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.

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.).

under Prof. S. Claesson. Holding 24 industrial patents and author of more than 120 publications, he has lectured at several European universities. His current interest is in the photochemistry and physical chemistry of polymers. He is a member of the Czech Chemical Society. Stefaan De Smedt was born in Geraardsbergen, Belgium, in 1967. He studied pharmacy at the University of Ghent, Belgium, and received his M.S. degree in pharmaceutical sciences in 1990. As a scholar of the Belgian Institute for the Encouragement of Scientific Research in Industry and Agriculture, he enrolled in a Ph.D. program at the University of Ghent, under the direction of Prof. J. Demeester. He studied rheology at the Catholic University of Leuven. He received the Scott Blair Biorheology Award in 1993−1995 for his work on the structural characterization of hyaluronan solutions. To study diffusion phenomena in polymer solutions, he collaborated with Prof. Y. Engelborghs at the Laboratory of Biomolecular Dynamics of the Catholic University of Leuven. In 1995, he joined the pharmaceutical development group of Janssen Research Foundation. Since 1997 he has been a postdoctoral fellow of F.W.O.-Vlaanderen at the Laboratory of General Biochemistry and Physical Pharmacy of the University of Ghent. He is a member of the Controlled Release Society, the European Federation for Pharmaceutical Sciences, the Belgian Society for Pharmaceutical Sciences, the European Society of Rheology, the Belgian Biophysical Society, and the Polymer Networks Group. He is a consultant to the Journal of Controlled Release and to Pharmaceutical Research. His current research interests include the mobility and interactions of macromolecular drugs in pharmaceutical polymer matrixes and biological polymer systems.

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

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.

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.

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.

The network structure of native and carbodiimide cross-linked gelatin A and B gels was studied based on their rheological behavior. Gelatin A and B contain different numbers of carboxylic acid groups caused by different preparation conditions and had previously shown different characteristics in controlled release applications. It was evaluated to which extent chemical cross-linking densified the network structure of physical gelatin gels. After normalization of the equilibrium shear modulus (G e) with respect to swelling (Q), it was observed that the normalized G e values largely depend on the way gelatin is prepared from collagen. At an equal number of chemical junctions, chemically cross-linked gelatin B gels had a lower elasticity modulus than chemically cross-linked gelatin A gels. This seemed contradictory as gelatin B contains more carboxylic acid groups, available for cross-linking, but is related to a higher probability for intramolecular cross-linking, as was validated quantitatively by chemical and rheological analysis of the number of cross-links. Assuming an ideal network, the average molecular weight of the elastic network chains (M c) was calculated for physical and chemical gelatin A and B networks, and on the basis of M c the mesh sizes of the gels were estimated. The calculated mesh sizes were experimentally confirmed by lysozyme and albumin diffusion. Chemical cross-linking increased the resistance of the gels toward thermal degradation, resulting in a more gradual disintegration of physical cross-links upon heating. Moreover, chemical cross-linking prevented recombination of these cross-links upon cooling.