Complexity in mood disorder diagnosis: fMRI connectivity networks predicted medication-class of response in complex patients.

OBJECTIVE: This study determined the clinical utility of an fMRI classification algorithm predicting medication-class of response in patients with challenging mood diagnoses. METHODS: Ninety-nine 16-27-year-olds underwent resting state fMRI scans in three groups-BD, MDD and healthy controls. A predictive algorithm was trained and cross-validated on the known-diagnosis patients using maximally spatially independent components (ICs), constructing a similarity matrix among subjects, partitioning the matrix in kernel space and optimizing support vector machine classifiers and IC combinations. This classifier was also applied to each of 12 new individual patients with unclear mood disorder diagnoses. RESULTS: Classification within the known-diagnosis group was approximately 92.4% accurate. The five maximally contributory ICs were identified. Applied to the complicated patients, the algorithm diagnosis was consistent with optimal medication-class of response to sustained recovery in 11 of 12 cases (i.e., almost 92% accuracy). CONCLUSION: This classification algorithm performed well for the know-diagnosis but also predicted medication-class of response in difficult-to-diagnose patients. Further research can enhance this approach and extend these findings to be more clinically accessible.

Therapeutic cell encapsulation techniques and applications in diabetes.

The encapsulation of therapeutic cells permits the implantation of allogeneic and xenogeneic cells for the regulation of certain physiological processes damaged by the death or senescence of host tissues. The encapsulation of pancreatic cells for the treatment of diabetes is emphasized; however, many of the techniques are applicable to a wide array of mammalian cell applications. The summary of both established and novel encapsulation techniques, clinical trials, and commercial product developments highlights the metered but steady pace of therapeutic cell encapsulation towards implementation.

Protein micro and nanoencapsulation within glycol-chitosan/Ca²+/alginate matrix by spray drying.

Encapsulation of therapeutic peptides and proteins into polymeric micro and nanoparticulates has been proposed as a strategy to overcome limitations to oral protein administration. Particles having diameter less than 5 µm are able to be taken up by the M cells of Peyer's patches found in intestinal mucosa. Current formulation methodologies involve organic solvents and several time consuming steps. In this study, spray drying was investigated to produce protein loaded micro/nanoparticles, as it offers the potential for single step operation, producing dry active-loaded particles within the micro to nano-range. Spherical, smooth surfaced particles were produced from alginate/protein feed solutions. The effect of operational parameters on particle properties such as recovery, residual activity and particle size was studied using subtilisin as model protein. Particle recovery depended on the inlet temperature of the drying air, and mean particle size ranged from 2.2 to 4.5 µm, affected by the feed rate and the alginate concentration in the feed solution. Increase in alginate:protein ratio increased protein stability. Presence of 0.2 g trehalose/g particle increased the residual activity up to 90%. Glycol-chitosan-Ca(2+)alginate particles were produced in a single step operation, with resulting mean diameter of 3.5 µm. Particles showed fluorescein isothiocyanate labeled bovine serum albumin (BSA)-protein entrapment with increasing concentration toward the particle surface. Similar, limited release profiles of BSA, subtilisin and lysozyme were observed in gastric simulation, with ultimate full release of the proteins in gastrointestinal simulation.

Insulin-loaded nanoparticles are prepared by alginate ionotropic pre-gelation followed by chitosan polyelectrolyte complexation.

Alginate nanoparticles were prepared from dilute alginate sol by inducing a pre-gel with calcium counter ions, followed by polyelectrolyte complex coating with chitosan. Particles in the nanometer size range were obtained with 0.05% alginate and 0.9 mM Ca2+. The mean particle size was influenced by time and stirring speed of nanoparticle preparation, by alginate guluronic acid content and chitosan molecular weight and by the initial alginate:chitosan mass ratio. The association efficiency of insulin into alginate nanoparticles, as well as loading capacity were mainly influenced by the alginate:chitosan mass ratio. Under optimized size conditions, the association efficiency and loading capacities were as high as 92% and 14.3%, respectively. Approximately 50% of the protein was partially retained by the nanoparticles in gastric pH environment up to 24 hours while a more extensive release close to 75% was observed under intestinal pH conditions. Mild formulation conditions, optimum particle size range obtained, high insulin entrapment efficiency, and resistance to gastrointestinal release seem to be synergic and promising factors toward development of an oral insulin delivery form.

Encapsulation of tannase for the hydrolysis of tea tannins.

Tannase was encapsulated in alginate, chitosan, carrageenan or pectin gel matrices, and in the case of alginate, coated with high or low molecular weight chitosan to reduce enzyme release. Cross-linking with glutaraldehyde also improved enzyme retention. Active enzyme preparations were obtained, although carrageenan gels were unstable in tea. Tannase activity was evaluated by reduction in centrifugable (flocculated) tea solids, and a reduction in tea cream measured turbidimetrically after removal of flocculated solids. Tannin interactions with the polysaccharide gels increased the level of centrifugable solids (flocculent) in the tea. An optimum bead formulation consisted of an alginate core, coated with chitosan and cross-linked with glutaraldehyde. Both core and coating materials contained active enzyme. Beads were prepared in a single step procedure involving extrusion of alginate/tannase solution into a hardening bath containing tannase-loaded, chitosan solution. Tannase retained hydrolytic activity through three successive batch cycles, for a total period of 39h processing, and tea cream was visibly removed by treatment with the immobilized tannase. Activity remained stable during 1-month bead storage under refrigeration.

Microencapsulation of lipophilic drugs in chitosan-coated alginate microspheres.

Chitosan-coated alginate microspheres containing a lipophilic marker dissolved in an edible oil, were prepared by emulsification/internal gelation and the potential use as an oral controlled release system investigated. Microsphere formation involved dispersing a lipophilic marker dissolved in soybean oil into an alginate solution containing insoluble calcium carbonate microcrystals. The dispersion was then emulsified in silicone oil to form an O/W/O multiple phase emulsion. Addition of an oil soluble acid released calcium from carbonate complex for gelation of the alginate. Chitosan was then applied as a membrane coat to increase the mechanical strength and stabilize the microspheres in simulated intestinal media. Parameters studied included encapsulation yield, alginate concentration, chitosan molecular weight and membrane formation time. Mean diameters ranging from 500 to 800 micron and encapsulation yields ranging from 60 to 80% were obtained. Minimal marker release was observed under simulated gastric conditions, and rapid release was triggered by transfer into simulated intestinal fluid. Higher overall levels of release were obtained with uncoated microspheres, possibly due to binding of marker to the chitosan membrane coat. However the slower rate of release from coated microspheres was felt better suited as a delivery vehicle for oil soluble drugs.

DNA encapsulation within co-guanidine membrane coated alginate beads and protection from extracapsular nuclease.

Co-guanidine membranes were shown to form intact, ionically complexed membranes on alginate beads, serving as an alternative to the commonly used polymers, poly-L-lysine and chitosan. DNA was encapsulated and membrane thickness, the level of DNA protection from nuclease diffusion and the degree of DNA-complexation with co-guanidine membranes were all shown to be dependent on both polymer concentration and coating time. The highest level of DNAse exclusion was possible within beads coated with a polymer concentration of 5 mg/ml. Recovery of double-stranded DNA after nuclease exposure for 60 min reached 90% of that initially encapsulated. The molecular weight cut-off for these co-guanidine membranes was approximately 31 kDa, sufficient to exclude extracapsular nuclease. The level of DNA protection was found to be comparable to high molecular weight poly-L-lysine membranes (197.1 kDa). Intracapsular DNA was accessible to the carcinogen ethidium bromide, which showed a 4-fold increase in uptake in uncoated beads and 2-fold uptake in co-guanidine coated beads compared to beads lacking in DNA. Co-guanidine membranes coating alginate result in a molecular weight cut-off sufficient to retain DNA and exclude 31 kDa DNAse, while providing access to the low molecular weight carcinogen, ethidium bromide.

Stability of chitosan and poly-L-lysine membranes coating DNA-alginate beads when exposed to hydrolytic enzymes.

Soluble chitosan and poly-L-lysine are readily hydrolysed using lysozyme or chitosanase for chitosan, and trypsin, chymotrypsin or proteinase K for poly-L-lysine. For similar amounts of enzyme, chitosanase hydrolysed 57% of the chitosan, compared to 35% for lysozyme. In the case of poly-L-lysine, chymotrypsin and trypsin exhibited similar activities, hydrolysing approximately 41% of the polymer compared to proteinase K at only 16%. In contrast, chitosan and poly-L-lysine membranes, coating alginate beads, were almost totally inert to the respective hydrolytic enzymes. Less than 2% of the membrane weight was hydrolysed. It appears that either membrane material would be stable for in vivo application, and in particular in the protection of DNA during gastrointestinal transit. At chitosanase concentrations of 1.4 mg/ml and in the presence of sodium ions, 20% of the total double-stranded DNA was released from chitosan coated beads. An exchange of calcium for sodium within the bead liquefied the alginate core releasing DNA. The presence of calcium stabilized the alginate bead, retaining all the DNA. Highly pure DNA was recovered from beads through mechanical membrane disruption, core liquefaction in citrate and use of DNA spin-columns to separate DNA/alginate mixtures in a citrate buffer. DNA recovery efficiencies as high as 94% were achieved when the initial alginate/DNA weight ratio was 1000.

Electrophoretic extraction and analysis of DNA from chitosan or poly-L-lysine-coated alginate beads.

Alginate beads containing entrapped DNA were produced using both external and internal calcium sources, and coated with chitosan or poly-L-lysine membranes. The beads were assayed with DNase nuclease to determine formulation conditions offering the highest level of DNA protection from nucleic acid hydrolysis, simulating gastrointestinal exposure. A method was developed to extract and assay intracapsular DNA through a modified agarose electrophoresis system. Both external and internally gelled beads were permeable to DNase (Mw = 31 kDa), indicated by the absence of DNA after nuclease exposure. At low levels of DNase exposure, coated high guluronic content alginate beads offered a higher level of DNA protection compared with coated beads with low guluronic alginate. No apparent correlation was found with chitosan membrane molecular weight and degree of deacetylation; however, increasing poly-L-lysine molecular weight appeared to increase DNase exclusion from beads. At elevated levels of DNase exposure, DNA hydrolysis was evident within all coated beads with the exception of those coated with the highest molecular weight poly-L-lysine (Mw = 197.1 kDa), which provided almost total nuclease protection. Optimal combination then for DNA protection from nucleases is a high guluronic alginate core, coated with high molecular weight poly-L-lysine.

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation.

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.

DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads.

DNA was immobilized within alginate matrix using an external or an internal calcium source, and then membrane coated with chitosan or poly-L-lysine. Membrane thickness increased with decreasing polymer molecular weight and increasing degree of deacetylation (chitosan). Beads were exposed to a 31,000 molecular weight nuclease to determine the levels of DNA protection offered by different membrane and matrix combinations. Almost total hydrolysis of DNA was observed in alginate beads following nuclease exposure. Less than 1% of total double-stranded DNA remained unhydrolyzed within chitosan- or poly-L-lysine-coated beads, corresponding with an increase in DNA residuals (i.e. double- and single-stranded DNA, polynucleotides, bases). Chitosan membranes did not offer sufficient DNA protection from DNase diffusion since all of the double-stranded DNA was hydrolyzed after 40 min of exposure. Both chitosan and poly-L-lysine membranes reduced the permeability of alginate beads, shown by enhanced retention of DNA residuals after DNase exposure. The highest level of DNA protection within freshly prepared beads was obtained with high molecular weight (197,100) poly-L-lysine membranes coated on beads formed using an external calcium source, where over 80% of the double-stranded DNA remained after 40 min of DNase exposure. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DS-DNA recoveries of 60% for chitosan-coated, 90% for poly-L-lysine-coated, and 95% for uncoated alginate beads.

DNA encapsulation by an air-agitated, liquid-liquid mixer.

Smooth and spherical alginate microspheres and nylon-membrane bound microcapsules were formed in an air-agitated, liquid-liquid mixer by emulsification/internal gelation and interfacial polymerization respectively. The mean diameter of the alginate microspheres ranged from 100 to 800 microm, and was controlled by process modifications. Increase in emulsifier concentration, gas flowrate, and emulsification time resulted in smaller microsphere size as did a decrease in liquid height. Increase in the dispersed phase viscosity resulted in a longer emulsification time required for approaching a minimum microsphere size. Microspheres could be formed with the proportion of dispersed phase approaching 30%. The yield of alginate microspheres was 70%, with losses attributed to incomplete recovery during washing and filtration operations. The yield of DNA encapsulation within the fraction of recovered microspheres, was 94%. The small loss was thought to occur by surface release during the washing of the microspheres. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 464-470, 1997.

Microencapsulation of lobster carotenoids within poly(vinyl alcohol) and poly(D,L-lactic acid) membranes.

The use of natural pigments such as lobster carotenoids in fish feed formulations offers advantages over the use of the synthetic alternatives. Microencapsulation of the pigments, with or without the addition of antioxidants to the formulation, may be of benefit in terms of stabilizing pigment colour. In the present study, lobster carotenoids were extracted from lobster shell into petroleum ether and microencapsulated by phase separation and salt coacervation within (poly vinyl alcohol) and poly(vinyl alcohol)/poly(D,L-lactic acid) membranes. Spherical microcapsules, with smooth, thin and resilient membranes were obtained with mean diameters ranging from 50 to 150 microns, depending on the membrane material, and source of pigment. The microcapsules were pink-orange in colour, and colour stability was followed spectrophotometrically. Enhanced stability was observed in both membrane materials, in comparison to the non-encapsulated control. Rates of discoloration were determined under a variety of storage conditions, including the absence of light, reduced temperatures and under nitrogen atmosphere. The best stability of lobster carotenoids was observed under a nitrogen atmosphere within PVA/PLA membranes, representing an 11-fold enhancement of pigment stability in comparison to the controls. Under ambient conditions, the enhancement in pigment stability was approximately 6-fold. The optimum concentration of PVA during microencapsulation was 3-4%, and the microencapsulated pigments appeared most stable under acidic conditions. The rate of discoloration appeared independent of pigment concentration.

Blood urea clearance with microencapsulated urease.

The response of a kidney patient to treatment using a microencapsulated urease artificial kidney (MUAK) system was modelled. The model was used to simulate the patient's response and reactor performance for an initial blood urea concentration of 10 mM and a MUAK void volume of 0.5. The performance of the reactor was strongly dependent on the enzyme activity. An optimal activity of 10 mM sec-1 was achieved in the analysis. After operation for 4 h at a flow rate of 200 ml min-1, the reduction in blood urea concentration for reactor dimensions of 2 x 10 cm, 2 x 20 cm, 4 x 10 cm and 4 x 20 cm were 38%, 52%, 60% and 62%, respectively. The effect of flow rate on the performance of urea removal was studied using the reactor dimensions of 4 x 10 cm (optimal design). The results for flow rates of 100, 200, 300 and 400 ml min-1 predicted blood urea reductions of 38, 60, 70 and 76%, respectively. Although, the conversion of urea in the reactor decreased from 100% to 96% for the respective flows of 100 to 400 ml min-1, the high turnover of reactor volume at a higher flow rate was responsible for the improved reduction of the patient's blood urea level. The model has the ability to predict the performance of the MUAK and the patient's blood urea level simultaneously, at various operating conditions.

Microencapsulation of DNA within alginate microspheres and crosslinked chitosan membranes for in vivo application.

Calf thymus DNA was microencapsulated within crosslinked chitosan membranes, or immobilized within chitosan-coated alginate microspheres. Microcapsules were prepared by interfacial polymerization of chitosan, and alginate microspheres formed by emulsification/internal gelation. Diameters ranged from 20 to 500 microns, depending on the formulation conditions. Encapsulated DNA was quantified in situ by direct spectrophotometry (260 nm) and ethidium bromide fluorimetry, and compared to DNA measurements on the fractions following disruption and dissolution of the microspheres. Approximately 84% of the DNA was released upon core dissolution and membrane disruption, with 12% membrane bound. The yield of encapsulation was 96%. Leakage of DNA from intact microspheres/capsules was not observed. DNA microcapsules and microspheres were recovered intact from rat feces following gavage and gastrointestinal transit. Higher recoveries (60%) and reduced shrinkage during transit were obtained with the alginate microspheres. DNA was recovered and purified from the microcapsules and microspheres by chromatography and differential precipitation with ethanol. This is the first report of microcapsules or microspheres containing biologically active material (DNA) being passed through the gastrointestinal tract, with the potential for substantial recovery.

Performance features for urea hydrolysis in a CSTR with microencapsulated urease.

The factors which influence the steady state performance of a CSTR operation with microencapsulated urease for the regeneration of a dialysated solution have been studied at various enzyme activities. The theoretical model considered the effect of microcapsule diameter, pH-dependent kinetics, and product inhibition and substrate depletion, in relation to urea conversion and the capsule effectiveness factor. The limiting effects of pH, product inhibition and substrate depletion were also studied individually and in combination under eight case studies. The base case which included these three limiting factors in the reaction process, predicted the lowest urea conversion values at the enzyme activities considered. However, the effectiveness factor for each case at a fixed microcapsule diameter depended on the enzyme activity. This behaviour was studied for two different microcapsule diameters, 5 microns and 500 microns. At enzyme activities lower than 1 mM/s, a model considering Michaelis-Menten kinetics alone, predicted the highest effectiveness factors. On the other hand, beyond 1 mM/s enzyme activity, the lowest effectiveness factors were predicted, although higher conversions than that of the base case were achieved. This might be due to the rapid depletion of substrate at high activities when Michaelis-Menten kinetics were considered, leads to residual substrate concentrations (Sr) lower than the Michaelis-Menten constant Km,o; a condition for a dramatic drop in the intraparticle reaction rate. The limiting factors in the base case held Sr at a relatively higher value than Km,o.

Microencapsulation of Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. cremoris was microencapsulated within alginate/poly-L-lysine (alg/PLL), nylon or crosslinked polyethyleneimine (PEI) membranes. Toxic effects were observed with solvents and reagents used in nylon and PEI membrane formation. Alg/PLL encapsulation resulted in viable and active cell preparations which acidified milk at a rate proportional to the cell concentration, but at rates less than that of free cell preparations. At 4 x 10(8) colony-forming units (cfu/ml milk), encapsulated cells took 17 per cent longer than free lactococci to reduce the pH of milk to 5.5. Similar activities of free and micro-encapsulated cells may be attained at higher cell concentrations (10(9) cfu/ml milk). The rate of lactic acid production was approximately 2 mmol/h at an encapsulated cell concentration of 4 x 10(8) cfu/ml.

Immobilization of cells for application in the food industry.

Immobilization of cells offers advantages to the food process industries, including enhanced fermentation productivity and cell stability and reduced downstream processing costs due to facilitated cell recovery and recycle. This article summarizes the varied immobilization methodologies, including adsorption, entrapment, covalent binding, and microencapsulation. Examples of interest to the food industry are provided, together with a review of the physiological effects of immobilization. Topics in process engineering include immobilized cell bioreactor configurations and the scale-up potential of the various immobilization techniques.

Microencapsulation within crosslinked polyethyleneimine membranes.

A microencapsulation technique is proposed involving the formation of a polyethyleneimine (PEI) membrane crosslinked by an acid dichloride. The membranes were formed at pH 8 in a non-polar solvent, conditions which are better suited for the encapsulation of biocatalysts or fragile biochemicals than those using polyamide membranes. The mean diameter and size distribution of the PEI microcapsules were similar to that observed with nylon membranes. The resultant microcapsules were spherical, free-flowing with a strong membrane. The mass of membrane was seen to be independent of the reaction time (1-4 min), insensitive to the PEI concentration and proportional to the concentration of crosslinking agent.

Membrane formation by interfacial cross-linking of chitosan for microencapsulation of Lactococcus lactis.

Lactic acid bacteria were microencapsulated within cross-linked chitosan membranes formed by emulsification/interfacial polymerization. The technique was modified and optimized to provide biocompatible conditions during encapsulation involving the use of mineral oils as the continuous phase and chitosan as the membrane material. Chitosan cross-linked with hexamethylene diisocyanate or glutaraldehyde resulted in strong membranes, with a narrow size distribution about a mean diameter of 150 mum. Cell viability and activity was demonstrated by the acidification of milk. Loss of acidification activity during microencapsulation was recovered in subsequent fermentations to levels similar to that of free cell fermentations.