Efficient intracellular siRNA delivery by ethyleneimine-modified amphiphilic macromolecules

New materials that can bind and deliver oligonucleotides such as short interfering RNA (siRNA) without toxicity are greatly needed to fulfill the promise of therapeutic gene silencing. Amphiphilic macromolecules (AMs) were functionalized with linear ethyleneimines to create cationic AMs capable of complexing with siRNA. Structurally, the parent AM is formed from a mucic acid backbone whose tetra-hydroxy groups are alkylated with 12-carbon aliphatic chains to form the hydrophobic component of the macromolecule. This alkylated mucic acid is then mono-functionalized with poly(ethylene glycol) (PEG) as a hydrophilic component. The resulting AM contains a free carboxylic acid within the hydrophobic domain. In this work, linear ethyleneimines were conjugated to the free carboxylic acid to produce an AM with one primary amine (1N) or one primary amine and four secondary amines (5N). Further, an AM with amine substitution both to the free carboxylic acid in the hydrophobic domain and also to the adjacent PEG was synthesized to produce a polymer with one primary amine and eight secondary amines (9N), four located on each side of the AM hydrophobic domain. All amine-functionalized AMs formed nanoscale micelles but only the 5N and 9N AMs had cationic zeta potentials, which increased with increasing number of amines. All AMs exhibited less inherent cytotoxicity than linear polyethyleneimine (L-PEI) at concentrations of 10 μM and above. By increasing the length of the cationic ethyleneimine chain and the total number of amines, successful siRNA complexation and cellular siRNA delivery was achieved in a malignant glioma cell line. In addition, siRNA-induced silencing of firefly luciferase was observed using complexes of siRNA with the 9N AM and comparable to L-PEI, yet showed better cell viability at higher concentrations (above 10 μM). This work highlights the promise of cationic AMs as safe and efficient synthetic vectors for siRNA delivery. Specifically, a novel polymer (9N) was identified for efficient siRNA delivery to cancer cells and will be further evaluated.     

First direct measurement of the parity-violating coupling of the Z0 to the s quark

We present the first direct measurement of A(s), the parity-violating coupling of the Z0 boson to the strange quark, using approximately 550 000 e(+)e(-)-->Z0-->hadrons events recorded by the SLC Large Detector with a polarized e(-) beam. We tagged Z0-->s&smacr; events by the absence of B or D hadrons and the presence in each hemisphere of a high momentum K+/- or K(0)(s). Fitting the polar angle distributions of the strangeness-signed thrust axis gave A(s) = 0.895+/-0.066(stat)+/-0.062(syst). The analyzing power and uu+d&dmacr; background were constrained using the data, greatly reducing any model dependence.     

Precise measurement of the b-quark fragmentation function in Z0 boson decays

We have developed a new technique for inclusive reconstruction of the energy of B hadrons. The excellent efficiency and resolution of this technique allow us to make the most precise determination of the b-quark fragmentation function, using e(+)e(-)-->Z0 decays recorded in the SLAC Large Detector experiment. We compared our measurement with the predictions of a number of fragmentation models. We excluded several of these models and measured the average scaled energy of weakly decaying B hadrons to be = 0.714+/-0. 005(stat)+/-0.007(syst)+/-0.002 (model dependence).     

Molecular distribution of some commercial nonylphenol ethoxylates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Nonylphenol ethoxylates (NPEs) belong to a group of nonionic surfactants that are collectively referred to as alkylphenol ethoxylates (APEs). APEs find widespread use in heavy-duty commercial and household cleaning formulations, shampoos, and industrial processing, i.e. textile manufacture. Their environmental impact depends on the molecular distribution and the extent of their biodegradation in municipal sewage systems, waterways and rivers. We have established two sample preparation methods that have enabled the determination of the molecular distributions of six commercial nonylphenol ethoxylates using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). In both methods, alpha-cyano-4-hydroxycinnamic acid, dissolved in acetonitrile/tetrahydrofuran, was used as the matrix. In one set of experiments, the NPEs were dissolved in an acetonitrile/tetrahydrofuran solvent system prior to mixing with the matrix solution, and the resulting MALDI-TOF mass spectra produced mostly sodiated molecules [M + Na](+). The NPEs, all having the formula 4-(C(9)H(19))-C(6)H(4)-(OCH(2)CH(2))(n)-OH, are Surfonic (R)N-95, N-100, N-102, N-120, N-150 and N-300. Surfonic N-95 and N-100 gave n values of 5-20; Surfonic N-102, N-120, N-150, and N-300 gave n values of 5-21, 5-22, 8-25 and 15-40, respectively. In order to develop a sample preparation method that could be used with less polar NPEs, we dissolved the NPEs (except N-300) in pentane prior to mixing with the matrix solution, and found that the MALDI spectral quality was unaffected by the solvent systems. Copyright 1999 John Wiley & Sons, Ltd.     

Effective eddy viscosity in stratified turbulence

This paper investigates the effective eddy viscosity inferred from direct numerical simulations of decaying stratified and non-stratified turbulence. It is shown that stratification affects the horizontal eddy viscosity dramatically, by increasing non-local energy transfer between large and small horizontal scales. This non-local horizontal energy transfer is around 20% of the local horizontal energy transfer at the cutoff wavenumber k_c = 40. The non-local horizontal energy transfer occurs at large vertical wavenumbers, which may be larger than the buoyancy wavenumber k_b = N/u_rms, where N is the buoyancy frequency and u_rms is the root-mean-square velocity. By increasing the value of the test cutoff wavenumber k_c from large scales to the dissipation range, the non-local horizontal eddy viscosity decreases and the local eddy viscosity is dominant. Overall, the presence of stratification can significantly change the features of subgrid-scale (SGS) motions. Current SGS models should, therefore, be modified for use in large-eddy simulation of stratified turbulence.     

Mechanism and kinetics of ligand exchange between ferric citrate and desferrioxamine B

The kinetics of ligand exchange between ferric citrate and desferrioxamine B (DFB) was investigated at pH 8.0 and high citrate/Fe molar ratios (500-5000) with particular attention given to understanding the precise mechanism of ligand exchange. Ferric citrate complexes present in a test solution and therefore involved in the reaction with the incoming ligand (DFB) were initially examined by evaluating ferric citrate speciation on the basis of published thermodynamic constants. The speciation analysis indicated that mononuclear (mono- and dicitrate) ferric complexes are the major species responsible for the ligand exchange with DFB under the conditions examined in the present work. Given the tendency of DFB to adjunctively associate with the ferric citrate complexes, we propose a kinetic model containing the following three mechanisms: (i) direct association of DFB to the ferric dicitrate complex prior to any dissociation of citrate molecules from the Fe center, (ii) adjunctive association of DFB toward ferric monocitrate complex following dissociation of one molecule of citrate from the parent complex, and (iii) complexation of hydrated Fe by DFB after sequential dissociation of two molecules of citrate from the Fe center. Overall rates for the ligand exchange were determined by spectrophotometrically monitoring the formation of ferrioxamine B. Further analysis in quantifying the rate of each mechanism by use of published and determined rate constants of relevant elemental reactions suggested that the first and second mechanisms were significant under our experimental conditions where [Cit] ? [DFB] with the relative importance of these two pathways depending on citrate concentration.