Stefaan C. De Smedt

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.).

Biodegradable polymers as non-viral carriers for plasmid DNA delivery

Gene therapy holds a great promise for the treatment of acquired and inherited diseases with a genetic origin that are currently incurable. Non-viral gene delivery systems are gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherently associated with the use of viral systems. Many cationic polymers have been studied both in vitro and in vivo for gene delivery purposes. However, in recent years there has been a focus on biodegradable carrier systems. The potential advantage of biodegradable carriers as compared to their non-degradable counterparts is their reduced toxicity and the avoidance of accumulation of the polymer in the cells after repeated administration. Also, the degradation of the polymer can be used as a tool to release the plasmid DNA into the cytosol. In this article the recent results obtained with two classes of degradable gene delivery systems, namely those based on water-soluble cationic polymers and on micro- and nanoparticles will be summarized and discussed.

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.

Advanced nanogel engineering for drug delivery

Nanosized hydrogels (nanogels) have attracted considerable attention as multifunctional polymer-based drug delivery systems. Their versatility is demonstrated both in drug encapsulation and drug release. Nanogels can be designed to facilitate the encapsulation of diverse classes of bioactive compounds. With optimization of their molecular composition, size and morphology, nanogels can be tailor-made to sense and respond to environmental changes in order to ensure spatial and stimuli-controlled drug release in vivo. This manuscript aims to highlight recent advances in the interface between biology and nanomedicine with the emphasis on nanogels as carriers for controlled drug delivery.

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.

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.

Polyelectrolyte microcapsules for biomedical applications

In this paper we review the recent contributions of polyelectrolyte microcapsules in the biomedical field, comprising in vitro and in vivodrug delivery as well as their applications as biosensors.

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.

mRNA as gene therapeutic: how to control protein expression.

For many years, it was generally accepted that mRNA is too unstable to be efficiently used for gene therapy purposes. In the last decade, however, several research groups faced this challenge and not only proved the feasibility of mRNA-mediated transfection with surprising results regarding transfection efficiency and duration of protein expression, but also were able to demonstrate major advantages over the use of pDNA. These advantages will be the first issue discussed in this review, which first of all addresses the notions that mRNA does not need to cross the nuclear barrier to exert its biological activity and in addition lacks CpG motifs, which reduces its immunogenicity. Secondly, it provides insight in the (in)stability of the mRNA molecule, in how mRNA can be modified to increase its half-life and in the necessities of exogenously produced mRNA to be successfully used in transfection protocols. Furthermore, this review gives an in-depth overview of the different techniques and vehicles for intracellular mRNA delivery exploited by us and other groups, comprising electroporation, gene gun injection, lipo- and polyplexes. Finally, it covers recent literature describing specific applications for mRNA based gene delivery, showing that until now most attention has been paid to vaccination strategies. This review offers a comprehensive overview of current knowledge of the major theoretical as well as practical aspects of mRNA-mediated transfection, showing both its possibilities and its pitfalls and should therefore be useful for a diverse scientific audience.