Xianwen Lou

Modified poly(propylene imine) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes).

The major bottleneck in gene therapy remains the issue of delivery. In this work, various modified poly(propylene imine) (PPI) dendrimers are introduced as gene transfection agents. Commercially available PPI-dendrimers have been modified (i) at the exterior primary amines with acetyl groups or glycol gallate (PEG-like) groups, and (ii) at the interior tertiary amines with methyl iodide (MeI) or MeCl to produce multiple quaternized cationic sites in the core of the dendrimer. The prepared materials have been tested with respect to their binding capabilities to DNA, their toxicity in cell cultures, their in vitro transfection efficiency and their in vivo delivery possibilities. In all cases, a 33-mer oligonucleotide (DNAzyme) was used. Polyacrylamide gel electrophoresis (PAGE) studies have demonstrated strong but reversible binding, where the quarternized and higher generation dendrimer species have shown more potent binding. Typically, for the modified fourth PPI-dendrimers, binding is observed at a concentration of about 4 μM DNA and a dendrimer-DNA charge ratio of around 2:1–1:1. All the tested PPI-dendrimers display a low cellular toxicity, especially when higher serum contents are used in the culture medium. For example, most of the prepared fourth generation PPI-dendrimers are not or hardly toxic up to at least 20 μM in 20% serum. An in vitro characterization has revealed a high dendrimer-mediated intracellular uptake of the DNAzyme: all the tested fourth generation PPI-dendrimers display transfection efficiencies close to or exceeding 80%, even when the concentration of serum in the medium is increased from 10 to 40%. Finally, the potential of using modified PPI-dendrimers for in vivo gene therapy experiments is demonstrated. Injecting a G4-PEG(MeI)–ssDNA complex intravenously into Nude mice has resulted in a high nuclear uptake as confirmed by co-localization studies.

A simple way to speed up separations by GC-MS using short 0.53 mm columns and vacuum outlet conditions

There is a constant drive to speed up GC separations. Shorter analysis times provide more analyses per day, which reduces cost. One approach is to reduce column length and column diameter and columns of 0.15 mm i.d. have indeed grown in popularity. However, the majority of applications are still done with 0.25 mm and 0.32 mm columns.

Off-line size-exclusion chromatographic fractionation-matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for polymer characterization. Theoretical and experimental study

The parameters affecting the fractionation performance in size-exclusion chromatography (SEC) of broad polymer samples were investigated. Some equations were derived which enable the prediction of polydispersity (PD) in an SEC fraction. Good agreements were obtained between the calculated data and the experimental values. Based on these equations, SEC fractionation conditions were optimized. In the off-line SEC-matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), two different modes can be employed, i.e., using MALDI-MS to provide an absolute calibration curve for SEC, or using SEC as a sample preparation step for MALDI-MS measurements. It was demonstrated that it is more reliable to use the latter combination, because most problems inherent in SEC can be circumvented. Some guidelines for the optimization of off-line SEC fractionation-MALDI-TOF-MS were given. It was found that under optimized conditions normally only a few SEC fractions are already sufficient to separate a highly polydisperse sample into portions of low PD that can accurately be measured by MALDI-TOF-MS.

Temperature and pressure effects on solubility in supercritical carbon dioxide and retention in supercritical fluid chromatography

Solubilities of some polycyclic aromatic hydrocarbons (PAHs) in supercritical carbon dioxide were measured with a procedure based on a direct on-line combination of a saturation cell to a flame ionization detector. Acenaphthene, anthrance and chrysene were selected as the test solutes. A method was developed and evaluated which enables the measurement of the contribution of solute vapor pressure to the overall solubility. The effects of temperature and pressure on solubility in supercritical carbon dioxide were investigated and discussed in detail. The trends of solubility changes in supercritical carbon dioxide and the variations in observed retention in supercritical fluid chromatography (SFC) were correlated. Equations were derived to estimate the effects of temperature on the solutes affinity for the stationary phase in SFC.

Direct sample fraction deposition using electrospray in narrow-bore size-exclusion chromatography/matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for polymer characterization

A direct sample fraction deposition method was developed for off-line size-exclusion chromatography (SEC)/matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. By using electrospray, the SEC eluent, together with a suitable matrix solution added coaxially, was directly deposited on the MALDI plate. Owing to the formation of very small droplets in electrospray, solvent evaporation is much faster. The fractionation volume in narrow-bore SEC, which can directly be collected in one MALDI spot, can easily be optimized in the range of a few microlitres. In addition, fairly homogeneous sample spots were obtained. The possible influence of composition variation of the SEC effluent on the analytical results using direct fraction deposition was investigated; no substantial effects were observed. The applicability of the method was demonstrated by characterizing a broad poly(methyl methacrylate) sample. Copyright 2000 John Wiley & Sons, Ltd.

Generation of CsI cluster ions for mass calibration in matrix-assisted laser desorption/ionization mass spectrometry

A simple method was developed for the generation of cesium iodide (CsI) cluster ions up to m/z over 20,000 in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Calibration ions in both positive and negative ion modes can readily be generated from a single MALDI spot of CsI3 with 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) matrix. The major cluster ion series observed in the positive ion mode is [(CsI)nCs]+, and in the negative ion mode is [(CsI)nI]−. In both cluster series, ions spread evenly every 259.81 units. The easy method described here for the production of CsI cluster ions should be useful for MALDI MS calibrations.

Effects of Modifier Addition and Temperature Variation in SFE of Polymeric Materials

The effects of modifier addition and temperature variation on the supercritical fluid extraction of nylon-6 and poly(1,4-butylene terephthalate) samples are discussed. The modifiers studied include hexane, chloroform, methanol, and benzene. The influence of experimental parameters, such as extraction temperature and pressure, static time, and supercritical fluid flow rate, are investigated systematically with both pure and modified carbon dioxide. The addition of a suitable modifier results in significantly enhanced extraction rates, particularly at low extraction temperatures. A simple experimental setup for the introduction of additional volumes of modifier during the extraction was developed and evaluated. Guidelines for the optimization of the supercritical fluid extraction of polymeric samples are given.

On the mechanism of dynamic polymerization via recycled ss-DNA templated assembly of non-natural bases

We report on the mechanism of DNA templated dynamic polymerization and show that DNA of sufficient length can serve as a template for the polymerization of non-natural base building blocks in which the template can be used again by a simple heating–cooling step.

Characterization of poly(butylene terephthalate) by size-exclusion chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Poly(butylene terephthalate) (PBT) samples have been analyzed with size-exclusion chromatography (SEC) using a mixed solvent of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and chloroform as the mobile phase. Several matrices and different sample deposition methods have been investigated to analyze PBT with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Optimum results have been acquired by depositing PBT on top of a 2,4,6-trihydroxyacetophenone matrix. The found MALDI-TOF-MS method can be used to analyze the end group functionalities of PBT, as demonstrated with the samples at hand. By combining SEC (off-line) with MALDI-TOF-MS, absolute molecular masses of PBT can be measured, and these have been found to be considerably lower than those determined with SEC using polystyrene standards.

Influence of the Solvent and the Enantiomeric Purity on the Transition between Different Supramolecular Polymers

The self-assembly of two enantiomerically pure hexa(oligo(p-phenylene vinylene))-substituted benzenes having 24 stereocenters was studied in pure methylcyclohexane (MCH) and in a mixture of MCH/toluene (4:1). Irrespective of the solvent a cooperative supramolecular polymerization mechanism was determined for these star-shaped molecules by using temperature-dependent CD and UV/Vis spectroscopy. Quite remarkably, a transition from one helical supramolecular state (A) to a second more thermodynamically stable supramolecular helical assembly (B) was observed. The rate of the A→B transition was strongly dependent on the nature of the solvent; being faster in the solvent mixture than in pure MCH. By using size exclusion chromatography we could relate the increased rate to a decreased stability of the supramolecular A state in the solvent mixture. Next, we mixed the two enantiomerically pure hexa-substituted benzene derivatives in a so-called majority-rules experiment, which lead to the anitcipated chiral amplification in the A state. More importantly it appeared that the A→B transition was significantly hampered in these mixed systems. Furthermore, the absence of chiral amplification in the B state revealed the formation of separated enantiomerically pure assemblies. Therefore, by using a wide variety of spectroscopic and chromatographic techniques we determined the influence of solvent and enantiomeric purity on the transition between different supramolecular states.