Impact Factor
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ABSTRACT: A method is presented for preparing core-shell silica-polypeptide composite particles with variable and controllable shell growth. The procedure is demonstrated using poly(carbobenzoxy-L-lysine) and poly(benzyl-L-glutamate); after deprotection, these can lead to the most common basic and acidic homopolypeptides, poly(L-lysine) and poly(L-glutamic acid). Control over shell thickness is made possible by sequential addition of N-carboxyanhydride peptide monomer to surfaces that have been functionalized with an amino initiator combined with a surface passivation agent. This results in a series of particles having different shell thicknesses. Variation of shell thickness was evident both in light scattering and in thermogravimetric assays. The shells were visible by transmission electron microscopy; these images along with light scattering measurements suggest the polymers in the shells are highly solvated.
Langmuir 10/2010; 26(19):15604-13. · 4.19 Impact Factor
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ABSTRACT: The 40-residue peptide isoform beta-amyloid (Abeta(1-40)) is associated with Alzheimer's disease. Although found in the tangles and fibrous mats that characterize the brain in advanced stages of the disease, the toxic form of Abeta is believed to be oligomers or "protofibrils". Characterization of these fairly small structures in solution, especially in the presence of the much larger assemblies they also form, is a daunting task. Additionally, little is known about the rate of Abeta assembly or whether it can be triggered easily. Perhaps most importantly, the conditions for reversing assembly are not fully understood. Fluorescence photobleaching with modulation detection of the recovery profile is a sensitive and materials-efficient way to measure diffusers over a wide range of hydrodynamic sizes. The method does require attachment of a fluorescent label. Experiments to validate the use of 5-carboxyfluorescein-labeled Abeta(1-40) as a representative of the unlabeled, naturally occurring material included variation of photobleaching time and mixture of labeled and unlabeled materials. A dialysis cell facilitated rapid in situ changes in pH and salt conditions. Multiple steps and complex protocols can be explored with relative ease. Oligomeric aggregates were found by fluorescence photobleaching recovery to respond readily to pH and salt conditions. Changing these external cues leads to formation or disassembly of aggregates smaller than 100 nm within minutes.
Biomacromolecules 02/2010; 11(2):341-7. · 5.48 Impact Factor
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ABSTRACT: The synthesis of organo-soluble hybrid particles with a hydrophobic, α-helical polypeptide shell and silica core has been achieved by combining ring-opening polymerization and click chemistry. Alkyne end-terminated poly(γ-stearyl-α-l-glutamate) (alkyne-PSLG) was prepared by the ring-opening polymerization of the N-carboxyanhydride of γ-stearyl-α-l-glutamate using propargylamine as initiator. The molecular weight and structure of this polypeptide were characterized by GPC and MALDI, and the α-helical nature was established by 1H NMR and FTIR. Azide-functionalized silica particles (azido-silica) were prepared by the functionalization of silica particles with 3-bromopropyltrichlorosilane followed by nucleophilic substitution with sodium azide. The azide functionalization was confirmed by FTIR and XPS. Alkyne-PSLG was coupled to azido-silica by click reaction in tetrahydrofuran or toluene in the presence of pentamethyldiethylenetriamine and copper(I) bromide. Further characterization of the product using XPS, FTIR, and 1H NMR revealed that the grafted polypeptide retained its α-helical nature and formed colloidal particles that readily dispersed in organic solvents. These hydrophobic, polypeptide-functionalized particles can serve as model systems in studies of colloid dynamics and/or crystallization. They may also find use in investigations designed to model enzyme activation or the properties of hydrophobic proteins in cell membranes.
11/2009;
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ABSTRACT: A series of tree-shaped, amphiphilic dendrimers was synthesized. The products belong to the family of one-directional arborols of the form ([9]-n), where the notation signifies that each molecule has nine hydroxyl groups ([9]-) as the hydrophilic head and an alkyl chain as the hydrophobic moiety (n = 6, 8, or 10 carbon atoms). The surfactant character changes dramatically as the number of methylene groups increases. The critical micelle concentration of [9]-6 was determined, and pressure-area isotherms of the less soluble [9]-8 and [9]-10 were obtained. Large structures existed atop the spread layers. Large structures were also found in solutions of [9]-6.
Langmuir 04/2008; 24(5):1858-62. · 4.19 Impact Factor
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ABSTRACT: Two-dimensional arborols are bolaform amphiphiles in which a central, hydrophobic spacer separates twin hydrophilic ends. Under appropriate conditions, these relatively small molecules assemble into very long fibers; subsequently, the system gels if the arborol concentration is sufficiently high. The diffusion of linear or slightly branched dextran probes in 3 and 6% arborol gels, as determined by fluorescence photobleaching recovery, resembles that of dextrans in water, suggesting a highly open network structure. Melting the gels produces almost no change in diffusion of the dextran probes. Small-angle X-ray scattering (SAXS) of wet arborol gels at different concentrations and temperatures reveals the diameter of the repeating unit of the fibers to be 8.26+/-0.68 nm. This dimension, which is independent of concentration and temperature, exceeds the length of a single arborol molecule by about a factor of 3. Rheological investigation identifies the linear response regime of the gels and permits an examination of the weak correlation between dextran probe diffusion and gel viscoelasticity.
The Journal of Physical Chemistry B 02/2008; 112(1):29-35. · 3.70 Impact Factor
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ABSTRACT: The beta-amyloid peptide aggregates via a nucleation pathway where micellar aggregates propagate to form oligomers (protofibrils), which then polymerize into insoluble fibrils. This fibrillogenic process has been linked to the pathogenesis associated with Alzheimer's disease. One purpose of this chapter is to provide a protocol for reliably producing monomeric Abeta as a starting point for physical and biological studies. Many research groups have used organic solvents to disaggregate pre-seeded Abeta in an attempt to acquire monomeric starting materials. Others have used instrumental techniques such as size exclusion chromatography to isolate monomer, structural intermediates, and fibrils and study their affects on Abeta nucleation. This chapter discusses a modified method of Abeta preparation using organic solvents followed by dissolution into aqueous phosphate buffer systems that renders monomeric Abeta starting solutions for kinetic experiments. Additionally, this chapter details a number of physical techniques such as scanning force microscopy, circular dichroism spectroscopy, transmission electron microscopy, fluorescence spectroscopy, fluorescence photobleaching recovery, and dynamic light scattering, together with physiological techniques such as cell viability assays to characterize Abeta nucleation, aggregation, and fibrillization and the potential biological activity of the various Abeta particles.
Methods in molecular biology (Clifton, N.J.) 02/2007; 386:203-25.
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ABSTRACT: Dynamic light scattering is a powerful, noninvasive technique for the study of particles and macromolecules in solution. While commercial instruments are widely available, at any one time most of them can only measure the signal from a single detector positioned at a particular angle. For many systems, such as gels, solutions of nonspherical particles with dimensions exceeding about 100 nm, and polydisperse samples containing large components, proper characterization requires measurements at multiple angles. Such complete experiments are tedious, requiring the detector to be moved to each new angle. Even if the detector is motorized, the process is slow and this limits the study of evolving systems. To improve the situation, a multicorrelator apparatus for simultaneous multiple angle dynamic light scattering has been constructed. Provision is made for depolarized dynamic light scattering, and it is shown that in favorable cases a particle size can be obtained without prior knowledge of viscosity. Once that particle size is determined, the viscosity of the suspension can be measured. The instrument is well suited to the observation of kinetics of aggregation, growth, or gelation, but its utility is demonstrated with simple particulate systems.
Review of Scientific Instruments 04/2006; 77(4):043902-043902-6. · 1.37 Impact Factor
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ABSTRACT: The diffusion of poly(styrenesulfonate) sodium salt (NaPSS) was investigated using dialysis dynamic light scattering (DLS) and dialysis fluorescence photobleaching recovery (FPR). Never-dried or “virgin” NaPSS was synthesized directly from 4-styrenesulfonic sodium salt to achieve 100% sulfonation. Upon reducing the ionic strength directly in the DLS cell by dialysis, a clean sample developed clearly distinct fast and slow modes that were first identified as an extraordinary phase in low-salt solutions of poly-l-lysine by Lin, Lee, and Schurr [Biopolymers 1978, 17, 1041]. This result complements published polyelectrolyte investigations in a high-dielectric constant organic solvent, and also studies where the degree of ionization was tuned, which confirms that hydrophobic patches along the polymer chain are not required for the extraordinary behavior. The fast mode dominated even at low ionic strength, with scattering amplitude exceeding 70% of the total. For the virgin NaPSS sample in the dialysis cell, there is no convincing evidence of a slow mode at high salt (≥200 mM NaCl). The appearance of distinct slow and fast modes proved reversible upon removing and adding salt by dialysis, without any other perturbation save restoration of the concentration by dialysis centrifugation. This suggests that the behavior represents a thermodynamically equilibrated state. Dialysis FPR measurements of aqueous solutions of a commercial NaPSS that was labeled with fluoresceinamine (LNaPSS) showed no obvious long-range ordering. A reversible decrease in the optical tracer self-diffusion coefficient of LNaPSS as salt is dialyzed out of the solution is instead attributed to chain expansion. Comparison of FPR and DLS on a mixed LNaPSS/NaPSS sample suggests that the residence time of a chain in temporal aggregates [Schmitz et al. Biopolymers 1984, 23, 1637] that are thought to be responsible for the DLS slow mode is shorter than the FPR time scale.
12/2005;
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11/2005;
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ABSTRACT: Depolarized dynamic light scattering is used to measure the translational and rotational diffusion of a rodlike probe, tobacco mosaic virus, in matrix solutions of dextran, an extended polymer with some branching, and globular Ficoll. Translation and rotation both decline almost exponentially as the concentration of either matrix rises. The ratio of rotational to translational diffusion is similar in dextran or Ficoll solutions and close to the values expected from continuum theories of the friction of rodlike particles. Reinforcing a continuum picture in which hydrodynamic effects surpass any due to topological constraints, the declines in rotational and translational motion are almost inversely proportional to the solution viscosity. Only modest and gradual deviations from Stokes−Einstein behavior are observed, even at high matrix concentrations. This stands in stark contrast to an earlier study by this group [Macromolecules 1997, 30, 4920−4926]. The difference may be traced to the subtle effects of optical rotation (dextran and Ficoll are chiral) on instrument alignment, coupled to the weak depolarization of the strongly scattering tobacco mosaic virus and the very slow rotations encountered at high matrix content. In optically inactive solutions, and even in optically active ones studied with the correct and tedious alignment, a particle the shape and size of tobacco mosaic virus can serve as an effective microrheological probe. Confirming this conclusion, the apparent microviscosity obtained by inverting rotational or translational diffusion coefficients reflected the molecular weight trend, at a particular concentration, of shear viscosity measured in a cone and plate device.
11/2004;
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Langmuir 02/2004; 20(1):266-9. · 4.19 Impact Factor
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ABSTRACT: Methods for the self-assembly, as well as directed construction, of hexaruthenium metallomacrocycles employing bisterpyridine building blocks are described. Self-assembly is effected by a combination of equimolar mixtures of bismetalated and nonmetalated bis(terpyridinyl) monomers each possessing the requisite planar, 60 degrees, terpyridine-metal-terpyridine connectivity. Stepwise synthesis of the identical hexamer is also discussed and used to aid in verification of the self-assembled product. Preparation and analysis of the related FeII metallomacrocycle are detailed and its TEM image confirms the hexameric structure. Characterization of the metalated products includes cyclic voltammetry along with the routine analytical techniques.
Chemistry 08/2002; 8(13):2946-54. · 5.93 Impact Factor
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04/2002;
