Steric Barrier Research Articles

Flow Improvement of Waxy Oils by Modulating Long-Chain Paraffin Crystallization with Comb Polymers: An Observation by X-ray Diffraction

To understand the flow improvement of waxy oils by comb-type polymer additives, the crystallization of long-chain n-paraffins from solutions of decane was studied by X-ray diffraction. The effects of poly(ethylene-butene) (PEB) and poly(maleic anhydride amide-co-α-olefin) (MAC) polymers on the crystallization of C28, C32, and C36 n-paraffins and their mixtures were studied. In the absence of the polymer additives, paraffin platelet crystals give well-resolved low-angle spectra. PEB and MAC reduce the low-angle scattering from the layered structure, which is expected since the polymers cocrystallize with the paraffin and the noncrystallizable portion of the chain provides a steric barrier against platelet stacking. This is consistent with observations by optical microscopy and differential scanning calorimetry and can account for the significant reduction of yield stresses of model waxy oils upon addition of these polymers. The assembly of PEB and MAC with paraffins during crystallization results in improv...

Ligand Conjugation to Bimodal Poly(ethylene glycol) Brush Layers on Microbubbles

Using microbubbles as model systems, we examined molecular diffusion and binding to colloidal surfaces in bimodal poly(ethylene glycol) (PEG) brush layers. A microbubble is a gaseous colloidal particle with a diameter of less than 10 mum, of which the surface comprises amphiphilic phospholipids self-assembled to form a lipid monolayer shell. Due to the compressible gas core, microbubbles provide a sensitive acoustic response and are currently used as ultrasound contrast agents. Similar to the design of long circulating liposomes, PEG chains are typically incorporated into the shell of microbubbles to form a steric barrier against coalescence and adsorption of macromolecules to the microbubble surface. We introduced a buried-ligand architecture (BLA) design where the microbubble surface was coated with a bimodal PEG brush. After microbubbles were generated, fluorescent ligands with different molecular weights were conjugated to the tethered functional groups on the shorter PEG chains, while the longer PEG chains served as a shield to protect these ligands from exposure to the surrounding environment. BLA microbubbles reduced the binding of macromolecules (>10 kDa) to the tethers due to the steric hindrance of the PEG overbrush while allowing the uninhibited attachment of small molecules (<1 kDa). Roughly 40% less fluorescein-conjugated streptavidin (SA-FITC) bound to BLA microbubbles compared to exposed-ligand architecture (ELA) microbubbles. The binding of SA-FITC to BLA microbubbles suggested a possible phase separation between the lipid species on the surface leading to populations of revealed and concealed ligands. Ligand conjugation kinetics was independent of microbubble size, regardless of ligand size or microbubble architecture. We observed, for the first time, streptavidin-induced surface structure formation for ELA microbubbles and proposed that this phenomenon may be correlated to flow cytometry scattering measurements. We therefore demonstrated the feasibility of postlabeling for small-molecule ligands to BLA microbubbles to generate stealth targeted ultrasound contrast agents.

Significance of host cell kinases in herpes simplex virus type 1 egress and lamin-associated protein disassembly from the nuclear lamina

The nuclear lamina is thought to be a steric barrier to the herpesvirus capsid. Disruption of the lamina accompanied by phosphorylation of lamina proteins is a conserved feature of herpesvirus infection. In HSV-1-infected cells, protein kinase C (PKC) alpha and delta isoforms are recruited to the nuclear membrane and PKC delta has been implicated in phosphorylation of emerin and lamin B. We tested two critical hypotheses about the mechanism and significance of lamina disruption. First, we show that chemical inhibition of all PKC isoforms reduced viral growth five-fold and inhibited capsid egress from the nucleus. However, specific inhibition of either conventional PKCs or PKC delta does not inhibit viral growth. Second, we show hyperphosphorylation of emerin by viral and cellular kinases is required for its disassociation from the lamina. These data support hypothesis that phosphorylation of lamina components mediates lamina disruption during HSV nuclear egress.

BETTER ROUTE TO HARD-TO-GET ISOMERS

A NEW SYNTHETIC path to atropisomers—a class of chiral compounds that includes drugs, natural products, and ligands for asymmetric catalysis—could open the door to creating a wider range of such compounds, which until now have been difficult to synthesize. Atropisomers are stereoisomers that can be enantiomeric, owing to a steric barrier to rotation about a single bond. Examples include biaryl compounds with bulky substituents, natural products such as the antibiotic vancomycin, and BINAP, a ligand widely used as a catalyst in asymmetric synthesis. Previous techniques for creating atropisomers relied on capturing them with selective reactions of rapidly equilibrating compounds or forging new bonds between biaryl fragments. But catalytic reactions that can produce specific atropisomeric enantiomers from equilibrating racemic mixtures have not been extensively developed. Now, synthetic chemist Scott J. Miller and coworkers Jeffrey L. Gustafson and Daniel Lim of Yale University have crafted what could prove ...

ChemInform Abstract: Host-Guest Chemistry of Calixarene Capsules

Reversible encapsulation is one of the more recent forms of molecular recognition. Small molecule targets are completely surrounded by larger molecular assemblies and steric barriers keep the guest from escaping the host. Calix[4]arenes make useful modules for capsule construction and the review traces their history. Applications in physical organic chemistry, materials science and spectroscopy are described.

Long circulating nanoparticles of etoposide using PLGA‐MPEG and PLGA‐pluronic block copolymers: characterization, drug‐release, blood‐clearance, and biodistribution studies

AbstractThe anti‐leukemic drug, etoposide (ETO), has variable oral bioavailability ranging from 24–74% with a short terminal half‐life of 1.5 h i.v. necessitating continuous infusion for 24–34 h for the treatment of leukemia. In the present study, etoposide‐loaded PLGA‐based surface‐modified nanoparticles (NPs) with long circulation were designed as an alternative to continuous i.v. administration. PLGA‐mPEG and PLGA‐PLURONIC copolymers were synthesised and used to prepared ETO‐loaded NPs by high‐pressure homogenization. The mean particle size of ETO‐loaded PLGA‐MPEG nanoparticles was 94.02±3.4 nm, with an Entrapment Efficiency (EE) of 71.2% and zeta potential value of −6.9±1.3 mV. ETO‐loaded PLGA‐pluronic nanoparticles had a mean particle size of 148.0±2.1 nm, an EE of 73.12±2.7%, and zeta potential value of −21.5±1.6 mV. In vitro release of the pure drug was complete within 4 h, but was sustained up to 7 days from PLGA‐mPEG nanoparticles and for 5 days from PLGA‐pluronic nanoparticles. Release was first order and followed non‐Fickian diffusion kinetics in both instances. ETO and ETO‐loaded PLGA nanoparticles labeled with 99mTc were used in blood clearance studies in rats where the two coated NPs, 99mTc‐ ETO‐PLGA‐PLU NP and 99mTc‐ ETO‐PLGA‐mPEG NP, were found to be available in higher concentrations in the circulation as compared to the pure drug. Biodistribution studies in mice showed that ETO‐loaded PLGA‐MPEG NP and PLGA‐PLURONIC NP had reduced uptake by the RES due to their steric barrier properties and were present in the circulation for a longer time. Moreover, the NPs had greater uptake in bone and brain where concentration of the free drug, ETO, was negligible. Drug delivered from these NPs could result in a single i.v. injection that would release the drug for a number of days, which would be potentially beneficial and in better control of leukemia therapy. Drug Dev Res 71: 228–239, 2010. © 2010 Wiley‐Liss, Inc.

Replica-exchange method in van der Waals radius space: Overcoming steric restrictions for biomolecules

We present a new type of the Hamiltonian replica-exchange method, where the van der Waals radius parameter and not the temperature is exchanged. By decreasing the van der Waals radii, which control spatial sizes of atoms, this Hamiltonian replica-exchange method overcomes the steric restrictions and energy barriers. Furthermore, the simulation based on this method escapes from the local-minimum free-energy states and realizes effective sampling in the conformational space. We applied this method to an alanine dipeptide in aqueous solution and showed the effectiveness of the method by comparing the results with those obtained from the conventional canonical and replica-exchange methods.

R‐ to T‐ Transition in Fructose‐1,6‐bisphosphatase: Loop Displacement is the Penultimate Step to Inactivation

AMP transforms fructose‐1, 6‐bisphosphatase (FBPase) from its active R‐state to its inactive T‐state. During this quaternary transition, a catalytically essential loop (residues 52–72) leaves the active site (engaged conformation) and moves to its inactive (disengaged) conformation. Structures of FBPase and molecular dynamic simulations reveal the mechanism of loop displacement. The AMP complex of Asp10 FBPase provides the first example of an engaged dynamic loop in a near‐T quaternary state. Subunit rotation forces loop 190 (from subunit C1) into contact with the hinge (residues 52–57) of the dynamic loop of subunit C2. Additional conformational changes in residues 264–274 of subunit C2 reduce space available to the engaged conformation of loop 52–72. Target Molecular Dynamics indicates the absence of steric barriers in subunit rotations but requires the displacement of the loop 50–72 from its engaged conformation in order to achieve the T‐state. Principal Component Analysis reveals anti‐correlated motions, which cause steric clashes consistent with the displacement of loop 52–72 from its engaged conformation. This research is supported by NIH grant NS 10546.

Interaction and dispersion stability of alumina suspension with PAA in N,N′-dimethylformamide

The alumina suspension in N,N′-dimethylformamide (DMF) was studied in the presence of a polyacrylic acid (PAA) dispersant. The sedimentation measurements of the suspensions show that PAA can impart favorable dispersion stability to alumina in DMF over a wide apparent pH (pH a) range. The adsorption of PAA on alumina and the zeta potential of the suspension are pH a dependent. FTIR-ATR spectroscopic studies reveal that the different interaction mechanisms between PAA and alumina work as a function of pH a. But the observable difference of dispersion between pH a values of 2.6–12.7 is small according to the sedimentation experiments. This can be explained by a combination of the static effect of PAA dispersant and different sizes of steric barrier resulting from the configurational variation of PAA with changing pH a values.

DNA binding by pixantrone

The binding of the anticancer drug pixantrone (6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione dimaleate) to the octanucleotide duplexes d(ACGATCGT)(2) and the corresponding C-5 methylated cytosine ((5Me)C) analogue d(A(5Me)CGAT(5Me)CGT)(2) has been studied by NMR spectroscopy and molecular modelling. The large upfield shifts observed for the resonances from the aromatic protons of pixantrone upon addition to either d(ACGATCGT)(2) or the corresponding (5Me)C analogue is consistent with the drug binding the octanucleotides by intercalation. The selective reduction in the sequential NOEs between the C(2)-G(3) and C(6)-G(7) nucleotides in NOESY spectra of either octanucleotide with added pixantrone confirms the intercalative binding mechanism. Strong NOEs from the side-chain ethylene protons of pixantrone to the H5 protons and the 5-CH(3) protons of the C(2) and C(6) residues of d(ACGATCGT)(2) and d(A(5Me)CGAT(5Me)CGT)(2), respectively, indicate that pixantrone predominantly intercalates from the DNA major groove at the 5'-CG and 5'-(5Me)CG sites. Simple molecular models based on the conclusions from the NMR experiments indicated that the (5Me)C groups do not represent a steric barrier to intercalation from the major groove. However, the observation of weak NOEs from the ethylene protons of pixantrone to a variety of minor groove protons from either octanucleotide suggests that the drug can also associate in the minor groove.

Stabilization of the nitric oxide (NO) prodrugs and anticancer leads, PABA/NO and Double JS-K, through incorporation into PEG-protected nanoparticles.

We report the stabilization of the nitric oxide (NO) prodrugs and anticancer lead compounds, PABA/NO (O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate) and "Double JS-K" 1,5-bis-{1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diol-2-ato}-2,4-dinitrobenzene, through their incorporation into polymer-protected nanoparticles. The prodrugs were formulated in block copolymer-stabilized nanoparticles with sizes from 220 to 450 nm by a novel rapid precipitation process. The block copolymers, with polyethylene glycol (PEG) soluble blocks, provide a steric barrier against NO prodrug activation by glutathione. Too rapid activation and NO release has been a major barrier to effective administration of this class of compounds. The nanoparticle stabilized PABA/NO are protected from attack by glutathione as evidenced by a significant increase in time taken for 50% decomposition from 15 min (unformulated) to 5 h (formulated); in the case of Double JS-K, the 50% decomposition time was extended from 4.5 min (unformulated) to 40 min (formulated). The more hydrophobic PABA/NO produced more stable nanoparticles and correspondingly more extended release times in comparison with Double JS-K. The hydrophobic blocks of the polymer were either polystyrene or polylactide. Both blocks produced nanoparticles of approximately the same size and release kinetics. This combination of PEG-protected nanoparticles with sizes appropriate for cancer targeting by enhanced permeation and retention (EPR) and delayed release of NO may afford enhanced therapeutic benefit.

Probing cellular mechanobiology in three-dimensional culture with collagen–agarose matrices

The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network.

Synthesis of macrocyclic compounds with steric barriers in macroheterocycle proceeding from (Z)-1,2,3,6-tetrahydrophthalic and phthalic acids

Using 1,2-dibromoethane, (E)-1,4-dibromo-2-butene, (Z)-1,2,3,6-tetrahydrophthalic and phthalic acids we obtained along [1+1] scheme uncommon macrocyclic compounds containing ester groups, multiple bonds, and amide moieties in the macroheterocycle. The neutralization of bis-acids with ethylenediamine gave the corresponding macrocyclic diammonium salts that on heating to the melting point were converted into the corresponding macrocyclic diamides. The structure of compounds obtained was established from the elemental analyses, IR and NMR spectra.

Dimer–tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacterium Chromohalobacter salexigens DSM3043: Both residues 134 and 136 are critical for the tetramer assembly

The subunit structure of Halomonas nucleoside diphosphate kinase (HaNDK) is a dimer, different from NDKs of other species. We have shown before that it is due to Glu134 in HaNDK, which results in steric hindrance and charge repulsion between two dimeric units and prevents further assembly into the tetramer and that changing the Glu134 to neutral Ala results in formation of a stable tetramer. To our surprise, both wild-type NDK from moderately halophilc Chromohalobacter salexigens (CsNDK/GNE: GNE represents Gly134-Asn135-Glu136) and mutant CsNDK/ ANE, both of which have a neutral amino acid at residue 134, were found to form a dimer. These constructs contain Glu136, which may also cause steric barrier and charge repulsion. A double mutant, CsNDK/ AN T, having Thr at 136 resulted in stable tetrameric assembly, supporting the above notion. A mutant CsNDK/ GN T reverted, however, to a dimer again, indicating that the introduced Ala residue at 134th in the double mutant generated a hydrophobic cluster consisting of the Ala residues and thereby stabilized dimer–dimer association of CsNDK assembly, while Gly destabilized it due to the loss of this cluster. Based on these observations, it is evident that both residues 134 and 136 contribute to the subunit assembly of CsNDK.

Redox-cleavable star cationic PDMAEMA by arm-first approach of ATRP as a nonviral vector for gene delivery

In this work, we synthesized a star cationic polymer(s-PDMAEMA) consisting of cleavable poly[ N, N-bis(acryloyl) cystamine](PBAC) crosslinked core and poly( N, N-dimethyl-ethylamine methacrylate) (PDMAEMA) arms by atomic transfer radical polymerization using one-pot “arm first” method. The s-PDMAEMA that was degradable in a mimic intracellular redox environment was more efficient in condensing DNA. It was shown that s-PDMAEMA achieved higher gene transfection levels relative to their linear precursors and s-PDMAEMA200 with longer and more arms exhibited superior transfection efficiencies and lower cytotoxicity compared to PEI25K. The buffer capacities were examined by acid–base titration; the pH-dependent morphological evolution and enzyme stability of PDMAEMA/DNA complexes were investigated by atomic force microscopy (AFM) and time-resolved fluorescence spectroscopy, respectively. The results indicated that the star polymers exhibited a stronger buffering ability than their linear precursors due to the increased inner osmotic pressure. By decreasing the pH from 7.4 to 5.0, the linear PDMEMA/DNA complexes became more compact; in contrast, s-PDMAEMA200/DNA complex adopted a loose morphology due to the steric barrier of inter-arms and outward extension of positively charged arms. Analysis of the fluorescence life times of free and intercalated ethidium bromide unveiled more effective protection of DNA afforded by s-PDMAEMA. The effect of medium pH on the star PDMAEMA system was smaller owing to the ability of densely tertiary amino groups along multiple arms to absorb more protons, which was favorable for endosomolytic escape of complexes.

Thermodynamically stable, size selective solubilization of carbon nanotubes in aqueous solutions of amphiphilic block copolymers

Two molecular modes of amphiphilic block copolymer-carbon nanotube interactions have been identified in the literature, one involving the adsorption of individual block copolymer molecules on the carbon nanotubes and the other involving the adsorption of multimolecular, spherical micelles. In both cases, the nature of stability imparted to the dispersion of carbon nanotubes in the aqueous medium is kinetic, controlled by the steric barrier imposed by the adsorbed individual block copolymer molecules or the adsorbed micelles. In this study, we propose another mode of molecular interaction, wherein the block copolymer molecules self-assemble around the nanotube, generating aggregates in which the nanotubes are solubilized. In this case, the resulting system is a thermodynamically stable nanocolloidal solution, similar to aqueous surfactant solutions, in contrast to the kinetically stabilized nanotube dispersions. To examine whether such solubilization of nanotubes is possible, we have constructed a simple phenomenological theory for the free energy change associated with solubilization and have performed illustrative numerical simulations based on the theory. The calculated results for the commercially available symmetric PEO-PPO-PEO triblock copolymers and for the PEO-PPO diblock copolymers having the same composition and molecular weight as the triblocks show that indeed the block copolymer molecules are capable of solubilizing the carbon nanotubes. While the block copolymers whose natural curvature is cylindrical are the best candidates to solubilize the nanotubes, other block copolymers whose natural curvature is spherical or lamellar, are also found capable of solubilizing the nanotubes. Most interestingly, the solubilization is found to be size specific suggesting that this can be developed into a practical method to fractionate carbon nanotubes by their diameter. These results are applicable to both single-walled and multiwalled carbon nanotubes and the general conclusions are valid also for other diblock and triblock copolymers.

Interaction Forces and Reversible Collapse of a Polymer Brush-Gated Nanopore

Nanopores are ubiquitous in nature and technology, yet relatively little is known about how surface-grafted polymers can affect the interaction forces at the pore. By fabricating Au nanorings on Si substrates, we have constructed a unique experimental platform that allows for direct atomic force microscope (AFM) measurements to be made on polyethylene glycol (PEG) chains locally anchored onto a geometric pore surface. Force measurements show that the PEG gives rise to a steric repulsive barrier that envelops the entire nanoring, signifying polymer brush formation. This is confirmed by a direct imaging of the PEG brush, which reversibly collapses by switching between poor and good solvent conditions to "open" and "close" the pore, respectively. From the view of interaction forces, these results highlight possible functionalities in which polymer brushes may play a role in minimizing fouling/clogging effects in synthetic nanopores and biological nuclear pore complexes (NPCs). By the mechanism of a reversible collapse, this work illustrates how polymer brush-gated nanopores may be used as nonfouling sieves for small molecules and/or solvent-controlled chemical valves that regulate solute traffic.

Adsorption and function of recombinant Factor VIII at the air–water interface in the presence of Tween 80

The air–water surface tension kinetic and steady state behavior exhibited by a recombinant Factor VIII were recorded in the presence of the surfactant Tween 80. The rate of surface tension decrease was determined to be greater for rFVIII–Tween mixtures than for Tween acting alone, at all Tween concentrations studied (8–108 ppm). In addition, steady state interfacial behavior was determined to be governed entirely by surfactant adsorption in mixtures with Tween concentrations greater than or equal to about 18 ppm. The formation of aggregates, tertiary structure change, and biological activity of rFVIII in agitated as well as unagitated samples were monitored using turbidity measurements, intrinsic fluorescence spectroscopy, and the activated partial thromboplastin time, respectively. The results are consistent with two mechanisms characterizing the surfactant-mediated stabilization of rFVIII: the rapid, preferential adsorption of Tween molecules relative to rFVIII molecules at the air–water interface, and Tween association with rFVIII in solution to provide a steric barrier to aggregation. While both mechanisms are relevant in the presence of an interface, evidence recorded here suggests that the preferential adsorption of Tween at the interface is the major stabilization mechanism while the effectiveness of Tween association with rFVIII is compromised in agitated samples. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3099–3107, 2009

Crystal structure of the complete integrin alphaVbeta3 ectodomain plus an alpha/beta transmembrane fragment.

We determined the crystal structure of 1TM-αVβ3, which represents the complete unconstrained ectodomain plus short C-terminal transmembrane stretches of the αV and β3 subunits. 1TM-αVβ3 is more compact and less active in solution when compared with ΔTM-αVβ3, which lacks the short C-terminal stretches. The structure reveals a bent conformation and defines the α–β interface between IE2 (EGF-like 2) and the thigh domains. Modifying this interface by site-directed mutagenesis leads to robust integrin activation. Fluorescent lifetime imaging microscopy of inactive full-length αVβ3 on live cells yields a donor–membrane acceptor distance, which is consistent with the bent conformation and does not change in the activated integrin. These data are the first direct demonstration of conformational coupling of the integrin leg and head domains, identify the IE2–thigh interface as a critical steric barrier in integrin activation, and suggest that inside-out activation in intact cells may involve conformational changes other than the postulated switch to a genu-linear state.

The use of PEGylated liposomes to prolong circulation lifetimes of tissue plasminogen activator

Tissue plasminogen activator (tPA), a widely used thrombolytic agent, has an application limit due to short half-life. To prolong the half-life of tPA, liposomes composed of egg phosphatidylcholine (EPC), cholesterol (CHOL) and sodium cholesterol-3-sulfate (CS) were prepared by lipid film method. In addition, distearolyphosphatidyl ethanolamine- N-poly(ethylene glycol) 2000 (DSPE–PEG 2000) was included to give steric barrier to liposomes. Physicochemical characteristics such as particle size, zeta potential, entrapment efficiency and long-term storage stability at 4 °C were investigated. The fibrinolytic activity of tPA-loaded in liposomes was confirmed by fibrin clot lysis assay. In vivo pharmacokinetic properties of tPA and the effect of PEG on the blood circulation of tPA-loaded in liposomes in circulation were also evaluated. Both conventional liposomes (EPCL) and PEGylated liposomes (EPC–PEGL) were proper as an injectable formulation with small particle size. Loading process of tPA into liposomes did not alter fibrinolytic activity of intact tPA. Encapsulation of tPA into EPCL and EPC–PEGL prolonged half-life of tPA by 16 and 21 folds compared with free tPA, respectively. Therefore, the use of liposomes could prolong the circulation lifetimes and longevity effect of liposomes on tPA was increased by PEG.