Connexin Hemichannels: Methods for Dye Uptake and Leakage.

It is now clear that connexin-based, gap junction "hemichannels" in an undocked state are capable of opening and connecting cytoplasm to the extracellular milieu. Varied studies also suggest that such channel activity plays a vital role in diverse cell processes and abnormal hemichannel activity contributes to pathogenesis. To pursue fundamental questions in this area, investigators require methods for studying hemichannel permeability and dynamics that are quantitative, sensitive, versatile, and available to most cellular and molecular laboratories. Here we first provide a theoretical background for this work, including the role of cellular membrane potentials. We then describe in detail our computer-assisted methods for both dye uptake and leakage along with illustrative results from different cell systems. A key feature of our protocol is the inclusion of a mechanical stimulation step. We describe dye uptake, interpreted as connexin dependent, that is shown to be enhanced with reduced extracellular Ca2+, mechanically responsive, inhibited by TPA, inhibited by EL186 antibodies for Cx43 and sustained for more than 15 min following mechanical stimulation. We describe dye leakage that displays these same properties, with estimates of hemichannel numbers per cell being derived from leakage rates. We also describe dye uptake that is shown to be unaffected by a reduction in external Ca2+, insensitive to EL186 antibodies and relatively short-lived following mechanical stimulation; this uptake may occur via pannexin 1 channels expressed in the cells studied here. It is unlikely that cell damage plays a significant role in dye uptake following mechanical stimulation, given compelling results from various control experiments.

In vitro and in vivo evaluation of biodegradable embolic microspheres with tunable anticancer drug release.

Natural polymer-derived materials have attracted increasing interest in the biomedical field. Polysaccharides have obvious advantages over other polymers employed for biomedical applications due to their exceptional biocompatibility and biodegradability. None of the spherical embolic agents used clinically is biodegradable. In the current study, microspheres prepared from chitosan and carboxymethyl cellulose (CMC) were investigated as a biodegradable embolic agent for arterial embolization applications. Aside from the enzymatic degradability of chitosan units, the cross-linking bonds in the matrix, Schiff bases, are susceptible to hydrolytic cleavage in aqueous conditions, which would overcome the possible shortage of enzymes inside the arteries. The size distribution, morphology, water retention capacity and degradability of the microspheres were found to be affected by the modification degree of CMC. An anticancer drug, doxorubicin, was successfully incorporated into these microspheres for local release and thus for killing cancerous cells. These microspheres demonstrated controllable degradation time, variable swelling and tunable drug release profiles. Co-culture with human umbilical vein endothelial cells revealed non-cytotoxic nature of these microspheres compared to monolayer control (P>0.95). In addition, a preliminary study on the in vivo degradation of the microspheres (100-300µm) was performed in a rabbit renal embolization model, which demonstrated that the microspheres were compatible with microcatheters for delivery, capable of occluding the arteries, and biodegradable inside arteries. These microspheres with biodegradability would be promising for embolization therapies.

Bioresorbable hydrogel microspheres for transcatheter embolization: preparation and in vitro evaluation.

PURPOSE: To develop and evaluate a bioresorbable spherical material for embolization. MATERIALS AND METHODS: New bioresorbable hydrogel microspheres were prepared from carboxymethyl cellulose and chitosan by using an inverse emulsion method. Size distribution of the microspheres was determined with a microscope, and the colorability was tested with Evans blue dye. The compressibility was examined with a texture analyzer. After sieving, the suspendability of the microspheres was tested in saline solution/contrast agent mixture in different ratios, and then the injectability was tested with microcatheters (lumen sizes of 0.0165, 0.019, and 0.027 inches). The in vitro degradability tests were performed in a lysozyme solution. Cell culture study of the microspheres was performed with human fibroblasts. RESULTS: The microspheres exhibit diameters of 100-1,550 µm with a transparent appearance. Their fracture force can reach 0.58-0.88 N, and fracture deformation varies from approximately 70% to 95% of their original size. These microspheres can be colored by Evans blue dye, and uniform subgroups of microspheres can be readily obtained by sieving. Within 3 minutes, the microspheres form a stable suspension in a 4:6 contrast agent/saline solution mixture, which can be easily injected through microcatheters without aggregating or clogging. These microspheres are biodegradable, with degradation times varying from 2 weeks to 1 month, depending on their composition. Cell culture studies reveal no adverse effect on the growth of human fibroblasts in the presence of the microspheres. CONCLUSIONS: A biodegradable and noncytotoxic microsphere was successfully prepared. It can be well suspended in the contrast solution and easily injected through a microcatheter.

Doxorubicin loading and eluting characteristics of bioresorbable hydrogel microspheres: in vitro study.

Non-bioresorbable drug eluting microspheres are being increasingly used for the treatment of unresectable liver tumors, whereas bioresorbable microspheres have not received much attention. In this study, bioresorbable microspheres prepared from chitosan and carboxymethyl cellulose were loaded with doxorubicin (Doxo) via ion-exchange interactions with carboxylic groups in the microspheres. With a 25-40% decrease in the microsphere size depending on their size ranges, the microspheres could load a maximum of 0.3-0.7 mg Doxo/mg dry spheres. As confirmed by confocal microscopy, Doxo was mainly concentrated in the outer 20±5 µm surface layer of the microspheres. The loaded microspheres were stable in aqueous dispersions without aggregation for a prolonged period of time but degradable in a lysozyme solution. Furthermore, the loaded microspheres exhibited a noticeable pH-sensitive behavior with accelerated release of Doxo in acidic environment due to the protonation of carboxylic groups in the microspheres. Compared to commercial non-resorbable drug eluting beads, the loaded bioresorbable microspheres showed a sustained release manner in phosphate buffered saline (PBS). The release data were fitted to an empirical relationship, which reveals a non-Fickian transport mechanism (n=0.55-0.59). These results demonstrate that the bioresorbable microspheres are promising as attractive carriers for Doxo.