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Biomedical subjects

Joseph Demeester

Publications and source records attributed to Joseph Demeester.

16 recordsLinked to original sources
PubMed2007

Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples.

We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.

Algorithms↗
PubMed2006

Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes.

Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.

Animals↗
PubMed2006

Ultrasound-responsive polymer-coated microbubbles that bind and protect DNA.

Ultrasound in combination with microbubbles has recently been considered by gene delivery scientists to be an interesting approach to enhance gene transfer into cells. Its low toxicity and simplicity to apply in vivo without major complications make this technology (sonoporation) especially attractive. Sonoporation of DNA has been evaluated in vivo by the injection of free plasmid DNA (pDNA) together with microbubbles (as used in diagnostic imaging) in the bloodstream. However, the in vivo gene-transfer efficiency in these experiments remained rather low. Both the enzymatic degradation of the injected pDNA as well as the low pDNA concentration in the neighborhood of sonoporated cell membranes may explain this low efficiency. Therefore, we developed polymer-coated microbubbles that can bind and protect the pDNA. Coating albumin-shelled microbubbles with poly(allylamine hydrochloride) (PAH) makes the surface charge of the microbubbles positive without drastically affecting the size distribution of the microbubbles, thereby not affecting the ultrasound responsiveness and injectability. The cationic coating allowed both to bind up to 0.1 pg of DNA per microbubble as well as to protect the bound DNA against nucleases. Finally, the PAH coating significantly increased the lifetime of the microbubbles (half-life approximately 7 h), making them more convenient for in vivo applications because more microbubbles are expected to reach the target organ. Binding and nuclease protection of DNA by polymer-coated diagnostic microbubbles has, to our knowledge, never been demonstrated. We conclude that these LbL-coated microbubbles might be significant in the further development of ultrasound-mediated gene delivery.

DNA↗
PubMed2006

Glucose-responsive polyelectrolyte capsules.

Phenylboronic acids are known to form covalent complexes with polyol compounds such as glucose. A novel polyelectrolyte, containing phenylboronic acid as a glucose-sensitive moiety, has been synthesized and used for the fabrication of glucose-sensitive hollow polyelectrolyte capsules using the layer-by-layer technique. The response to glucose was observed as a rather fast dissolution of the capsules when brought into contact with a glucose-containing medium. These polyelectrolyte capsules are the first polyelectrolyte capsules able to respond to a stimulus that can be provided by the human body (i.e., an increase in glucose concentration). Therefore, the concept we present has promising applications in the biomedical field for the controlled delivery of insulin.

Boronic Acids↗
PubMed2006

Self-exploding lipid-coated microgels.

Self-exploding microparticles show potential for advanced delivery of certain therapeutics. This study evaluates (1) whether degrading biodegradable dextran hydroxyethyl methacrylate (dex-HEMA) microgels can be coated by a lipid membrane and (2) whether the surrounding membrane can be ruptured by the increasing swelling pressure of the degrading microgel. We found that adsorption of charged liposomes to oppositely charged dex-HEMA microgels provides efficient coating of the microgels, whereby microparticles with a "core-shell" structure were clearly obtained. Especially, we could confirm experimentally that the swelling pressure increase of degrading dex-HEMA microgels can destroy the lipid membrane surrounding the microgels.

Adsorption↗
PubMed2005

Vitreous: a barrier to nonviral ocular gene therapy.

PURPOSE: Intravitreal injection of therapeutic DNA, complexed to nonviral carriers such as cationic liposomes, may be promising in the treatment of many severe retinal eye diseases. However, after intravitreal injection, such DNA/cationic liposome complexes-called lipoplexes (LPXs)-which are typically hundreds of nanometers in size, must first diffuse through the vitreous before they can reach the retina. The aim of this study was to elucidate whether vitreous is a barrier for the LPXs and to find strategies to overcome this barrier. METHODS: Fluorescent polystyrene nanospheres and LPXs were mixed with vitreous, and their mobility was monitored by fluorescence recovery after photobleaching (FRAP), a microscopy-based technique. The stability of LPXs and naked plasmid DNA in vitreous was studied by gel electrophoresis. RESULTS: We showed that polystyrene nanospheres, in our first experiments used as a model for the LPXs, do not diffuse freely into the vitreous but adhere to fibrillar structures in the vitreous, most likely to collagen fibers. Making the surfaces of the polystyrene nanospheres hydrophilic by attaching hydrophilic polyethylene glycol (PEG) chains at their surfaces circumvented the binding to fibrillar structures in the vitreous. FRAP revealed that "pegylated" polystyrene nanospheres, as long as they are smaller than 500 nm, are indeed mobile in the vitreous. It was further demonstrated that LPXs severely aggregate in vitreous and strongly bind to biopolymers in the vitreous, which immobilizes them completely. However, as observed for the polystyrene nanospheres, coating of the LPXs with PEG averted their aggregation in the vitreous and their binding to fibrillar structures. CONCLUSIONS: Modifying the surfaces of LPXs with hydrophilic PEG chains prevents them from aggregating in vitreous. In this way, LPXs are obtained that can freely move in vitreous, an absolute criterion for reaching the retina after intravitreal injection.

Animals↗
PubMed2004

On the biological activity of anti-ICAM-1 oligonucleotides complexed to non-viral carriers.

An important challenge in antisense technology remains the adequate delivery of the oligonucleotides (ON) to individual cells. Understanding the subcellular distribution of ONs and their carrier is essential to explain the (lack of) biological activity. The ability of several cationic carriers to efficiently deliver anti-ICAM-1 oligonucleotides to their site of action was studied using a cell-based assay. In this assay we evaluated the ability of the ONs to downregulate the expression of the ICAM-1-protein in A549 cells. To understand why some carrier/ONs combinations showed biological activity while others failed, flow cytometry and confocal laser scanning microscopy (CLSM) measurements were used to study cellular uptake and intracellular distribution of the (fluorescently labeled) ONs. We showed that free ONs (both PS-ONs and PO-ONs) and ONs complexed to pEGpEI failed to decrease the ICAM-1 protein level. This was due to the inability of the (free or complexed) ONs to enter the cell, as shown by flow cytometry and CLSM. Flow cytometry and CLSM showed cellular uptake when PO-ONs and PS-ONs were complexed to graft-pDMAEMA and Lipofectin. However, while the uptake and intracellular localization seemed similar for ONs complexed to, respectively, graft-pDMAEMA and Lipofectin, the biological activity of the ONs was clearly dependent on their carrier: both PO-ONs and PS-ONs complexed to graft-pDMAEMA reduced the ICAM-1 expression; however, when complexed to Lipofectin only PS-ONs showed biological activity. Also, PS-ONs complexed to graft-pDMAEMA were more active than PO-ONs complexed to graft-pDMAEMA which could not be explained by the results from CLSM and flow cytometry. While the ICAM-1 assay proves whether a certain pharmaceutical carrier successfully delivers ONs or not, it does not answer the important question why one carrier is successful while another one fails. Also, our study shows that flow cytometry and CLSM, although useful techniques, failed to clearly explain the difference in transfection behavior between graft-pDMAEMA and Lipofectin. As ONs become susceptible to degradation by cytosolic DNase as soon as they are released from their carrier, one could argue that a better understanding of the time and (intracellular) place at which the dissociation of the complexes occurs could be crucial to fully explain our observations.

Cell Line, Tumor↗
PubMed2004

"Programmed polymeric devices" for pulsed drug delivery.

Pharmaceutical research strives to design drug delivery systems that respond to therapeutic needs. Considering the facts that physiologic parameters (e.g., heart rate, blood pressure, and plasma concentration of hormones, plasma proteins, and enzymes) display constancy over time, drug delivery systems with a constant release profile have been designed. However, because of circadian rhythms in physiologic parameters and pathologic conditions (e.g., asthma, angina pectoris), the conventional paradigm concerning drug concentrations "the flatter the better" may not be what the organism may need. Instead, to correlate with our biological needs, "precisely timed drug delivery," which could be accomplished with "programmable dosage forms," is required. Precisely timed drug delivery may maximize therapeutic efficacy, may minimize dose frequency, and may reduce toxicity by avoiding side effects and drug tolerance. This paper outlines the concepts that have been proposed to release drugs in a pulsed manner from pharmaceutical devices.

Drug Delivery Systems↗
PubMed2003

Mobility and stability of gene complexes in biogels.

The tenacious secretions lining the conductive airways of cystic fibrosis (CF) patients may pose a significant barrier to successful gene therapy to the lung. In this work, we evaluated the diffusion of nanospheres and cationic DOTAP lipoplexes through CF sputum and the influence of CF mucus components on the physicochemical properties and gene expression of cationic DOTAP lipoplexes and neutral, pegylated GL67 lipoplexes. The number of particles transported through the sputum was extremely low (<0.5%) and strongly depending on the size of the particles, with almost no transport for the largest nanospheres (560 nm). For small particles (<150 nm), the low transport was primarily due to the long distance they have to travel through the sputum, while for larger particles also sterical obstruction was responsible for the low transport. Upon exposure of the cationic DOTAP lipoplexes to albumin, linear DNA or mucin (at concentration ratios expected to occur in vivo) a significant decrease in gene transfection activity was observed. This was primarily due to aggregation of the lipoplexes. However, exposure of pegylated GL67 lipoplexes to the same components did not affect their gene transfection activity. Indeed, it was determined that CF mucus components did not interact significantly with these neutral, pegylated GL67 lipoplexes.

Biological Transport↗
PubMed2003

Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope.

Confocal scanning laser microscopes (CSLMs) are equipped with the feature to photobleach user-defined regions. This makes them a handy tool to perform fluorescence recovery after photobleaching (FRAP) measurements. To allow quantification of such FRAP experiments, a three-dimensional model has been developed that describes the fluorescence recovery process for a disk-shaped geometry that is photobleached by the scanning beam of a CSLM. First the general mathematical basis is outlined describing the bleaching process for an arbitrary geometry bleached by a scanning laser beam. Next, these general expressions are applied to the bleaching by a CSLM of a disk-shaped geometry and an analytical solution is derived that describes three-dimensional fluorescence recovery in the bleached area as observed by the CSLM. The FRAP model is validated through both the Stokes-Einstein relation and the comparison of the measured diffusion coefficients with their theoretical estimates. Finally, the FRAP model is used to characterize the transport of FITC-dextrans through bulk three-dimensional biological materials: vitreous body isolated from bovine eyes, and lung sputum expectorated by cystic fibrosis patients. The decrease in the diffusion coefficient relative to its value in solution was dependent on the size of the FITC-dextrans in vitreous, whereas it was size-independent in cystic fibrosis sputum.

Algorithms↗
PubMed2003

Encoding microcarriers by spatial selective photobleaching.

Bead-based assays on very large numbers of molecules in gene expression studies, drug screening and clinical diagnostics, require the encoding of each of the microspheres according to the particular ligand bound to its surface. This allows mixing the uniquely encoded microspheres and subjecting them to an assay simultaneously. When a particular microsphere gives a positive reaction, the substance on its surface can be identified by reading the code. Previously reported techniques for colour encoding polymer microspheres only allow for a limited number of unique codes. Graphical encoding methods use metallic particles, which are rather uncommon in screening applications. Here, we demonstrate a new approach to encode polymer microspheres that are commonly used in screening applications, such as polystyrene microspheres, with a method that provides a virtually unlimited number of unique codes. Patterns can be written in fluorescently dyed microspheres by 'spatial selective photobleaching' and can be identified by confocal microscopy. Such encoded microparticles can find broad application in the collection and analysis of genetic information, high-throughput screening, medical diagnostics and combinatorial chemistry, and can also be used for labelling of consumer goods or as security labels to prevent counterfeiting.

Microspheres↗
PubMed2002

A comparison between the use of dynamic mechanical analysis and oscillatory shear rheometry for the characterisation of hydrogels.

In this study the use of dynamic mechanical analysis (DMA) for the mechanical characterisation of pharmaceutical hydrogels was evaluated. DMA was used in two different modes, the "controlled force" and the "multi-strain" (MS). The results obtained on dextran methacrylate hydrogels of various compositions were compared to those obtained using an oscillatory shear rheometer. The best agreement was found between the MS-DMA and the rheometer results. The moduli measured in MS-DMA were extrapolated towards zero compression to obtain the modulus of the hydrogels. This procedure resulted in good agreement with the data obtained with the rheometer. Hydrogels were analysed after swelling to equilibrium with both methods, DMA and rheology. A scaling between the elastic modulus (G') and the equilibrium swollen polymer volume fraction (v(2,s)) could be found, although the best correlation between G' and v(2,s) was obtained with the rheometer.

Hydrogels↗
PubMed2002

On the transport of lipoplexes through cystic fibrosis sputum.

PURPOSE: The aim of this study was to examine the extent to which plasmid DNA (pDNA) complexed to cationic liposomes diffuse through cystic fibrosis (CF) sputum. The influence of the physical and chemical properties of the sputa was evaluated. We further investigated whether degradation of the sputa by recombinant human DNase I (rhDNase I) enhances the transport. METHODS: The transport of lipoplexes was studied through layers of CF sputa placed between the donor and acceptor compartment of vertical diffusion chambers. The content of the acceptor compartment was analyzed by confocal fluorescence microscopy, gel electrophoresis and Southern blotting. The influence of linear DNA present in the CF sputa on the size, surface charge and gene expression of the lipoplexes was evaluated by dynamic light scattering, particle electrophoresis and transfection experiments. RESULTS: Lipoplexes were observed in the acceptor compartments. However, the percent of diffused lipoplexes was low: 0.05/% +/- 0.01%. It was found that both steric obstruction by the sputa as well as the long" distance the lipoplexes have to travel were responsible for this low transport. Surprisingly, the transport occurred better through more viscoelastic sputa. The DNA in the CF sputa also retarded the transport, which was attributed to aggregation of the lipoplexes by the DNA. Finally, rhDNase I moderately enhanced the diffusion of lipoplexes. CONCLUSIONS: CF sputum drastically retards the diffusion of lipoplexes. DNA in the sputa aggregates the lipoplexes. This may lower the transport of lipoplexes through the sputa and gene expression. Pretreatment of CF patients with rhDNase I may enhance the efficiency of CF gene therapy, as it allows a better transport of the lipoplexes through the sputum and as it partly removes the sputum which will result in a thinner sputum layer on top of the epithelial cells.

Analysis of Variance↗
PubMed2002

Encoding microcarriers: present and future technologies.

In answer to the ever-increasing need to carry out many assays simultaneously in drug screening and drug discovery, several microcarrier-based multiplex technologies have arisen in the past few years. The compounds to be screened are attached to the surface of microcarriers, which can be mixed together in a vessel that contains the target analyte. Each microcarrier has to be encoded to know which compound is attached to its surface. In this article, the methods that have been developed for the encoding of microcarriers are reviewed and discussed.

Biological Assay↗
PubMedDate not supplied

Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein.

In this study we examine the implications of excitation saturation on fluorescence recovery after photobleaching (FRAP) experiments. In particular we present both experimental and theoretical evidence that fluorescein, one of the most frequently used fluorophores in FRAP, does not always comply with the basic assumptions that are made in many FRAP models: an invariant bleaching illumination intensity distribution (BID) in combination with first-order photobleaching kinetics. High light intensity levels, which are typical for the photobleaching phase of FRAP experiments, can cause excitation saturation of fluorescein in the excited triplet state. We show by experiments and computer simulations that under such saturating conditions the higher-order diffraction maxima of the BID substantially contribute to the photobleaching process and can no longer be neglected. As a result, the bleached regions are larger than expected theoretically from the FRAP models. Although this effect is not always directly evident from the FRAP experiments, neglecting it may shift the calculated diffusion coefficient by as much as over one order of magnitude. We present a discussion on the implications of this saturation effect on various types of FRAP models.

Algorithms↗