Joseph Demeester

Cationic Polymer Based Gene Delivery Systems

Gene transfer to humans requires carriers for the plasmid DNA which canefficiently and safely carrythe gene into the nucleus of the desired cells. A series of chemically differentcationic polymers arecurrently being investigated for these purposes. Although many cationic polymersindeed condense DNAspontaneously, which is a requirement for gene transfer in most types of cells,the physicochemical andbiopharmaceutical behavior of the current generation of polyplexes severelylimits an efficient genetransfer in vitro and especially in vivo. This papersummarizes recent physicochemical and biologicalinformation on polyplexes and aims to provide new insights with respect to thistype of gene deliverysystem. Firstly, the chemical structure of frequently studied cationic polymersis represented. Secondly,the parameters influencing condensation of DNA by cationic polymers aredescribed. Thirdly, the surfaceproperties, solubility, aggregration behavior, degradation and dissociation ofpolyplexes are considered.The review ends by describing the in vitro and in vivo genetransfection behavior of polyplexes.

Release mechanisms for polyelectrolyte capsules

Polyelectrolyte capsules have recently been introduced as new microscopic vehicles which could have high potential in the biomedical field. In this critical review we give an introduction to the layer-by-layer (LbL) technique which is used to fabricate these polyelectrolyte capsules as well as to the different triggers that have been exploited to obtain drug release from these capsules. Furthermore, other types of triggered delivery systems are compared and critically discussed with regard to their clinical relevance. (171 references.).

The Use of Inhibitors to Study Endocytic Pathways of Gene Carriers: Optimization and Pitfalls

Nonviral gene complexes can enter mammalian cells through different endocytic pathways. For efficient optimization of the gene carrier it is important to profile its cellular uptake, because this largely determines its intracellular processing and subsequent transfection efficiency. Most of the current information on uptake of these gene-delivery vehicles is based on data following the use of chemical inhibitors of endocytic pathways. Here, we have performed a detailed characterization of four commonly used endocytosis inhibitors [chlorpromazine, genistein, methyl-beta-cyclodextrin (MbetaCD), and potassium depletion] on cell viability and endocytosis in five well-described cell lines. We found that chlorpromazine and to a lesser extent MbetaCD significantly decreased cell viability of some cell lines even after short incubation periods and at concentrations that are routinely used to inhibit endocytosis. Through analyzing the uptake and subcellular distribution of two fluorescent endocytic probes transferrin and lactosylceramide (LacCer) that are reported to enter cells via clathrin-dependent (CDE) and clathrin-independent (CIE) mechanisms, respectively, we showed poor specificity of these agents for inhibiting distinct endocytic pathways. Finally, we demonstrate that any inhibitory effects are highly cell line dependent. Overall, the data question the significance of performing endocytosis studies with these agents in the absence of very stringent controls.

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.

Design and Evaluation of Doxorubicin-containing Microbubbles for Ultrasound-triggered Doxorubicin Delivery: Cytotoxicity and Mechanisms Involved

Drug delivery with microbubbles and ultrasound is gaining more and more attention in the drug delivery field due to its noninvasiveness, local applicability, and proven safety in ultrasonic imaging techniques. In this article, we tried to improve the cytotoxicity of doxorubicin (DOX)-containing liposomes by preparing DOX-liposome-containing microbubbles for drug delivery with therapeutic ultrasound. In this way, the DOX release and uptake can be restricted to ultrasound-treated areas. Compared to DOX-liposomes, DOX-loaded microbubbles killed at least two times more melanoma cells after exposure to ultrasound. After treatment of the melanoma cells with DOX-liposome-loaded microbubbles and ultrasound, DOX was mainly present in the nuclei of the cancer cells, whereas it was mainly detected in the cytoplasm of cells treated with DOX-liposomes. Exposure of cells to DOX-liposome-loaded microbubbles and ultrasound caused an almost instantaneous cellular entry of the DOX. At least two mechanisms were identified that explain the fast uptake of DOX and the superior cell killing of DOX-liposome-loaded microbubbles and ultrasound. First, exposure of DOX-liposome-loaded microbubbles to ultrasound results in the release of free DOX that is more cytotoxic than DOX-liposomes. Second, the cellular entry of the released DOX is facilitated due to sonoporation of the cell membranes. The in vitro results shown in this article indicate that DOX-liposome-loaded microbubbles could be a very interesting tool to obtain an efficient ultrasound-controlled DOX delivery in vivo.

Controlled release of proteins from dextran hydrogels

Dextran hydrogels were investigated as matrices for the controlled release of proteins. The hydrogels were prepared by a free radical polymerization of aqueous solutions of glycidyl methacrylate derivatized dextran (dex-GMA). The release of three model proteins (lysozyme, BSA and IgG) from hydrogels varying in water content and degree of GMA-substitution was studied. The release rate was dependent on the size of the proteins and the equilibrium water content of the gels. It was shown that the release of the proteins was independent of the degree of GMA substitution of gels at high equilibrium water contents. On the other hand, the release was strongly affected by the degree of GMA substitution of the gels at low water contents. Some of these gels did not show any significant protein release, which suggests that the hydrogel mesh size was smaller than the protein diameter. In hydrogels where no screening occurred, the diffusion of the proteins could be effectively described by the free volume theory. Hydrogel mesh sizes were estimated from swelling data using the Flory-Rehner theory. This approach, however, resulted in an underestimation of the actual hydrogel mesh as derived from release experiments. Possible explanations for this discrepancy are discussed.

Fluorescence Recovery After Photobleaching: A Versatile Tool for Mobility and Interaction Measurements in Pharmaceutical Research

This review introduces the basics of fluorescence recovery after photobleaching (FRAP) from a theoretical and an instrumentational approach. The most interesting and innovative applications with a pharmaceutical point of view are briefly discussed and possible future applications are suggested. These future applications include research on the mobility of macromolecular drugs in macro- or microscopic pharmaceutical dosage forms, mobility, and binding of antitumor drugs in tumor tissue, intracellular trafficking of gene complexes and mobility of drugs in membranes prior to transmembrane penetration. The paper is also intended to be an introductory guideline to those who would like to get involved in FRAP related experimental techniques. Therefore, comprehensive details on different setups and data analysis are given, as well as a brief outline of the problems that may be encountered when performing FRAP. Overall, this review shows the great potential of FRAP in pharmaceutical research. This is complemented by our own results illustrating the possibility of performing FRAP in microscopic dosage forms (microspheres) using a high resolution variant of FRAP.

Extracellular barriers in respiratory gene therapy

Abstract Respiratory gene therapy has been considered for the treatment of a broad range of pulmonary disorders. However, respiratory secretions form an important barrier towards the pulmonary delivery of therapeutic nucleic acids. In this review we will start with a brief description of the biophysical properties of respiratory mucus and alveolar fluid. This must allow the reader to gain insights into the mechanisms by which respiratory secretions may impede the gene transfer efficiency of nucleic acid containing nanoparticles (NANs). Subsequently, we will summarize the efforts that have been done to understand the barrier properties of respiratory mucus and alveolar fluid towards the respiratory delivery of therapeutic nucleic acids. Finally, new and current strategies that can overcome the inhibitory effects of respiratory secretions are discussed.

An oral controlled release matrix pellet formulation containing nanocrystalline ketoprofen.

A controlled release pellet formulation using a NanoCrystal colloidal dispersion of ketoprofen was developed. In order to be able to process the aqueous NanoCrystal colloidal dispersion into a hydrophobic solid dosage form a spray drying procedure was used. The in vitro dissolution profiles of wax based pellets loaded with nanocrystalline ketoprofen are compared with the profiles of wax based pellets loaded with microcrystalline ketoprofen and of a commercial sustained release ketoprofen formulation. Pellets were produced using a melt pelletisation technique. All pellet formulations were composed of a mixture of microcrystalline wax and starch derivatives. The starch derivatives used were waxy maltodextrin and drum dried corn starch. Varying the concentration of drum dried corn starch increased the release rate of ketoprofen but the ketoprofen recovery remained problematic. To increase the dissolution yield surfactants were utilised. The surfactants were either added during the production process of the NanoCrystal colloidal dispersion (sodium laurylsulphate) or during the pellet manufacturing process (Cremophor RH 40). Both methods resulted in a sustained but complete release of nanocrystalline ketoprofen from the matrix pellet formulations.

Self-assembled liposome-loaded microbubbles: the missing link for safe and efficient ultrasound triggered drug-delivery

Liposome-loaded microbubbles have been recently introduced as a promising drug delivery platform for ultrasound guided drug delivery. In this paper we design liposome-loaded (lipid-shelled) microbubbles through the simple self-assembly of the involved compounds in a single step process. We thoroughly characterized the liposome-loading of the microbubbles and evaluated the cell killing efficiency of this material using doxorubicin (DOX) as a model drug. Importantly, we observed that the DOX liposome-loaded microbubbles allowed killing of melanoma cells even at very low doses of DOX. These findings clearly prove the potential of liposome-loaded microbubbles for ultrasound targeted drug delivery to cancer tissues.