Granulation of subtilisin by internal gelation of alginate microspheres for application in detergent formulation

Subtilisin was encapsulated within impact-resistant alginate granules produced by emulsification, internal gelation, and acetone extractive drying. The mechanical and controlled release properties of the granules were modified by adding to the alginate varying levels of formulation excipients, including titanium dioxide, polyvinyl alcohol, microcrystalline cellulose, starch and sucrose. Optimum protease activity and mass yields of 83 and 88%, respectively (mg active subtilisin/g granules), occurred for granules formulated with 3% alginate, 10% starch, 10% titanium dioxide, and 3% subtilisin. Mass losses occurred primarily during the gelation step. Maximum encapsulation efficiency is achieved by using higher molecular weight alginate, increasing the alginate concentration, and carefully controlling process temperature and pH. The strongest granules were obtained at the higher concentrations of medium-G or high-G alginate, while fastest granule dissolution was achieved when a lower concentration of alginate was used in combination with polyvinyl alcohol or microcrystalline cellulose as dispersants. Mechanical properties of alginate granules were found to be unaffected by the different cations employed in matrix gel formation.     

Evaluation of Chitosan/Alginate Beads Using Experimental Design: Formulation and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization

Bovine serum albumin-loaded beads were prepared by ionotropic gelation of alginate with calcium chloride and chitosan. The effect of sodium alginate concentration and chitosan concentration on the particle size and loading efficacy was studied. The diameter of the beads formed is dependent on the size of the needle used. The optimum condition for preparation alginate–chitosan beads was alginate concentration of 3% and chitosan concentration of 0.25% at pH 5. The resulting bead formulation had a loading efficacy of 98.5% and average size of 1,501 μm, and scanning electron microscopy images showed spherical and smooth particles. Chitosan concentration significantly influenced particle size and encapsulation efficiency of chitosan–alginate beads (p < 0.05). Decreasing the alginate concentration resulted in an increased release of albumin in acidic media. The rapid dissolution of chitosan–alginate matrices in the higher pH resulted in burst release of protein drug.     

Roller Compaction, Granulation and Capsule Product Dissolution of Drug Formulations Containing a Lactose or Mannitol Filler, Starch, and Talc

This study investigated the influence of excipient composition to the roller compaction and granulation characteristics of pharmaceutical formulations that were comprised of a spray-dried filler (lactose monohydrate or mannitol), pregelatinized starch, talc, magnesium stearate (1% w/w) and a ductile active pharmaceutical ingredient (25% w/w) using a mixed-level factorial design. The main and interaction effects of formulation variables (i.e., filler type, starch content, and talc content) to the response factors (i.e., solid fraction and tensile strength of ribbons, particle size, compressibility and flow of granules) were analyzed using multi-linear stepwise regression analysis. Experimental results indicated that roller compacted ribbons of both lactose and mannitol formulations had similar tensile strength. However, resulting lactose-based granules were finer than the mannitol-based granules because of the brittleness of lactose compared to mannitol. Due to the poor compressiblility of starch, increasing starch content in the formulation from 0% to 20% w/w led to reduction in ribbon solid fraction by 10%, ribbon tensile strength by 60%, and granule size by 30%. Granules containing lactose or more starch showed less cohesive flow than granules containing mannitol and less starch. Increasing talc content from 0% to 5% w/w had little effect to most physical properties of ribbons and granules while the flow of mannitol-based granules was found improved. Finally, it was observed that stored at 40 °C/75% RH over 12 weeks, gelatin capsules containing lactose-based granules had reduced dissolution rates due to pellicle formation inside capsule shells, while capsules containing mannitol-based granules remained immediate dissolution without noticeable pellicle formation.     

The plasticizing effect of alginate on the thermoplastic starch/glycerin blends

Corn starch and corn starch–alginate (5–15%) blends plasticized with 35% glycerin were prepared, water was intentionally excluded from the formulations. Torque rheometry measurements were carried out during the processing of the blends in a batch counterrotating twin screw mixer. A progressive decrease in the plasticization energy of the blends was observed as the alginate content was increased, with a 5-fold decrease for the blend with the higher alginate content (15%). The steady state torque of the plasticized melted blends also showed a decrease as alginate content was increased; with a drastic drop occurring for the formulation with higher alginate content. After mixing, test specimens for mechanical, thermal and microstructural testing were made by compression molding. A decrease in the elastic properties and an increase in elongation at break and impact resistance was observed when alginate content was increased in the blends. The transition of the materials towards a more viscous behavior, as alginate content was increased, was confirmed by differential scan calorimetric analysis. For the corn starch–alginate blends glass transitions were detected in the temperature range −60 to −90 °C. Scanning electron microscopy was used to examine the morphology of cryofractured surfaces of the molded test specimens. A reduction of the granular crystalline structures typical of corn starch was observed as alginate content was increased in the blends. The experimental evidence presented in this work indicates that, when water is excluded from thermoplastic corn starch preparation, alginate acts synergistically with glycerin increasing the degree and efficiency of the plasticization process.     

Preparation and characterization of novel polymeric microcapsules for live cell encapsulation and therapy

This article describes the preparation and in vitro characterization of novel genipin cross-linked alginate-chitosan (GCAC) microcapsules that have potential for live cell therapy applications. This microcapsule system, consisting of an alginate core with a covalently cross-linked chitosan membrane, was formed via ionotropic gelation between calcium ions and alginate, followed by chitosan coating by polyelectrolyte complexation and covalent cross-linking of chitosan by naturally derived genipin. Results showed that, using this design concept and the three-step procedure, spherical GCAC microcapsules with improved membrane strength, suppressed capsular swelling, and suitable permeability can be prepared. The suitability of this novel membrane formulation for live cell encapsulation was evaluated, using bacterial Lactobacillus plantarum 80 (pCBH1) (LP80) and mammalian HepG2 as model cells. Results showed that capsular integrity and bacterial cell viability were sustained 6 mo postencapsulation, suggesting the feasibility of using this microcapsule formulation for live bacterial cell encapsulation. The metabolic activity of the encapsulated HepG2 was also investigated. Results suggested the potential capacity of this GCAC microcapsule in cell therapy and the control of cell signaling; however, further research is required.     

Alginate microparticles as novel carrier for oral insulin delivery

Alginate microparticles produced by emulsification/internal gelation were investigated as a promising carrier for insulin delivery. The procedure involves the dispersion of alginate solution containing insulin protein, into a water immiscible phase. Gelation is triggered in situ by instantaneous release of ionic calcium from carbonate complex via gentle pH adjustment. Particle size is controlled through the emulsification parameters, yielding insulin-loaded microparticles. Particle recovery was compared using several washing protocols. Recovery strategies are proposed and the influence on particle mean size, morphology, recovery yield (RY), encapsulation efficiency, insulin release profile, and structural integrity of released insulin were evaluated. Spherical micron-sized particles loaded with insulin were produced. The recovery process was optimized, improving yield, and ensuring removal of residual oil from the particle surface. The optimum recovery strategy consisted in successive washing with a mixture of acetone/hexane/isopropanol coupled with centrifugation. This strategy led to small spherical particles with an encapsulation efficiency of 80% and a RY around 70%. In vitro release studies showed that alginate itself was not able to suppress insulin release in acidic media; however, this strategy preserves the secondary structure of insulin. Particles had a mean size lower than the critical diameter necessary to be orally absorbed through the intestinal mucosa followed by their passage to systemic circulation and thus can be considered as a promising technology for insulin delivery.     

Dose-dependent suppression of hunger by a specific alginate in a low-viscosity drink formulation

Addition of specific types of alginates to drinks can enhance postmeal suppression of hunger, by forming strong gastric gels in the presence of calcium. However, some recent studies have not demonstrated an effect of alginate/calcium on appetite, perhaps because the selected alginates do not produce sufficiently strong gels or because the alginates were not sufficiently hydrated when consumed. Therefore, the objective of the study was to test effects on appetite of a strongly gelling and fully hydrated alginate in an acceptable, low-viscosity drink formulation. In a balanced order crossover design, 23 volunteers consumed a meal replacement drink containing protein and calcium and either 0 (control), 0.6, or 0.8% of a specific high-guluronate alginate. Appetite (six self-report scales) was measured for 5 h postconsumption. Relevant physicochemical properties of the drinks were measured, i.e., product viscosity and strength of gel formed under simulated gastric conditions. Hunger was robustly reduced (20-30% lower area under the curve) with 0.8% alginate (P < 0.001, analysis of covariance), an effect consistent across all appetite scales. Most effects were also significant with 0.6% alginate, and a clear dose-response observed. Gastric gel strength was 1.8 and 3.8 N for the 0.6 and 0.8% alginate drinks, respectively, while product viscosity was acceptable (<0.5 Pa.s at 10 s(-1)). We conclude that strongly gastric-gelling alginates at relatively low concentrations in a low-viscosity drink formulation produced a robust reduction in hunger responses. This and other related studies indicate that the specific alginate source and product matrix critically impacts upon apparent efficacy.     

Studies on Mefenamic Acid Microparticles: Formulation, In Vitro Release, and In Situ Studies in Rats

In this study, we investigated the in vitro characteristics of mefenamic acid (MA) microparticles as well as their effects on DNA damage. MA-loaded chitosan and alginate beads were prepared by the ionotropic gelation process. Microsponges containing MA and Eudragit RS 100 were prepared by quasi-emulsion solvent diffusion method. The microparticles were characterized in terms of particle size, surface morphology, encapsulation efficiency, and in vitro release profiles. Most of the formulation variables manifested an influence on the physical characteristics of the microparticles at varying degrees. We also studied the effects of MA, MA-loaded microparticles, and three different polymers on rat brain cortex DNA damage. Our results showed that DNA damage was higher in MA-loaded Eudragit microsponges than MA-loaded biodegradable chitosan or alginate microparticles.     

Alteration of the properties of Aspergillus sp. K-27 glucoamylase on limited proteolysis with subtilisin

An active derivative (mol. wt. 48,000) of Aspergillus sp. K-27 glucoamylase (mol. wt. 76,000) was obtained by limited proteolysis with subtilisin. The amino acid sequences of native and modified enzymes at the N-termini were Ala-Gly-Gly-Thr-Leu-Asp and Ala-Val-Leu, respectively. The proteolysis greatly decreased the affinity of the enzyme for amylopectin and glycogen, but not for oligosaccharides. It also reduced the ability of the enzyme to degrade raw starch, abolished the ability of the enzyme to adsorb onto starch granules, and eliminated the synergistic action of the enzyme in the hydrolysis of starch granules with alpha-amylase. These findings imply that the enzyme has a specific affinity site for polysaccharide substrates besides the catalytic site, i.e., a starch-binding site, and that the former is removed by proteolysis. The extent of the reduction in the activity for raw starches caused by the modification varied with the starch source, as the modified enzyme digested raw potato starch better than either raw corn or sweet potato starches. A new method for evaluation of the raw starch-digesting activity of glucoamylase is described.     

Starch-branching enzymes preferentially associated with A-type starch granules in wheat endosperm

Two starch granule-bound proteins (SGP), SGP-140 and SGP-145, were preferentially associated with A-type starch granules (>10 microm) in developing and mature wheat (Triticum aestivum) kernels. Immunoblotting and N-terminal sequencing suggested that the two proteins were different variants of SBEIc, a 152-kD isoform of wheat starch-branching enzyme. Both SGP-140 and SGP-145 were localized to the endosperm starch granules but were not found in the endosperm soluble fraction or pericarp starch granules younger than 15 d post anthesis (DPA). Small-size starch granules (<10 microm) initiated before 15 DPA incorporated SGP-140 and SGP-145 throughout endosperm development and grew into full-size A-type starch granules (>10 microm). In contrast, small-size starch granules harvested after 15 DPA contained only low amounts of SGP-140 and SGP-145 and developed mainly into B-type starch granules (<10 microm). Polypeptides of similar mass and immunologically related to SGP-140 and/or SGP-145 were also preferentially incorporated into A-type starch granules of barley (Hordeum vulgare), rye (Secale cereale), and triticale (x Triticosecale Wittmack) endosperm, which like wheat endosperm have a bimodal starch granule size distribution.     

Properties of spherical calcium pectate and alginate gels and their use in diffusion chromatography,solids separations and immobilization of enzymes and cells

Water-soluble acidic polysaccharides—deesterified pectins and carboxy-derivatives of starch—precipitated with calcium ions were tested as precursors of spherical calcium gels. Pectates prepared from apple or citrus pectin, similarly to alginates, are compounds forming spherical calcium gels stable in aqueous medium which have a relatively highly reproducible mass, particle size, water content, shape, mechanical strength and shearing. Both the liquid-solid partition of low- and high-molar-mass solutes and its kinetics proved to be reasonable features. Distribution of pore size in the above materials was estimated. Detailed pictures of surface and of the interior of calcium beads in the scanning electron microscope are presented. The possible use of calcium beads as enzyme carriers, as affinity matrixes and entrapment materials for diffusion chromatography, solids separations and bioindication of a specific water pollution was evaluated. Calcium alginate beads were always used as reference material.     

Ionic and acid gel formation of epimerised alginates; the effect of AlgE4

AlgE4 is a mannuronan C5 epimerase converting homopolymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 resulted in different amounts of alternating sequences. Both ionically cross-linked alginate gels as well as alginic acid gels were prepared from the epimerised alginates. Gelling kinetics and gel equilibrium properties were recorded and compared to results obtained with the original non-epimerised alginates. An observed reduced elasticity of the alginic acid gels following epimerisation by AlgE4 seems to be explained by the generally increased acid solubility of the alternating sequences. Ionically (Ca(2+)) cross-linked gels made from epimerised alginates expressed a higher degree of syneresis compared to the native samples. An increase in the modulus of elasticity was observed in calcium saturated (diffusion set) gels whereas calcium limited, internally set alginate gels showed no change in elasticity. An increase in the sol-gel transitional rate of gels made from epimerised alginates was also observed. These results suggest an increased possibility of creating new junction zones in the epimerised alginate gel due to the increased mobility in the alginate chain segments caused by the less extended alternating sequences.     

Formulation and drying of alginate beads for controlled release and stabilization of invertase

Several alternatives to the conventional alginate beads formulation were studied for encapsulation of invertase. Pectin was added to the alginate/enzyme solution while trehalose and β-cyclodextrin were added to the calcium gelation media. The effect of composition changes, freezing, drying methods (freeze, vacuum, or air drying), and thermal treatment were evaluated on invertase stability and its release kinetics from beads. The enzyme release mechanism from wet beads depended on pH. The addition of trehalose, pectin, and β-cyclodextrin modified the bead structure, leading in some cases to a release mechanism that included the relaxation of the polymer chains, besides Fickian diffusion. Enzyme release from vacuum-dried beads was much faster than from freeze-dried beads, probably due to their higher pore size. The inclusion of β-cyclodextrin and especially of pectin prevented enzyme activity losses during bead generation, and trehalose addition was fundamental for achieving adequate invertase protection during freezing, drying, and thermal treatment. Present results showed that several alternatives such as drying method, composition, as well as pH of the relese medium can be managed to control enzyme release.     

Fluidized Bed Hot-Melt Granulation as a Tool to Improve Curcuminoid Solubility

Curcumin is the main bioactive component of Curcuma longa L. and has recently aroused growing interest from the scientific community. Unfortunately, the medicinal properties attributed to curcuminoids are impaired by their low oral bioavailability or low solubility in aqueous solutions. Many strategies have been studied to improve curcumin solubility; however, the preparation of granules using hydrophilic materials has never been attempted. The aim of this work was to develop curcumin granules by fluidized bed hot-melt granulation using the hydrophilic carrier Gelucire® 50:13. A two-level factorial design was used to verify the influence of Gelucire® 50:13 and lactose contents found in the granules on their size, morphology, bulk and tapped densities, flow, moisture content, and water activity. The granules obtained were also evaluated by differential scanning calorimetry, thermogravimetric analysis, X-ray powder diffraction, and infrared spectrometry. The curcumin solubility and dissolution rates in water were determined by liquid chromatography. The best formulation provides an increase of curcumin solubility of 4642-fold and 3.8-fold compared to the physical mixture. The dissolution tests showed a maximum drug release from granules after 45 min of 70% at pH 1.2 and 80% at pH 5.8 and 7.4, while for non-granulated curcumin, the release was below 20% in all pH. The solid-state characterization and solubility measurement showed good stability of granules over 9 months. The results attest that the fluidized bed hot-melt granulation with hydrophilic binders is an attractive and promising alternative to obtain solid forms of curcumin with enhanced bioavailability.     

Production of cell-enclosing hollow-core agarose microcapsules via jetting in water-immiscible liquid paraffin and formation of embryoid body-like spherical tissues from mouse ES cells enclosed within these microcapsules

We developed agarose microcapsules with a single hollow core templated by alginate microparticles using a jet-technique. We extruded an agarose aqueous solution containing suspended alginate microparticles into a coflowing stream of liquid paraffin and controlled the diameter of the agarose microparticles by changing the flow rate of the liquid paraffin. Subsequent degradation of the inner alginate microparticles using alginate lyase resulted in the hollow-core structure. We successfully obtained agarose microcapsules with 20-50 microm of agarose gel layer thickness and hollow cores ranging in diameter from ca. 50 to 450 microm. Using alginate microparticles of ca. 150 microm in diameter and enclosing feline kidney cells, we were able to create cell-enclosing agarose microcapsules with a hollow core of ca. 150 microm in diameter. The cells in these microcapsules grew much faster than those in alginate microparticles. In addition, we enclosed mouse embryonic stem cells in agarose microcapsules. The embryonic stem cells began to self-aggregate in the core just after encapsulation, and subsequently grew and formed embryoid body-like spherical tissues in the hollow core of the microcapsules. These results show that our novel microcapsule production technique and the resultant microcapsules have potential for tissue engineering, cell therapy and biopharmaceutical applications.     

Formation and oral administration of alginate microspheres loaded with pDNA coding for lymphocystis disease virus (LCDV) to Japanese flounder

Oral delivery of plasmid DNA (pDNA) is a desirable approach for fish immunization in intensive culture. However, its effectiveness is limited because of possible degradation of pDNA in the fish's digestive system. In this report, alginate microspheres loaded with pDNA coding for fish lymphocystis disease virus (LCDV) and green fluorescent protein were prepared with a modified oil containing water (W/O) emulsification method. Yield, loading percent and encapsulation efficiency of alginate microspheres were 90.5%, 1.8% and 92.7%, respectively. The alginate microspheres had diameters of less than 10 microm, and their shape was spherical. As compared to sodium alginate, a remarkable increase of DNA-phosphodiester and DNA-phosphomonoester bonds was observed for alginate microspheres loaded with pDNA by Fourier transform infrared (FTIR) spectroscopic analysis. Agarose gel electrophoresis showed a little supercoiled pDNA was transformed to open circular and linear pDNA during encapsulation. The cumulative release of pDNA in alginate microspheres was or=0.3) for anti-LCDV antibody from week 3 to week 16 for fish orally vaccinated with alginate microspheres loaded with pDNA, in comparison with fish orally vaccinated with naked pDNA. Our results display that alginate microspheres obtained by W/O emulsification are promising carriers for oral delivery of pDNA. This encapsulation technique has the potential for DNA vaccine delivery applications due to its ease of operation, low cost and significant immune effect.     

Microbial and algal alginate gelation characterized by magnetic resonance

Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca(2+) ions generates gels of different bulk homogeneities dependent on the alginate structure. Shorter spin-spin T(2) magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.     

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.     

Eudragit E as excipient for production of granules and tablets from phyllanthus niruri L spray-dried extract

The aim of this study was to investigate the feasibility of using Eudragit E as a granulating agent for a spray-dried extract fromPhyllanthus niruri to obtain tablets containing a high dose of this product. The granules were developed by wet granulation and contained 2.5%, 5.0%, and 10.0% Eudragit E in the final product concentration. The tablets were produced on a single-punch tablet press by direct compression of granules using 0.5% magnesium stearate as a lubricant. The tablets were elaborated following a 2×3 factorial design, where Eudragit E concentration and compression force were the in-dependent variables, and tensile strength and the extract release of the tablets were the dependent variables. All granules showed better technological properties than the spray-dried extract, including less moisture sorption. The characteristics of the granules were directly dependent on the proportion of Eudragit E in the formulation. In general, all tablets showed high mechanical resistance with less than 1% friability, less moisture sorption, and a slower extract release profile. The Eudragit E concentration and compression force of the tablets significantly influenced both dependent variables studied. In conclusion, Eudragit E was efficient as a granulating agent for the spray-dried extract, but additional studies are needed to further optimize the formuations in order to achieve less water sorption and improve the release of the extract from the tablets. Published: April 27, 2007