Capsule permeability via polymer and protein ingress/egress.

Static incubation tests, where microcapsules and beads are contacted with polymer and protein solutions, have been developed for the characterization of permselective materials applied for bioartificial organs and drug delivery. A combination of polymer ingress, detected by size-exclusion chromatography, and protein ingress/ egress, assessed by gel electrophoresis, provides information regarding the diffusion kinetics, molar mass cutoff(MMCO) and permeability. This represents an improvement over existing permeability measurements that are based on the diffusion of a single type of solute. Specifically, the permeability of capsules based on alginate, cellulose sulfate, polymethylene-co-guanidine were characterized as a function of membrane thickness. Solid alginate beads were also evaluated. The MMCO of these capsules was estimated to be between 80 and 90 kDa using polymers, and between 116-150 kDa with proteins. Apparently, the globular shape of the proteins (radius of gyration (Rg) of 4.2-4.6 nm) facilitates their passage through the membrane, comparatively to the polysaccharide coil conformation (Rg of 6.5-8.3 nm). An increase of the capsule membrane thickness reduced these values. The MMCO of the beads, which do not have a membrane limiting their permselective properties, was higher, between 110 and 200 kDa with dextrans, and between 150 and 220 kDa with proteins. Therefore, although the permeability estimated with biologically relevant molecules is generally higher due to their lower radius of gyration, both the MMCO of synthetic and natural watersoluble polymers correlate well, and can be used as in vitro metrics for the immune protection ability of microcapsules and microbeads. This article shows, to the authors' knowledge, the first reported concordance between permeability measures based on model natural and biological macromolecules.

Neurolysis using a carbohydrate polymer gel for the treatment of postoperative neuropathic pain.

Perineural and intraneural fibrosis is thought to be the main cause of failure of the many surgical treatments of neuropathic pain. We have used Adcon-T/N carbohydrate polymer gel for prevention of perineural fibrosis in several parts of the body. In this retrospective study, 54 patients who presented with postoperative neuropathic pain had microsurgical epineural neurolysis and relocation of a terminal neuroma. In 19 of them, the carbohydrate gel was applied at the same time. The mean follow-up was four years and the nerve distribution varied. Postoperative improvement in pain scores (visual analogue scale (VAS) and neuropathic pain scale inventory (NPSI)), sensitivity, overall improvement and satisfaction were equivalent in the two groups, with pain relief in about 80% of the patients. There was no significant beneficial effect in the carbohydrate gel group. Patients treated with this device had a higher infection rate (21 compared with 0, p = 0.01) and delayed wound healing (31.6 compared with 11.8, p = 0.2). We conclude that good long-term pain relief is obtained postoperatively independently of the addition of carbohydrate gel. There was a slight but not significant trend towards profound pain relief with the gel.

Development of a coculture model of encapsulated cells.

In the whole animal, metabolic regulations are set by reciprocal interactions between various organs, via the blood circulation. At present, analyses of such interactions require numerous and uneasily controlled in vivo experiments. In a search for an alternative to in vivo experiments, our work aims at developing a coculture system in which different cell types are isolated in polymer capsules and grown in a common environment. The signals exchanged between cells from various origins are, thus, reproducing the in vivo intertissular communications. With this perspective, we evaluated a new encapsulation system as an artificial housing for liver cells on the one hand and adipocytes on the other hand. Murine hepatocytes were encapsulated with specially designed multicomponent capsules formed by polyelectrolyte complexation between sodium alginate, cellulose sulphate and poly(methylene-coguanidine) hydrochloride, of which the permeability has been characterized. We demonstrated the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Encapsulated hepatocytes retain their specific functions--transaminase activity, urea synthesis, and protein secretion--during the first four days of culture in minimum medium. Mature adipocytes, isolated from mouse epidydimal fat, were embedded in alginate beads. Measurement of protein secretion shows an identical profile between free and embedded adipocytes. We finally assessed the properties of encapsulated hepatocytes, cryopreserved over a periods of up to four months. The perspective of using encapsulated cells in coculture are discussed, since this system may represent a promising tool for fundamental research, such as analyses of drug metabolism, intercellular regulations, and metabolic pathways, as well as for the establishment of a tissue bank for storage and supply of murine hepatocytes.

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity.

Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

Coating carbon nanotubes with a polystyrene-based polymer protects against pulmonary toxicity.

BACKGROUND: carbon nanotubes (CNT) can have adverse effects on health. Therefore, minimizing the risk associated with CNT exposure is of crucial importance. The aim of this work was to evaluate if coating multi-walled CNT (MWCNT) with polymers could modify their toxicity, thus representing a useful strategy to decrease adverse health effects of CNT. We used industrially-produced MWCNT uncoated (NT1) or coated (50/50 wt%) with acid-based (NT2) or polystyrene-based (NT3) polymer, and exposed murine macrophages (RAW 264.7 cell line) or Balb/c mice by intratracheal administration. Biological experiments were performed both in vitro and in vivo, examining time- and dose-dependent effects of CNT, in terms of cytotoxicity, expression of genes and proteins related to oxidative stress, inflammation and tissue remodeling, cell and lung tissue morphology (optical and transmission electron microscopy), and bronchoalveolar lavage fluid content analysis.RESULTS: extensive physico-chemical characterization of MWCNT was performed, and showed, although similar dimensions for the 3 MWCNT, a much smaller specific surface area for NT2 and NT3 as compared to NT1 (54.1, 34 and 227.54 m(2)/g respectively), along with different surface characteristics. MWCNT-induced cytotoxicity, oxidative stress, and inflammation were increased by acid-based and decreased by polystyrene-based polymer coating both in vitro in murine macrophages and in vivo in lung of mice monitored for 6 months.CONCLUSIONS: these results demonstrate that coating CNT with polymers, without affecting their intrinsic structure, may constitute a useful strategy for decreasing CNT toxicity, and may hold promise for improving occupational safety and that of general the user.

Symmetry-breaking in cumulative measures of shapes of polymer models.

Using numerical simulations we investigate shapes of random equilateral open and closed chains, one of the simplest models of freely fluctuating polymers in a solution. We are interested in the 3D density distribution of the modeled polymers where the polymers have been aligned with respect to their three principal axes of inertia. This type of approach was pioneered by Theodorou and Suter in 1985. While individual configurations of the modeled polymers are almost always nonsymmetric, the approach of Theodorou and Suter results in cumulative shapes that are highly symmetric. By taking advantage of asymmetries within the individual configurations, we modify the procedure of aligning independent configurations in a way that shows their asymmetry. This approach reveals, for example, that the 3D density distribution for linear polymers has a bean shape predicted theoretically by Kuhn. The symmetry-breaking approach reveals complementary information to the traditional, symmetrical, 3D density distributions originally introduced by Theodorou and Suter.

Oxygen uptake of isolated toad skin epithelium: micromeasurement and effect of ionic acclimation.

Oxidative metabolism of isolated toad skin epithelium (Bufo viridis) was investigated in vitro under open-circuit conditions using the spectrophotometric oxyhemoglobin micromethod. This highly sensitive technique has been adapted for studying several epithelia in parallel and for detecting possible regional variations of oxygen uptake in individual epithelium. Changes in the proportion of mitochondria-rich cells (MRC) by ionic acclimation affected oxidative metabolism under nontransporting condition. After acclimation of animals to either NaNO3 or NaCl solutions (100 mmol/l, for greater than 2 wk), the number of MRC per square millimeter in epithelia from nonacclimated and NaNO3- and NaCl-acclimated animals was 350 +/- 113, 460 +/- 196, and 107 +/- 52, respectively. O2 uptake of nonacclimated and NaNO3-acclimated epithelia was significantly higher than that of NaCl-acclimated epithelia (i.e., 0.89 and 0.90 vs. 0.57 nmol O2.h-1.mm-2, respectively). The correlation established between O2 uptake and number of MRC allowed evaluation of the respiration rate of one single MRC, i.e., approximately 1 pmol O2/h. The lowest mitochondrial oxidative activity was found in the epithelia from NaCl-acclimated toads where the uncoupler 2,4-dinitrophenol (50 mumols/l) had the highest relative stimulatory effect (+114%). Acetazolamide (50 mumols/l), a potent inhibitor of carbonic anhydrase mainly present in the MRC, reduced selectively by 31% O2 uptake of the MRC-rich epithelia (NaNO3 acclimated). O2 uptake increased significantly by approximately 80% when basolateral pH increased from 5.8 to 7.8, but did not depend on apical pH. These findings indicate that under nontransporting (open-circuit) conditions, aerobic metabolism of the isolated toad skin epithelium is related to the density and/or characteristics of the MRC.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopolymers

Plants naturally synthesize a variety of polymers that have been used by mankind as a source of useful biomaterials. For example, cellulose, the main constituent of plant cell wall and the most abundant polymer on earth, has been used for several thousand years as a source of fibers for various fabrics. Similarly, rubber extracted from the bark of the tree Hevea brasiliensis, has been a major source of elastomers until the development of similar synthetic polymers. In the last century, the usefulness of plant polymers as biomaterials has been expanded through the chemical modification of the natural polymers. For example, a number of plastics have been made by substituting the hydroxyl groups present on the glucose moiety of cellulose with larger groups, such as nitrate or acetate, giving rise to materials such as cellulose acetate, a clear plastic used in consumer products such as toothbrush handles and combs. Similarly, starch has been used in the manufacture of plastics by either using it in blends with synthetic polymers or as the main constituent in biodegradable plastics. The advent of transformation and expres- sion of foreign genes in plants has created the possibility of expanding the usefulness of plants to include the synthesis of a range of biomolecules. In view of the capacity of certain crops to produce a large quantity of organic raw material at low cost, such as oils and starch, it is of interest to explore the possibility of using transgenic plants as efficient vectors for the synthesis of biopolymers. Such plant based biopolymers could replace, in part, the synthetic plastics and elastomers produced from petroleum, offering the advantage of renewability and sustainability. Furthermore, being natural pro- ducts, biopolymers are usually biodegradable and can thus contribute to alleviate problems associated with the management of plastic waste. In this article, the emphasis will be on the use of transgenic plants for the synthesis of two novel classes of industrially useful polymers, namely protein based polymers made from natural or artificial genes, and polyhydroxyalkanoates, a family of bacterial poly- esters having the properties of biodegradable plastics and elastomers.

Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study.

Huntington's disease (HD) is a monogenic neurodegenerative disease that affects the efferent neurons of the striatum. The protracted evolution of the pathology over 15 to 20 years, after clinical onset in adulthood, underscores the potential of therapeutic tools that would aim at protecting striatal neurons. Proteins with neuroprotective effects in the adult brain have been identified, among them ciliary neurotrophic factor (CNTF), which protected striatal neurons in animal models of HD. Accordingly, we have carried out a phase I study evaluating the safety of intracerebral administration of this protein in subjects with HD, using a device formed by a semipermeable membrane encapsulating a BHK cell line engineered to synthesize CNTF. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle; the capsule was retrieved and exchanged for a new one every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed; however, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous cell survival, however, stresses the need for improving the technique.

Monoclonal antibodies to carcinoembryonic antigen: ionic strength as a factor in the selection of antibodies for immunoscintigraphy.

As part of an ongoing effort to improve the technique of immunoscintigraphy for the detection of human carcinomas with radiolabeled monoclonal antibodies (MABs) to carcinoembryonic antigen (CEA), we have developed a series of MABs to CEA and have studied the effects of low- and physiological molarity buffers on their CEA binding and affinity, as well as their cross-reactivity with granulocyte glycoprotein(s). These in vitro results in different buffer systems were then correlated with the use of these MABs to CEA in the detection of human colon carcinoma grafts in nude mice. Our results show that the binding of CEA by some MABs is influenced by ionic strength and that this may be an important factor in their successful use for the immunolocalization of carcinomas in vivo.

Micro-patterning of biomolecules on polymer substrates.

UV−excimer laser photoablation was used, in combination with surface blocking techniques, to pattern proteins on the surfaces of polyimide and poly(ethylene terephthalate). This technique involves physical adsorption of avidin through laser-defined openings in low-temperature laminates or adsorbed protein blocking layers. Visualization of biomolecular patterns were monitored using avidin and fluorescein-labeled biotin as a model receptor−ligand couple. Adsorbed proteins could be shown to bind to UV-laser-treated polymer surfaces up to three times higher than on commercially available polymers. UV-laser photoablation was also used for the generation of three-dimensional structure, which leads to the possibility of biomolecule patterning within polymer-based microanalytical systems. The simplicity and easy handling of the described technique facilitate its application in microdiagnostic devices.

Combined Electrostatic and Covalent Polymer Networks for Cell Microencapsulation

Two types of hydrogel microspheres have been developed. Fast ionotropic gelation of sodium alginate (Na-alg) in the presence of calcium ions was combined with slow covalent cross-linking of poly(ethylene glycol) (PEG) derivatives. For the first type, the fast obtainable Ca-alg hydrogel served as spherical matrix for the simultaneously occurring covalent cross-linking of multi-arm PEG derivative. A two-component interpenetrating network was formed in one step upon extruding the mixture of the two polymers into the gelation bath. For the second type, heterobifunctional PEG was grafted onto Na-alg prior to gelation. Upon extrusion of the polymer solution into the gelation bath, fast Ca-alg formation ensured the spherical shape and was accompanied by cross-linker-free covalent cross-linking of the PEG side chains. Thus, one-component hydrogel microspheres resulted. We present the physical properties of the hydrogel microspheres and demonstrate the feasibility of cell microencapsulation for both types of polymer networks.

Effects of polymer-coated multi-wall carbon nanotubes on mouse RAW 264.7 macrophages

Surface characteristics (area, chemical reactivity) play an important role in cell response to nanomaterials. The aim of this study was to evaluate the oxidative and inflammatory effects of multi−wall carbon nanotubes (MWCNT) uncoated (P0) or coated with carboxylic polyacid or polystyrene polybutadiene polymetacrylate of methyl polymers (P1 and P2 respectively) on murine macrophages (RAW 264.7 cell line). Carbon black nanoparticles (CB, diameter 95 nm) and crocidolite fibers (diameter: 80 nm, length: < 10 μm) were used as controls. Surface functional groups present on MWCNTs were analyzed by Knudsen flow reactor. The amount of acidic sites was P1> P0> P2, for basic sites was P0> P1>> P2 and for oxidizable sites was P0> P2> P1. In contact with cells, P2 formed smaller aggregates than P0 and P1, which were of similar size. Optical microscopy showed the formation of vacuoles after exposure only to P0, P1 and crocidolite. Incubation of cells with P0, P1 and crocidolite fibers induced a significant and similar decrease in metabolic activity, whereas P2 and CB had no effect. Cell number and membrane permeability were unmodified by incubation with the different particles. Incubation of macrophages with P0, P1 and crocidolite induced a dose− and time−dependent increase in mRNA expression of oxidative stress marker (HO−1, GPX1) and inflammatory mediators (TNF−a, MIP−2). No such responses were observed with P2 and CB. In conclusion, MWCNT coated with a carboxylic polyacid polymer exerted similar oxidative and inflammatory effects to uncoated MWCNT. By contrast, no such effects were observed with MWCNT coated with a polystyrene−based polymer. This kind of coating could be useful to decrease MWCNT toxicity.