Determination of pore diameter and molecular weight cut-off of hydrogel-membrane liquid-core capsules for immunoisolation

The exclusion limit expressed as the largest pore size of capsules composed of hydrogel Ca-alginate membrane and hydroxy-propyl-ammonium starch liquid core considered as the immunoprotective system has been determined by means of inverse size exclusion chromatography with dextran molecular weight standards. The exclusion limits of the capsules were not influenced by the change of starch concentration in the core solution from 4 to 6% but were influenced by the change in alginate concentration in the membrane from 0.5 to 1.0%, causing the membranes to be less permeable. It was found that the diameter of the largest pores in hydrogel membranes was in the range 7.2-8.0 nm. Based on the relationship between solute size and its molecular weight, the capsules had an approximate exclusion limit of 21-25 kD for dextran and 78-103 kD for protein, which is sufficient to block the antibodies penetrating through the membrane.     

Effect of preparation conditions on properties and permeability of chitosan-sodium hexametaphosphate capsules

Capsules were obtained by interpolymer complexation between chitosan (polycation) and sodium hexametaphosphate (SMP, oligoanion). The effect of the preparation conditions on the capsule characteristics was evaluated. Specifically, the influence of variables such as pH, ionic strength, reagent concentration, and additives on the capsule permeability properties was investigated using dextran as a model permeant. The capsule membrane permeability was found to increase by decreasing the chitosan/SMP ratio as well as adding mannitol to the oligoanion recipient bath. Increasing the ionic strength or the pH of the initial chitosan solution was also found to enhance the membrane permeability, moving the membrane exclusion limit to higher values. Generally, the capsules prepared tinder all tested conditions had a relatively low permeability which rarely exceeded a molecular cut-off of 40 kD based on dextran standards. Furthermore, the diffusion rate showed a strong temporal dependence, indicating that the capsules prepared under various conditions exhibit different apparent pore size densities on the surface. The results indicated that, in order to obtain the desired capsule mass-transfer properties, the preparation conditions should be carefully considered and adjusted. Adding a polyol as well as low salt amount (less than 0.15%) is preferable as a means of modulating the diffusion characteristics, without disturbing the capsule mechanical stability.     

Effect of preparation conditions on properties and permeability of chitosan–sodium hexametaphosphate capsules

Capsules were obtained by interpolymer complexation between chitosan (polycation) and sodium hexametaphosphate (SMP, oligoanion). The effect of the preparation conditions on the capsule characteristics was evaluated. Specifically, the influence of variables such as pH, ionic strength, reagent concentration, and additives on the capsule permeability properties was investigated using dextran as a model permeant. The capsule membrane permeability was found to increase by decreasing the chitosan/SMP ratio as well as adding mannitol to the oligoanion recipient bath. Increasing the ionic strength or the pH of the initial chitosan solution was also found to enhance the membrane permeability, moving the membrane exclusion limit to higher values. Generally, the capsules prepared under all tested conditions had a relatively low permeability which rarely exceeded a molecular cut-off of 40 kD based on dextran standards. Furthermore, the diffusion rate showed a strong temporal dependence, indicating that the capsules prepared under various conditions exhibit different apparent pore size densities on the surface. The results indicated that, in order to obtain the desired capsule mass-transfer properties, the preparation conditions should be carefully considered and adjusted. Adding a polyol as well as low salt amount (less than 0.15%) is preferable as a means of modulating the diffusion characteristics, without disturbing the capsule mechanical stability.     

Controlled Molecular Release Using Nanoporous Alumina Capsules

The use of mechanically robust nanoporous alumina capsules, with highly uniform pores of 25 nm to 55 nm, for controled drug delivery is demonstrated. The nanoporous alumina capsules were fabricated by anodization of an aluminum tube, resulting in a highly uniform, large surface area, relatively inexpensive device suitable for biofiltration applications. Characterization of diffusion from the nanoporous capsules using fluorescein isothiocyanate and dextran conjugates of varying molecular weight, showed that molecular transport could be readily controlled by selection of capsule pore size. A branched membrane structure, with a stepwise change in pore size from large to small, is used to provide small pore-sized membranes with sufficient mechanical strength for handling.     

Characterization of neutrophil-mediated degradation of human C-reactive protein and identification of the protease.

Human C-reactive protein (CRP) is shown to mediate a release of enzymatic activity from neutrophils which promotes its own degradation in the extracellular medium. This egress of proteolytic activity, which was upregulated by phorbol 12-myristate 13-acetate (PMA), was found to occur from both the cytoskeleton and membrane fractions of neutrophils and was dependent on the time of incubation of CRP with the cells and the concentration of CRP. Neutrophil kinases activated by PMA are found to be involved in upregulating the activity of the CRP-degrading protease. The apparent molecular weight of the CRP-degrading protease associated with the conditioned medium from PMA-stimulated neutrophils and neutrophil membrane and cytoskeleton preparations, was found by size exclusion chromatography to be 600 kD and migrated on 3-13% SDS-PAGE as four discrete bands to positions corresponding to apparent molecular weights of 209 kD, 316 kD, 398 kD and 501 kD.     

Characteristics of Poly-L-Ornithine-coated alginate microcapsules

Poly-L-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-L-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean +/- SEM diameter of PLL-coated microcapsules increased from 718+/-17 to 821 +/- 17 microm (p < 0.05) during 14 days incubation at 37 degrees C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.7 +/- 4.9%) compared to 3.1 +/- 2.05% for PLO capsules (p < 0.0001,n = 6). We conclude that PLO-coated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules.     

Determination of human lens capsule permeability and its feasibility as a replacement for Bruch's membrane

We have investigated human anterior lens capsule as a potential replacement for Bruch's membrane as a treatment for age-related macular degeneration. Any substrate to replace Bruch's membrane should possess certain characteristics to maintain proper function of the overlying retina. One of the important properties of Bruch's membrane is allowing the flow of nutrients and waste between the retinal pigment epithelium and the choriocapillaris. Here, we measured the permeability of the lens capsule by studying the diffusion of various molecular weight FITC-dextran molecules. Expressions for extraction of diffusion coefficients from concentration vs. time data from a blind-well chamber apparatus were derived for both a single and double membrane experiments. The diffusion coefficients in the lens capsule were found to be in the range of 10(-6) to 10(-10)cm2/s. We demonstrated a power law relationship, with the diffusion coefficient possessing a -0.6 order dependence on molecular weight. The molecular weight exclusion limit was determined to be 150+/-40 kDa. We have compared this value with reported values of Bruch's membrane molecular weight exclusion limit and find that the lens capsule has the potential to act as a substitute Bruch's membrane.     

Biocompatibility of alginates for grafting: impact of alginate molecular weight

Optimising microencapsulation technology towards the effective clinical transplantation has created the need for highly biocompatible alginates. Therefore, in this study the biocompatibility of different beads prepared from alginates with varying average molecular weight was examined. In some experiments the beads were covered with a multilayer membrane surrounded by an alginate layer. First of all, we found that beads made of a lower weight average alginate elicted a much stronger fibrotic response compared to beads made of a higher weight average alginate (LV-alginate > MV-alginate). The results were confirmed by the observation that the extent of tissue fibrosis was significantly increased in multilayer capsules made of an alginate with a lower weight average (core and surface LV-alginate, Mw 0.7-1 * 10(6) g/mol, viscosity of a 0.1% solution 1-2.5 mPa s(-1)) compared to multilayer capsules made of an alginate with a higher weight average (core and surface MV-alginate; Mw 1.2-1.3 * 10(6) g/mol, viscosity of a 0.1% solution 5-7 mPa s(-1)). It should be stressed, that the pro-fibrotic effect of the LV-alginate alginate in the core was only partially reversed by a MV-alginate on the surface of the multilayer capsules. On the basis of the raised data, it can be assumed that the molecular weight average of the alginates have an decisive effect on the biocompatibility. Therefore, it seems to be recommendable to reduce the low molecular weight fractions of the alginate during the purification process to improve the biocompatibility.     

Statistical approach in alginate membrane formulation for cell encapsulation in a GMP-based cell factory

A cell encapsulation technology in alginate has been developed with the aim of obtaining cell controlled release or three-dimensional cultures. The aim of this work is to verify the predictability of alginate capsules for large-scale production by Good Manufacturing Practice (GMP) standardized procedures in a cell factory. A cell-free capsule model was performed following the GMP guidelines: an opaque agent suspension in a bivalent cation solution (Ca(2+), Ba(2+), Sr(2+)) was dropped in a sodium alginate solution, obtaining capsules presenting a liquid core surrounded by a gel alginate membrane. The concentration of the ion, and the treatment with protamine, can considerably vary the characteristics of the capsules (weight, whole diameter, core diameter, gel capsule thickness, capsule strength). It is therefore possible to optimize the performance of the capsules, relating the molecular structure and size of the polymeric membrane to the desired functional properties. Technological resources are available for large-scale cell encapsulation intended for advanced therapies (gene therapy, somatic cell therapy and tissue engineering) in a cell factory, following GMP guidelines.     

Preparation and characterization of alpha-amylase-immobilized thermal-responsive composite hydrogel membranes.

Composite hydrogel membranes of crosslinked poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyet hyl methacrylate) [P(NIPAAm-NAS-HEMA)] with starch, as a macropore forming agent, on nonwoven polyester was prepared. The membranes could swell and de-swell around the characteristic lower critical solution temperature (LCST) of poly(N-isopropylacrylamide) (PNIPAAm). It was demonstrated that the presence of macropores in the membranes could improve the immobilization efficiency as well as lead to a short responding time upon temperature change across the LCST. Immobilized alpha-amylase could retain as high as 33% of the activity of the free enzyme with a loading level of 0.60-0.65 mg/cm2 when the membrane preparation and enzyme immobilization conditions were optimized. The half time (T0.5) for the swelling or de-swelling response of the gel phase within the membranes was less than 2 min, and the 90% time (T0.9) was less than 6 min. The permeability for maltose through the membranes could change as much as 4.9-fold when the temperature was raised above or reduced below the LCST.     

Stability assessment of chitosan–sodium hexametaphosphate capsules

The assessment of the stability of capsules based on chitosan-sodium hexametaphosphate complex formation has been carried out using two independent methods - compression and osmotic swelling, and the influence of the preparation variables was evaluated. The formulation containing 1.5% core polymer (chitosan) and 1.5% oligophosphate, in the absence of salt or at low ionic strength (0.15% NaCl) was found to provide the best membrane resistance. A higher concentration of crosslinker (2.25%) produced stable capsules only in absence of electrolyte. Mannitol, a porogen added to the preparation solutions, did not affect the stability of the obtained membranes. At elevated polyol (1%) and crosslinker levels (2.25%), and at 0% salt, membranes with decreased elasticity were obtained, having lower compression and osmotic bursting values and lower deformation at the breaking points. A significant influence of salt amount on the capsule stability was also found. This was attributed to changes in the membrane formation process resulting in membranes with different thickness and structure. Membrane compression stability was found to be dependent on the pH of both oligophosphate and chitosan solutions, as well as on the reaction time. The bursting force values decreased for capsule diameters below 1.6 mm. The increased membrane/capsule volume ratio for the small capsules decreased the capsule deformation freedom and caused capsule rupture at low force values. The capsules made at low salt amounts showed very good storage stability over time and at elevated temperatures. The results demonstrated that the capsules could be formulated with controlled properties for various biomedical applications.     

Steady-state diffusion of water through soft-contact-lens materials

Water transport through soft contact lenses (SCL) is important for acceptable performance on the human eye. Chemical-potential gradient-driven diffusion rates of water through SCL materials are measured with an evaporation-cell technique. Water is evaporated from the bottom surface of a lens membrane by impinging air at controlled flow rate and humidity. The resulting weight loss of a water reservoir covering the top surface of the contact-lens material is recorded as a function of time. New results are reported for a conventional hydrogel material (SofLens One Day, hilafilcon A, water content at saturation w10 = 70 weight %) and a silicone hydrogel material (PureVision, balafilcon A, w10 = 36%), with and without surface oxygen plasma treatment. Also, previously reported data for a conventional 2-hydroxyethyl methacrylate (HEMA)-SCL (w10 = 38%) hydrogel are reexamined and compared with those for SofLens One Day and PureVision hydrogels. Measured steady-state water fluxes are largest for SofLens One Day, followed by PureVision and HEMA. In some cases, the measured steady-state water fluxes increase with rising relative air humidity. This increase, due to an apparent mass-transfer resistance at the surface (trapping skinning), is associated with formation of a glassy skin at the air/membrane interface when the relative humidity is below 55-75%. Steady-state water fluxes are interpreted through an extended Maxwell-Stefan diffusion model for a mixture of species starkly different in size. Thermodynamic nonideality is considered through Flory-Rehner polymer-solution theory. Shrinking/swelling is self-consistently modeled by conservation of the total polymer mass. Fitted Maxwell-Stefan diffusivities increase significantly with water concentration in the contact lens.     

Surface modification of chitosan membranes by complexation-interpenetration of anionic polysaccharides for improved blood compatibility in hemodialysis

Chitosan membrane surface was modified by cornplexation and interpenetration of anionic polysaccharides - heparin and dextran sulfate - for improved blood compatibility in hemodialysis. Electron spectroscopy for chemical analysis results showed a characteristic sulfur (S) and sodium (Na) peaks after modification with dextran sulfate. The sulfur/carbon (S/C) atomic composition ratio increased from 0.03 to 0.08 when the bulk dextran sulfate concentration used for modification was increased from 2.5 to 10 mg ml^-1 . The permeability of urea and creatinine did not change significantly upon modification with heparin or dextran sulfate. Surface modification, however, did decrease the permeability coefficients of glucose, vitamin B-2, and vitamin B-12. Unlike Cuprophan, chitosan and surface-modified chitosan membranes did not significantly activate the complement system as measured by the serum iC3b concentration. Compared to forty and sixty fully-activated platelets present on control surfaces, surface modification with heparin and dextran sulfate significantly reduced the number of adherent platelets per 25 000 μm² area and the extent of platelet activation. Surface modification with anionic polysaccharides, however, did significantly shorten the plasma recalcification time leading to fibrin clot formation. The results of this study show that chitosan membrane surface can be modified by complexation-interpenetration of anionic modifying agents. The modified membranes do resist complement activation and platelet adhesion and activation.     

Some structural properties of thymus leukemia antigen (TL) solubilized with detergent.

Plasma membranes were isolated from the leukemia cell line ASL1w and extracted with detergent (DOC). DOC solubilized more TL activity than could be detected on isolated membranes. However, extraction of membranes with LDS or EDTA solubilized only 17% and 4%, respectively, of the activity. This indicated that TL was not loosely associated with the membrane but rather was integrated into the lipid bilayer. At low concentrations of DOC (0.05%), TL was found to be largely aggregated and was also prone to autolysis. Neither aggregation nor autolysis was observed at a higher DOC concentration (0.5%). The apparent molecular weight of TL in 0.5% DOC was determined by Sephadex G-200 chromatography to be about 65,000-70,000. Digestion of a 0.5% DOC extract of TL with either papain or trypsin produced a fragment of TL of about 35,000 molecular weight. These fragments were similar in size to a fragment produced by autolysis. These data suggested that a region of the TL molecule was very prone to proteolytic attack. The 35,000 molecular weight proteolytic fragments bound specifically to lentil lectin affinity columns, which indicated that they retained at least part of the carbohydrate present on the native molecule.     

聚己内酯埋植剂的制备及药物通透性研究

目的制备聚己内酯（PCL）长效药物缓释制剂,优化药物缓释效果,并研究PCL埋植剂的药物通透性.方法 合成分子质量为（8～16）×104 u含有普朗尼克F68的PCL,用挤出法制备不同壁厚的PCL长效缓释埋植剂.将左炔诺孕酮（LNG）装入PCL管封装,制成LNG药囊.通过高效液相色谱法测定LNG的释放效果,考察PCL的分子质量及PCL管壁厚度对LNG通透性的影响.结果 当PCL分子质量为（8～16）×104u时,对LNG通透性无显著影响.PCL管壁厚在0.20～0.40 mm时,对LNG通透性亦无显著影响;当壁厚小于0.15 mm时,LNG通透性显著提高.结论 PCL埋植剂对LNG具有通透性,可通过改变PCL管壁厚度增加药物通透性.     

Release of protein from highly cross-linked hydrogels of poly(ethylene glycol) diacrylate fabricated by UV polymerization

Highly crosslinked hydrogel spheres were fabricated using UV photopolymerization of poly(ethylene glycol) diacrylate (PEG-DA) and pentaerythritol triacrylate (PETA) with 2,2'-dimethoxy-2-phenyl-acetophenone (DMPA) as the photoinitiator. Spheres were fabricated both with and without one of three comonomers: acrylic acid, acrylamide or allylamine. Photopolymerization rates and polymer morphology were determined using attenuated total reflectance/Fourier transform infrared spectroscopy and electron microscopy, respectively. These gels were further characterized for volume change, equilibrium water content, diffusivity of the expanding gel, molecular weight between crosslinks and polymer mesh size. Hydrogels with comonomers generally demonstrated an increase in equilibrium water content, average molecular weight between crosslinks and mesh size. Bovine serum albumin was incorporated into the hydrogel to simulate delivery of a model protein drug. The protein diffusion coefficients, based a Fickian release model, were calculated to be between 10(-10) and 10(-12) cm2/s with slight variance due to PETA concentration and the type of comonomer used.     

Role of electrophoretic mobility of protein on its retention by an ultrafiltration membrane. Comparison to chromatography mechanisms

Lysozyme and lactoferrin, two globular proteins, were first studied separately in order to elaborate a strategy for the improvement of their separation by ultrafiltration (UF) with zirconia-based membranes of different charge sign and pore radius. The electrophoretic mobility (mu) at fixed pH and variable ionic strength was used for the characterisation of both proteins and zirconia particles, similar to the active layer of the membrane during the UF run. Specific adsorption of phosphate ions was shown for both proteins resulting in new isoelectric points. The occurrence of electrostatic exclusion mechanism in addition to the molecular sieving in UF of charged solutes was shown for: * Low molecular weight solute: multivalent citrate at pH 6 was specifically adsorbed on zirconia and its transmission through the membrane (defined as the ratio of the concentration in the permeate to that of the feed solution) was reduced in the range 0.001-0.01 mol l(-1) of citrate concentration * Proteins: their transmissions increase when the ionic strength increases (ion-exchange is not the relevant mechanism because transmission is irrespective of the initial charge of the membrane compared with the protein charge). A model based on convection, diffusion and electrophoretic migration mechanisms (CDE model) was proposed to take into account this behaviour. The CDE model predicts the possible existence of a depleted sub-layer of the charged protein in the concentration polarisation layer, located in the close vicinity of the membrane surface. A strategy for the separation of two proteins in mixed solution was proposed by varying both the physico-chemical environment in the feed solution (pH, ionic strength, chemical nature of the electrolyte) and the membrane pore radius. Maximum selectivity was obtained when the target protein (to be transmitted in the permeate side) is close to being uncharged due to specific adsorption of electrolyte ions. Ultrafiltration selectivity is enhanced with membrane of large pore radius, which provides high transmission of the target protein and efficient electrostatic exclusion of the solute to be retained in the retentate side. This UF approach corresponds roughly to the separation of one uncharged and one charged protein from a mixed solution by size exclusion chromatography of the uncharged protein combined with electrostatic exclusion of the charged protein due to packing of similar charge.     

Protective effect of dextrans on glucose oxidase denaturation and inactivation

In the present study, the stabilizing effect of dextrans as additives on the denaturation and inactivation of glucose oxidase (GOD) was investigated. Three different molecular weighted dextrans (M(w) 17.5, 75, 188 kD) were used with different concentrations. Dramatically increased enzyme activities were measured after one hour of incubation of enzyme with additives between 25-40°C in water bath. Highest activity value was measured with 75 kDa molecular weighted dextran (in concentration 30% w/v) at pH 5. Dextran as an additive supplied a long shelf-life to the enzyme at 4°C. In the presence of the 75 kDa dextran, the enzyme was more stable and its activity was increased 2.7-fold at 30°C. In addition, dextran protected GOD against inactivation by a n-heptane/aqueous buffer-stirred system.     

Stability assessment of chitosan-sodium hexametaphosphate capsules

The assessment of the stability of capsules based on chitosan-sodium hexametaphosphate complex formation has been carried out using two independent methods--compression and osmotic swelling, and the influence of the preparation variables was evaluated. The formulation containing 1.5% core polymer (chitosan) and 1.5% oligophosphate, in the absence of salt or at low ionic strength (0.15% NaCl) was found to provide the best membrane resistance. A higher concentration of cross-linker (2.25%) produced stable capsules only in absence of electrolyte. Mannitol, a porogen added to the preparation solutions, did not affect the stability of the obtained membranes. At elevated polyol (1%) and cross-linker levels (2.25%), and at 0% salt, membranes with decreased elasticity were obtained, having lower compression and osmotic bursting values and lower deformation at the breaking points. A significant influence of salt amount on the capsule stability was also found. This was attributed to changes in the membrane formation process resulting in membranes with different thickness and structure. Membrane compression stability was found to be dependent on the pH of both oligophosphate and chitosan solutions, as well as on the reaction time. The bursting force values decreased for capsule diameters below 1.6 mm. The increased membrane/capsule volume ratio for the small capsules decreased the capsule deformation freedom and caused capsule rupture at low force values. The capsules made at low salt amounts showed very good storage stability over time and at elevated temperatures. The results demonstrated that the capsules could be formulated with controlled properties for various biomedical applications.     

Investigation of the allergenic activity of isolated fractions of a standardized partly purified whole mite body (Dermatophagoides pteronyssinus) extract

Twelve fractions of a molecular weight range of 1.35-670.00 kilodaltons (kD) were isolated from a biologically standardized partly purified whole mite body extract (Dermatophagoides pteronyssinus) by preparative size exclusion high-performance liquid chromatography. The allergenic activity and the antigen and allergen patterns of the isolated fractions were investigated by RAST, RAST inhibition and crossed (radio)immunoelectrophoresis (CIE/CRIE). By CRIE, each of the fractions showed allergen patterns, mostly of different compositions. Each fraction showed allergenic activity of different degrees by RAST inhibition. The highest allergenic activity could be measured by RAST inhibition with fractions which contained predominantly the major allergens Der pI and PY as detected by CRIE. Also proteins of higher molecular weights (greater than 158 kD) showed IgE-binding capacities. Nearly all antigens detected by CIE could be identified as allergens using CRIE.