Macrophage Depletion Attenuates Extracellular Matrix Deposition and Ductular Reaction in a Mouse Model of Chronic Cholangiopathies.

Chronic cholangiopathies, such as primary and secondary sclerosing cholangitis, are progressive disease entities, associated with periportal accumulation of inflammatory cells, encompassing monocytes and macrophages, peribiliary extracellular matrix (ECM) deposition and ductular reaction (DR). This study aimed to elucidate the relevance of macrophages in the progression of chronic cholangiopathies through macrophage depletion in a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) mouse model. One group of mice received a single i.p. injection of Clodronate encapsulated liposomes (CLOLipo) at day 7 of a 14 day DDC treatment, while control animals were co-treated with PBSLipo instead. Mice were sacrificed after 7 or respectively 14 days of treatment for immunohistochemical assessment of macrophage recruitment (F4/80), ECM deposition (Sirius Red, Laminin) and DR (CK19). Macrophage depletion during a 14 day DDC treatment resulted in a significant inhibition of ECM deposition. Porto-lobular migration patterns of laminin-rich ECM and ductular structures were significantly attenuated and a progression of DR was effectively inhibited by macrophage depletion. CLOLipo co-treatment resulted in a confined DR to portal regions without amorphous cell clusters. This study suggests that therapeutic options selectively directed towards macrophages might represent a feasible treatment for chronic cholestatic liver diseases.

Liposome based systems for systemic siRNA delivery: stability in blood sets the requirements for optimal carrier design.

siRNA therapeutics are currently regarded as promising candidates to make a leap forward in the search for treatments of various hard to cure diseases. In order to exploit the full potential of siRNA based therapeutics, development of delivery systems that can efficiently guide the siRNA molecules to their target without major side effects will be the key to success. Lipid based delivery systems, originating from earlier research in the fields of gene delivery, are the most studied candidates for siRNA delivery. Here we discuss the requirements that need to be met by these siRNA delivery systems to ensure adequate stability after systemic application and subsequent deposition in the target tissue. The encountered hurdles in the blood stream and the solutions proposed in literature are discussed.

Silica-based monolithic columns versus conventional particle-packed columns for liquid chromatographic analysis of tetracycline, oxytetracycline and chlortetracycline.

The rise of monolithic stationary phases offers to routine and research laboratories several advantages. In spite of their recent discovery, they have rapidly become highly popular separation media for liquid chromatography. Time reduction and economic reasons like e.g. a diminished use of mobile phase are the most important ones. At the same time, it was reported that these columns offer a faster and better separation. The aim of this article was to investigate the transferability of methods originally developed on conventional particle-packed C(18) columns (XTerra RP18 and Zorbax RX), onto the more recent monolithic columns. Both types, conventional particle-packed and monolithic columns, were able to separate tetracycline, oxytetracycline and chlortetracycline from their respective impurities with sufficient resolution, but showed remarkably shorter analysis times and lower backpressures, improving the lifetime of the column.

Can ultrasound solve the transport barrier of the neural retina?

PURPOSE: Intravitreal injection of nonviral gene complexes may be promising in the treatment of retinal diseases. This study investigates the permeation of lipoplexes and polystyrene nanospheres through the neural retina and their uptake by the retinal pigment epithelium (RPE) either with or without ultrasound application. MATERIALS AND METHODS: Anterior parts and vitreous of bovine eyes were removed. The neural retina was left intact or peeled away from the RPE. (Non)pegylated lipoplexes and pegylated nanospheres were applied. After 2 h incubation, the RPE cells were detached and analyzed for particle uptake by flow cytometry and confocal microscopy. RESULTS: The neural retina is a significant transport barrier for pegylated nanospheres and (non)pegylated lipoplexes. Applying ultrasound improved the permeation of the nanoparticles up to 130 nm. CONCLUSIONS: Delivery of liposomal DNA complexes to the RPE cells is strongly limited by the neural retina. Ultrasound energy may be a useful tool to improve the neural retina permeability, given the nucleic acid carriers are small enough. Our results underline the importance to design and develop very small carriers for the delivery of nucleic acids to the neural retina and the RPE after intravitreal injection.

Wanted and unwanted properties of surface PEGylated nucleic acid nanoparticles in ocular gene transfer.

Ocular gene therapy may offer new hope for severe eye diseases. Many of these ocular diseases are due to a gene defect in the retina, a multi-layered sensory tissue that lines the back of the eye. However, it is well known that the blood-retina barrier and sclera prevent hydrophilic and high molecular weight drugs to reach the retina after systemic or topical application. Therefore, intravitreal injection of non-viral nucleic acid nanoparticles has been considered as a safe and promising approach in ocular gene transfer. However, after intravitreal injection the non-viral nucleic acid nanoparticles should be stable and mobile in the vitreous. In this overview we focus on the behavior of non-viral nucleic acid nanoparticles (lipoplexes) in vitreous and on PEGylation strategies that improve their behavior in vitreous, but that do not affect their transfection capacity.

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.

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.

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.