Characterisation of small molecules diffusion in hydrogel-membrane liquid-core capsules

The overall diffusion coefficients for several low molecular weight solutes, such as glucose, fructose, sucrose, lactose, and vitamin B(12) have been determined in Ca-alginate membrane liquid-core capsules using the unsteady-state method following the release of solutes from the capsules to a well-stirred solution of limited volume. The diffusion coefficients obtained for saccharides were 5-20% lower than the corresponding diffusivity in water while for vitamin B(12) about 50% that of water. The diffusion coefficients of the investigated capsules were not influenced by the change in alginate concentration in the capsule membrane from 0.5 to 1.0%. Lower diffusivities and higher deviations from the diffusivity in water were obtained for higher molecular weight solutes.     

Locally controlled release of basic fibroblast growth factor from multilayered capsules

Biodegradable multilayered capsules encapsulating basic fibroblast growth factor (bFGF) were developed as a cytokine release carrier for drug delivery systems. The multilayered hollow capsules were fabricated via the layer-by-layer (LbL) assembly of chitosan (CT) and dextran sulfate (Dex). The bFGF was encapsulated into the CT/Dex multilayered capsules by controlling the membrane permeability, and the local and sustained release of bFGF from the capsules was examined. At pH < 8.0, the capsule membrane tightened, and FITC-dextran ( Mw = 4000) could not enter the capsules. However, FITC-dextran ( M w = 250000) easily entered the capsules at pH > 8.0, which can be attributed to the electrostatic repulsion of Dex caused by the deprotonation of the amine group in CT. After treatment with acetic acid buffer (pH 5.6), FITC-dextran or bFGF was successfully encapsulated into the capsules. The amount of encapsulated bFGF was approximately 34 microg/1 mg of capsule. Initially, about 30% of the encapsulated bFGF was released in serum-free medium within a few hours, however, the release was sustained over 70 h. When the bFGF encapsulating capsules were added to cell culture medium (serum-free), the mouse L929 fibroblast cells proliferated well for 2 weeks as compared to cultures, where bFGF was added to the medium or where bFGF and empty hollow capsules were added separately. The proliferation is due to the local and sustained release of bFGF from the adsorbent capsule to the cell surface.     

Diffusion characteristics of substrates in Ca-alginate gel beads

The diffusion characteristics of several substrates of varying molecular sizes into and from Ca-alginate gel beads in well-stirred solutions were investigated. The values of the diffusion coefficient (D) of substrates such as glucose, L-tryptophan, and alpha-lactoalbumin [with molecular weight (MW) less than 2 x 10(4)] into and from the gel beads agreed with those in the water system. Their substrates could diffuse freely into and from the gel beads without disturbance by the pores in the gel beads. The diffusion of their substrates into and from the gel beads was also not disturbed by increasing the Ca-alginate concentration in the beads and the CaCl(2) concentration used in the gel preparation. In the case of higher molecular weight substances such as albumin (MW = 6.9 x 10(4)), gamma-globulin (MW = 1.54 x 10(5)) and fibrinogen (MW = 3.41 x 10(5)), the diffusion behaviors of the substrates into and from the gel beads were very different. No diffusion of their substrates into the gel beads from solutions was observed, and only albumin was partly absorbed on the surface of the gel beads. The values of D of their substrates from the gel beads into their solutions were smaller than their values in the water system, but all their substrates could diffuse from the gel beads. The diffusion of high molecular weight substrates was limited more strongly by the increase of Ca-alginate concentration in the gel beads than by the increase of the CaCl(2) concentration used in the gel preparation. Using these results, the capacity of Ca-alginate gel as a matrix of immobilization was discussed.     

大孔型NaCS-PDMDAAC微胶囊固定化细胞在摇床和鼓泡塔中的培养研究

NaCS-PDMDAAC生物微胶囊囊膜较为致密,影响胶囊内外物质的交换,从而影响胶囊内细胞的生长。利用淀粉酶对致孔剂淀粉的降解作用制备了一种大孔型的纤维素硫酸钠_聚二甲基二烯丙基氯化铵（NaCS-PDMDAAC）生物微胶囊,实验表明胶囊的孔径和通透性能都有了很大的提高。将酵母和大肠杆菌作为模型细胞包埋于胶囊中分别通过摇瓶和鼓泡塔半连续培养,在鼓泡塔中胶囊内细胞的密度要高于摇床,表明氧气的传递是胶囊内好氧细胞生长的限制因素,大孔胶囊由于囊膜孔径变大,氧气的传递更为快速,在鼓泡塔中大孔型胶囊内的最大细胞密度比常规胶囊要高出20%～110%。由于对氧气的需求量的不同,大肠杆菌菌浓提高的程度要高于酵母。     

Poly(carbamoylsulfonate), a material for immobilization: Synthesis,diffusion- and mechanical properties

Summary A simple method is described for the synthesis of a material for immobilization, Poly(carbamoylsulfonate) (PCS). PCS is based on the synthesis of a hydrophilic polyether polyol and a polyisocyanate to the isocyanate-pre-polymer. The real diffusion coefficients of the PCS hydrogel are comparable with the values of Ca-alginate, the molecular weight cut-off can be found between myoglobine (MW 17000 g/mol) and albumine (MW 67000 g/mol). PCS hydrogels have optimal elastic properties (higher elongation at break and lower modulus of elasticity than other natural hydrogels) and therefore can be used for a matrix of biocatalysts in an agitated reactor as well as in a fluid-bed reactor.     

Use of holographic laser interferometry to study the diffusion of polymers in gels

The aim of this study was to demonstrate the potential for holographic interferometry to be used for diffusion studies of large molecules in gels. The diffusion and partitioning of BSA (67,000 g/mol) and pullulans (5,900-112,000 g/mol) in agarose gel were investigated. The gel diffusion coefficients obtained for BSA were higher when distilled water was used as a solvent compared to those obtained with 0.1 M NaCl as the solvent. Furthermore, the gel diffusion coefficient increased with increasing BSA concentration. The same trend was found for liquid BSA diffusion coefficients obtained by DLS. BSA partition coefficients obtained at different agarose gel concentrations (2-6%, w/w) decreased slightly with increasing gel concentration. However, all BSA gel diffusion coefficients measured were significantly lower than those in pure solvent and they decreased with increasing agarose concentration. The gel diffusion coefficients obtained for pullulans decreased with increasing pullulan molecular weight. The same effect from increased molecular weight was seen in the liquid diffusion coefficients measured by DLS. The pullulan partition coefficients obtained decreased with increasing molecular weight. However, pullulans with a larger Stokes' radius than BSA had partition coefficients that were higher or approximately the same as BSA. This implied that the pullulan molecules were more flexible than the BSA molecules. The results obtained for BSA in this study agreed well with other experimental studies. In addition, the magnitude of the relative standard deviation was acceptable and in the same range as for many other methods. The results thereby obtained showed that holographic interferometry is a suitable method for studying diffusion of macromolecules in gels.     

Zonal rate model for axial and radial flow membrane chromatography. Part I: Knowledge transfer across operating conditions and scales

The zonal rate model (ZRM) has previously been applied for analyzing the performance of axial flow membrane chromatography capsules by independently determining the impacts of flow and binding related non‐idealities on measured breakthrough curves. In the present study, the ZRM is extended to radial flow configurations, which are commonly used at larger scales. The axial flow XT5 capsule and the radial flow XT140 capsule from Pall are rigorously analyzed under binding and non‐binding conditions with bovine serum albumin (BSA) as test molecule. The binding data of this molecule is much better reproduced by the spreading model, which hypothesizes different binding orientations, than by the well‐known Langmuir model. Moreover, a revised cleaning protocol with NaCl instead of NaOH and minimizing the storage time has been identified as most critical for quantitatively reproducing the measured breakthrough curves. The internal geometry of both capsules is visualized by magnetic resonance imaging (MRI). The flow in the external hold‐up volumes of the XT140 capsule was found to be more homogeneous as in the previously studied XT5 capsule. An attempt for model‐based scale‐up was apparently impeded by irregular pleat structures in the used XT140 capsule, which might lead to local variations in the linear velocity through the membrane stack. However, the presented approach is universal and can be applied to different capsules. The ZRM is shown to potentially help save valuable material and time, as the experiments required for model calibration are much cheaper than the predicted large‐scale experiment at binding conditions. Biotechnol. Bioeng. 2013; 110: 1129–1141. © 2012 Wiley Periodicals, Inc.     

Polymethyl-methacrylate-sorbitol-based capsules as local drug delivery vehicles: an in vitro antibiotic elution study

A PMMA (polymethyl-methacrylate)-sorbitol-based capsule system was recently developed, and the permeability of 16 types of capsules with different wall thicknesses and sorbitol contents tested. By optimizing these two parameters, we showed that capsule permeability could be controlled. Promising preliminary data obtained using BPB (Bromophenol Blue) as diffusion marker prompted us to further investigate the antibiotic release of capsules showing the most appropriate release characteristics. PMMA-sorbitol capsules were prepared with wall thickness of 0.5 or 0.6 mm and 60 or 70 w/w% (weight percent) of sorbitol content. In vitro gentamicin, amikacin, tobramycin releases were determined by using a microbiological agar plate diffusion assay. Capsules released 70-100% of their gentamicin load, substantially superior to Septopal, and showed preferable, extended release profiles when compared with the beads. The release kinetics of amikacin and tobramycin closely resembled those of gentamicin. PMMA-sorbitol capsules have been developed and tested, which make them promising devices for local antibiotic delivery.     

Characterizing molecular diffusion in the lens capsule

The lens capsule compartmentalizes the cells of the avascular lens from other ocular tissues. Small molecules required for lens cell metabolism, such as glucose, salts, and waste products, freely pass through the capsule. However, the lens capsule is selectively permeable to proteins such as growth hormones and substrate carriers which are required for proper lens growth and development. We used fluorescence recovery after photobleaching (FRAP) to characterize the diffusional behavior of various sized dextrans (3, 10, 40, 150, and 250 kDa) and proteins endogenous to the lens environment (EGF, γD-crystallin, BSA, transferrin, ceruloplasmin, and IgG) within the capsules of whole living lenses. We found that proteins had dramatically different diffusion and partition coefficients as well as capsule matrix binding affinities than similar sized dextrans, but they had comparable permeabilities. We also found ionic interactions between proteins and the capsule matrix significantly influence permeability and binding affinity, while hydrophobic interactions had less of an effect. The removal of a single anionic residue from the surface of a protein, γD-crystallin [E107A], significantly altered its permeability and matrix binding affinity in the capsule. Our data indicated that permeabilities and binding affinities in the lens capsule varied between individual proteins and cannot be predicted by isoelectric points or molecular size alone.     

Chitosan hollow fiber membranes

Chitosan hollow fibers were produced by wet spinning, taking advantage of the unique rheological properties of highly viscous chitosan solutions in acetic acid. The mechanical and separation properties of hollow fibers were tested. The mechanical properties were determined by measuring tensile force, tensile stress, elongation, and initial elasticity module. The separation properties were specified by determining retention coefficients of particular blood components and determining cut-off of the membrane by the analysis of dextran molecular weight distribution in the feed and permeate using a technique of gel chromatography (GPC)-Shimadzu gel chromatograph.     

Diffusive and convective transport through hollow fiber membranes for liver cell culture

For an efficient membrane bioreactor design, transport phenomena determining the overall mass flux of metabolites, catabolites, cell regulatory factors, and immune-related soluble factors, need to be clarified both experimentally and theoretically. In this work, experiments and calculations aimed at discerning the simultaneous influence of both diffusive and convective mechanisms to the transport of metabolites. In particular, the transmembrane mass flux of glucose, bovine serum albumin (BSA), APO-transferrin, immunoglobulin G, and ammonia was experimentally measured, under pressure and concentration gradients, through high-flux microporous hydrophilic poly-ether-sulphone (PES-HFMs) and poly-sulphone hollow fiber membranes (PS-HFMs). These data were analyzed by means of a model based on the mechanism of capillary pore diffusion, assuming that solute spherical molecules pass through an array of solvent-filled cylindrical pores with a diffusive permeation corrected for friction and steric hindrances. Additionally, resistances to the mass transfer were taken into account. Convective permeation data were discussed in terms of morphological properties of the polymeric membranes, molecular Stokes radius, and solute-membrane interactions according to information given by contact angle measurements. The observed steady-state hydraulic permeance of PS-HFMs was 0.972 L/m2hmbar, about 15.6-fold lower than that measured for PES-HFMs (15.2 L/m2h); in general, PS-HFMs provided a significant hindrance to the transport of target species. Diffusion coefficients of metabolites were found to be similar to the corresponding values in water through PES-HFMs, but significantly reduced through PS-HFMs (D(Glucose)(Membrane)=2.8x10(-6)+/-0.6x10(-6)cm2/s, D(BSA)(Membrane)=6.4 x 10(-7)+/-1 x 10(-7)cm(/s, D(Apotransferrin)(Membrane)=2.3 x 10(-7)+/-0.25 x 10(-7)cm2/s).     

Determination of diffusion coefficients and diffusion characteristics for chlorferon and diethylthiophosphate in Ca-alginate gel beads

Diffusion characteristics of chlorferon and diethylthiophosphate (DETP) in Ca-alginate gel beads were studied to assist in designing and operating bioreactor systems. Diffusion coefficients for chlorferon and DETP in Ca-alginate gel beads determined at conditions suitable for biodegradation studies were 2.70 x 10(-11) m(2)/s and 4.28 x 10(-11) m(2)/s, respectively. Diffusivities of chlorferon and DETP were influenced by several factors, including viscosity of the bulk solution, agitation speed, and the concentrations of diffusing substrate and immobilized cells. Diffusion coefficients increased with increasing agitation speed, probably due to poor mixing at low speed and some attrition of beads at high speeds. Diffusion coefficients also increased with decreasing substrate concentration. Increased cell concentration in the gel beads caused lower diffusivity. Theoretical models to predict diffusivities as a function of cell weight fraction overestimated the effective diffusivities for both chlorferon and DETP, but linear relations between effective diffusivity and cell weight fraction were derived from experimental data. Calcium-alginate gel beads with radii of 1.65-1.70 mm used in this study were not subject to diffusional limitations: external mass transfer resistances were negligible based on Biot number calculations and effectiveness factors indicated that internal mass transfer resistance was negligible. Therefore, the degradation rates of chlorferon and DETP inside Ca-alginate gel beads were reaction-limited.     

Diffusion characteristics and controlled release of bacterial fertilizers from modified calcium alginate capsules

An indigenous Cellulosimicrobium cellulans GS6 isolate able to solubilize insoluble phosphate complexes in soil is a potential bacterial fertilizer. Enclosure of the phosphate-solubilizing bacterium (PSB) in biodegradable capsules may protect the PSB cells inoculated into soil and, in the meantime, enable the control of cell release that confers long-term fertilizing effects. In this study, calcium alginate (CA) was used as the core matrix to encapsulate cells of C. cellulans GS6. The cell-liberating properties of the CA-based capsules were modified by blending with a variety of supplemental materials (SM), including chitin, cellulose, olive oil, and gelatin. The experimental results showed that the maximum cell-release percentage (MCR%) of the capsules decreased in the order of CA-cellulose>CA-olive oil>CA-chitin>CA-gelatin>CA. Furthermore, a mass transport model was developed to accurately describe the kinetics of cell release results for each capsule. The diffusion coefficient (D(e)) of each capsule was also determined from the model simulation. We found that the estimated D(e) values are positively correlated to the release rate with rare exceptions. Lastly, as our results underscored the crucial roles that the type of capsules plays in the rate and amount of cell release, controlled release of the bacterial fertilizer (C. cellulans GS6 cells) may be achieved via the design of capsule materials.     

Expanded and packed bed albumin adsorption on fluoride modified zirconia

The expanded bed characteristics of 75-103microm fluoride-modified zirconia (FmZr) particles synthesized by a fed batch oil emulsion process were investigated. These particles are distinguished from commercially available expanded-bed adsorbents by virtue of their high density (2.8 g/cc) and the mixed mode protein retention mechanism which allows for the retention of both cationic and anionic proteins. The linear velocity versus bed porosity data agree with the Richardson-Zaki relationship with the terminal velocity in infinite medium of 2858.4 cm/h and a bed expansion index of 5.1. Residence time distribution (RTD) studies and bovine serum albumin (BSA) adsorption studies were performed as a function of the height of the settled bed to the column diameter (H:D) ratio and degree of bed expansion with superficial velocities of 440 to 870 cm/h. The settled bed, a 2x expanded bed, and a 3x expanded bed were studied for the H:D ratios of 1:1, 2:1, and 3:1. The dynamic binding capacity (DBC) at 5% breakthrough was low (2-8 mg BSA/mL settled bed) and was independent of the H:D ratio or the degree of bed expansion. The saturation DBC was 32.3 +/- 7.0 mg BSA/mL settled bed. The adsorption-desorption kinetics and intraparticle diffusion for protein adsorption on FmZr (38-75 micrometer) were investigated by studying the packed bed RTD and BSA adsorption as a function of temperature and flow rate. The data show that the adsorption-desorption kinetics along with intraparticle diffusion significantly influence protein adsorption on FmZr. Low residence times ( approximately 0.8 min) of BSA result in a DBC at 5% breakthrough which is 3.5-fold lower compared to that at 6-fold higher protein residence time. At low linear velocity (45 cm/h) the breakthrough curve is nearly symmetrical and becomes asymmetrical and more dispersed at higher linear velocity (270 cm/h) due to the influence of slow adsorption-desorption kinetics and intraparticle diffusion.Copyright 1998 John Wiley & Sons, Inc. Bioeng 60: 333-340, 1998.     

Demonstration of efficient trichloroethylene biodegradation in a hollow‐fiber membrane bioreactor

Rapid cometabolism of trichloroethylene (TCE) by pure cultures of Methylosinus trichosporium OB3b PP358 was demonstrated in a two‐stage hollow‐fiber membrane bioreactor over the course of 3 weeks. PP358 was grown in a continuous‐flow chemostat and circulated through the shell of a hollow‐fiber membrane module (HFMM), while TCE contaminated water (160 to 1450 μg/L) was pumped through the fiber lumen (fiber interior). In parallel‐flow HFMM biological experiments, 82% to 89% of the influent TCE was removed from the lumen (5.1‐min residence time) with 99% of the transferred TCE undergoing biodegradation. Biological experiments in a larger capacity baffled radial‐flow HFMM resulted in 66% to 99% TCE transferred and 93% to 96% TCE biodegradation at lumen residence times of between 1.5 and 3.7 min. Biodegradation was maintained throughout the experiments at pseudo‐first‐order biodegradation rate constants of 0.41 to 2.8 L/mg TSS/day. Best‐fit computer modeling of the baffled radial‐flow biological process estimated mass transfer coefficients as large as 2.7 × 10⁻² cm/min. The computer model was also shown to simulate the experimental results quite well. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 681–692, 1999.     

Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump

A parallel chromatographic procedure for the purification of milligram amounts of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quarternary ammonium group with respect to elution and regeneration properties and the 75 cm(2) Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimised. By introducing this step into the protocol, endotoxin levels could be reduced approximately 100-fold to 相似文献   

Determination of glucose and ethanol effective diffusion coefficients in Ca-alginate gel.

Glucose and ethanol diffusion coefficients in 2% Ca-alginate gel were measured using the experimental technique based on solute diffusion into or out of gel beads in a well-stirred solution. The aim of the study was to make the measurements under typical conditions found in alcoholic fermentations, such as the concentrations of glucose (100 g l-1) and ethanol (50 g l-1), the simultaneous counter-diffusion of glucose and ethanol, and the presence of cells in the gel beads at a level of 10(9) cells g-1 of beads. Previously, an evaluation of the error associated with the methodology used indicated how the experimental procedure would minimize the error. The individual measurement of glucose and ethanol coefficients in 2% Ca-alginate with no cells gave values of 5.1 and 9.6 x 10(-6) cm2 s-1, respectively, which are lower than those in water. When the effect of counter-diffusion was investigated, both coefficients decreased: glucose by 14% and ethanol by 28%. When cells were incorporated into the beads, only the ethanol coefficient decreased significantly, while the glucose coefficient apparently increased its value to 6.9 10(-6) cm2 s-1.     

SELECTIVE PERMEABILITY OF THE EXTRACELLULAR ENVELOPE OF THE MICROALGA SPONDYLOSIUM PANDURIFORME (CHLOROPHYCEAE) AS REVEALED BY ELECTRON PARAMAGNETIC RESONANCE

The aim of this work was to investigate the role of the polysaccharide sheath of the microalga Spondylosium panduriforme (Chlorophyceae, Desmidiaceae) in the selective permeability and transport of molecules into the interior of the cell. We have used the electron paramagnetic resonance (EPR) technique applied to a variety of spin labels of a hydrophobic nature with different substitutents on the ring (−OH, =O, −N=C=S, −NH₃⁺, and others). The spin label EPR signals were destroyed as a consequence of metabolic processes once the spin probes had entered the cells. The decay time of the EPR signal was regulated by the diffusion mechanism across the polysaccharide sheath, cell wall, and membrane. To discriminate the effect of the polysaccharide sheath from that of the cell wall and membrane, the polysaccharide sheath was removed by ultrasonic treatment. The decay times for the cells without capsule were faster than those for intact cells, and a possible mechanism of interaction involving hydrogen bonds between the spin labels and the −OH groups of the polysaccharide sheath is presented. These were expressed by their diffusion and friction coefficients as derived from Ficks' Second Law and the Einstein-Stokes equation and were summarized in terms of diffusion coefficients ( D ₁) for the capsule medium in the order: =O < −OH < −phe < −H < −N=C=S; and for cell wall and membrane ( D ₂): −OH < −H < =O < −NH₃⁺≅−phe < −N=C=S. For the friction coefficients ( f ₁ and f ₂), the order was inverted. These results suggest the capsule plays a role in selectivity as a result of polar interactions with the spin labels.     

Albumin/asparaginase capsules prepared by ultrasound to retain ammonia

Asparaginase reduces the levels of asparagine in blood, which is an essential amino acid for the proliferation of lymphoblastic malign cells. Asparaginase converts asparagine into aspartic acid and ammonia. The accumulation of ammonia in the bloodstream leads to hyperammonemia, described as one of the most significant side effects of asparaginase therapy. Therefore, there is a need for asparaginase formulations with the potential to reduce hyperammonemia. We incorporated 2 % of therapeutic enzyme in albumin-based capsules. The presence of asparaginase in the interface of bovine serum albumin (BSA) capsules showed the ability to hydrolyze the asparagine and retain the forming ammonia at the surface of the capsules. The incorporation of Poloxamer 407 in the capsule formulation further increased the ratio aspartic acid/ammonia from 1.92 to 2.46 (and 1.10 from the free enzyme), decreasing the levels of free ammonia. This capacity to retain ammonia can be due to electrostatic interactions and retention of ammonia at the surface of the capsules. The developed BSA/asparaginase capsules did not cause significant cytotoxic effect on mouse leukemic macrophage cell line RAW 264.7. The new BSA/asparaginase capsules could potentially be used in the treatment of acute lymphoblastic leukemia preventing hyperammonemia associated with acute lymphoblastic leukemia (ALL) treatment with asparaginase.     

Oxygen diffusivity in gel beads containing viable cells

This article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (D(e)) in gel beads entrapping viable cells. We applied this method to the measurement of D(e) in Ca- and Ba-alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca-alginate gel beads was superior to that of the Ba-alginate gel beads, and the D(e) in the Ca-alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca-alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.