Microflow system promotes acetaminophen crystal nucleation

It is known that interfaces have various impacts on crystallization from a solution. Here, we describe crystallization of acetaminophen using a microflow channel, in which two liquids meet and form a liquid–liquid interface due to laminar flow, resulting in uniform mixing of solvents on the molecular scale. In the anti‐solvent method, the microflow mixing promoted the crystallization more than bulk mixing. Furthermore, increased flow rate encouraged crystal formation, and a metastable form appeared under a certain flow condition. This means that interface management by the microchannel could be a beneficial tool for crystallization and polymorph control.     

Formation of bioerodible polymeric microspheres and microparticles by rapid expansion of supercritical solutions.

Polyhydroxy acids [poly(L-lactic acid) (L-PLA), poly(D,L-lactic acid) (DL-PLA), and poly-(glycolic acid) (PGA)], biocompatible and bioerodible polymers that are being investigated for controlled delivery of pharmaceuticals and are approved by the Food and Drug Administration for in vivo sutures and bone repair implants, have been dissolved in supercritical CO2 and precipitated by rapid expansion of the resulting supercritical solutions (RESS). The formation of these microparticles and microspheres is a first step toward the goal of producing, in a single processing step, drug-loaded polymeric microspheres for use in controlled release applications. Nucleation of poly(L-lactic acid) from CO2 and CO2-acetone mixtures produced microparticles and microspheres ranging from 4 to 25 microns. Microspheres (2-20 microns) were also obtained with chlorotrifluoromethane as solvent. Commercial L-PLA precipitated after extraction of low molecular weight oligomers showed degradation kinetics similar to that of the starting material. The precipitation of DL-PLA from CO2 produced irregular-sized particles (10-20 microns). PGA, a polymer insoluble in most organic solvents, was found to be soluble in supercritical CO2. Nucleation of PGA from CO2 produced both regular-sized particles and needles of 10-40-microns length. The total solubility of commercial L-PLA in supercritical CO2 at 250 bar and 55 degrees C decreased from 0.14 wt % to less than 0.05 wt % and then leveled off as the cumulative flow of CO2 per unit mass of L-PLA loaded in the extractor increased beyond 20 standard L of CO2/g of L-PLA. Use of acetone (1 wt %) as a cosolvent increased L-PLA solubility by approximately 500%.     

The crystalline polymorphism of mannan in plant cell walls and after recrystallisation

Mannan-rich plant cell walls were mechanically disintegrated and chemically extracted in order to ascertain their morphology and structure by electron microscopy and electron diffraction. For Acetabularia crenulata and Codium fragile, the cell-wall fragments were found to consist of alkali-resistant fibrillar mannan II encrusted with alkali-soluble granular mannan I. In the case of ivory nuts (Phytelephas macrocarpa) there is, in addition, a microfibrillar cellulose component which was also identified. The mannan I—mannan II polymorphism was also obtained when various mannan fractions were recrystallized from solution. In these recrystallizations, the occurrence of one or the other polymorph was found to depend on several parameters: the molecular weight of the mannan, the temperature of crystallization and the polarity of the crystallization medium.Abbreviations DP degree of polymerization - EDTA ethylenediaminetetraacetic acid Affiliated with the Scientific and Medical University of Grenoble     

Production of micronic particles of biocompatible polymer using supercritical carbon dioxide

Three micronization techniques, based on the use of supercritical carbon dioxide, were investigated to produce microspheres of a natural biocompatible polysaccharide. Particles smaller than 20 mum were obtained by means of the rapid expansion of a supercritical solution method (RESS), both with and without cosolvents. The mean diameter of the particles was reduced to about 0.5 mum when a solution of the polymer in an organic solvent was expanded by using carbon dioxide as a supercritical antisolvent (SAS). The SAS process was operated both in a continuous and in a batch mode. The former leads to aggregated structures and fibers, and the latter to the formation of micronic spherical particles. It was found that the experimental temperature did not substantially affect the shape and dimension of the particles. A stronger dependence is shown with respect to the solute concentration in the starting solution. The proposed method is attractive as the basis of a new process for the preparation of drug delivery systems. (c) 1997 John Wiley & Sons, Inc.     

Microparticles of soy lecithin formed by supercritical processes

Finely divided particles of phospholipids are used to form controlled drug delivery systems called liposomes. Conventional physicochemical methods for preparing these microparticles are hampered by a major drawback-the use of organic solvents that remain at few but inhibitory concentration in the final product. This study aimed to propose an alternative method for preparing microparticles of phospholipids starting from soy lecithin-the process had to be free of solvent or at least, the solvent had to be nontoxic. Two micronization techniques based on the use of supercritical carbon dioxide were investigated: the RESS and the SAS processes. The RESS process failed to separate the particles formed from the cosolvent. Performing the SAS process with ethanol as auxiliary solvent, enabled fine particles to form with size ranging from 1 to 40 microm. Particles were spherical and partly agglomerated and seemed to be free of solvent as shown by preliminary infrared analysis.     

Trehalose amorphization and recrystallization

The stability of the amorphous trehalose prepared by using several procedures is presented and discussed. Amorphization is shown to occur by melting (T(m)=215 degrees C) or milling (room temperature) the crystalline anhydrous form TRE-beta. Fast dehydration of the di-hydrate crystalline polymorph, TRE-h, also produces an amorphous phase. Other dehydration procedures of TRE-h, such as microwave treatment, supercritical extraction or gentle heating at low scan rates, give variable fractions of the polymorph TRE-alpha, that undergo amorphization upon melting (at lower temperature, T(m)=130 degrees C). Additional procedures for amorphization, such as freeze-drying, spray-drying or evaporation of trehalose solutions, are discussed. All these procedures are classified depending on the capability of the undercooled liquid phase to undergo cold crystallization upon heating the glassy state at temperatures above the glass transition temperature (T(g)=120 degrees C). The recrystallizable amorphous phase is invariably obtained by the melt of the polymorph TRE-alpha, while other procedures always give an amorphous phase that is unable to crystallize above T(g). The existence of two different categories is analyzed in terms of the transformation paths and the hypothesis that the systems may exhibit different molecular mobilities.     

Study of the RESS Process for Producing Beclomethasone-17,21-Dipropionate Particles Suitable for Pulmonary Delivery

The purpose of this research was to micronize beclomethasone-17,21-dipropionate (BDP), an anti-inflammatory inhaled corticosteroid commonly used to treat asthma, using the rapid expansion of supercritical solution (RESS) technique. The RESS technique was chosen for its ability to produce both micron particles of high purity for inhalation, and submicron/nano particles as a powder handling aid for use in next generation dry powder inhalers (DPIs). Particle formation experiments were carried out with a capillary RESS system to determine the effect of experimental conditions on the particle size distribution (PSD). The results indicated that the RESS process conditions strongly influenced the particle size and morphology; with the BDP mean particle size decreasing to sub-micron and nanometer dimensions. An increase in the following parameters, i.e. nozzle diameter, BDP mol fraction, system pressure, and system temperature; led to larger particle sizes. Aerodynamic diameters were estimated from the SEM data using three separate relations, which showed that the RESS technique is promising to produce particles suitable for pulmonary delivery.     

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.     

Biliary cholesterol crystallization characterized by single-crystal cryogenic electron diffraction

Cholesterol crystals are the building blocks of cholesterol gallstones. The exact structure of early-forming crystals is still controversial. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers (Biophysical J., In press) in cholesterol monolayers compressed on the air-water interface. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. In conclusion, classic ChM is the predominant and stable form in native and model biles. However, under certain (low phospholipid) conditions, transient intermediate polymorphs may form. These findings, documenting single-crystal analysis in bulk solution, provide an experimental approach to investigating factors influencing biliary cholesterol crystal nucleation and growth as well as other processes of nucleation and crystallization in liquid systems.     

Evaluation and modelling the utility of SCCO2 to support efficient lipase mediated esterification

Supercritical fluids offer environmental advantages over chemical solvents, while providing enhanced separation and chemical selectivity. The use of supercritical fluids for the recovery of products from biomass and the transformation of selected molecules (to add value) was studied. Free fatty acids were bio-catalytically transformed to fatty acid esters using lipase within a supercritical fluid environment. A central composite rotatable design was used to evaluate the influence of operating conditions on the enzymatic esterification process and a response surface equation was optimized to identify the most favourable process conditions for maximum free fatty acid conversion. Based on the model equation the process conditions under which it was predicted a yield of 100% esters could be obtained were: pressure 200 bar, temperature 60 °C, ethanol concentration 2.0 M, enzyme concentration 11 wt.% and time 60 min. Experiments conducted under these conditions gave an ester yield of 94.3% (close to predicted results). The activity per unit mass of biocatalyst was found to be 1585 μmol/min/g_cat. The results support the use of supercritical fluids for process integration.     

Downstream processing and purification of eicosapentaenoic (20:5n-3) and arachidonic acids (20:4n-6) from the microalga Porphyridium cruentum

Eicosapentaenoic acid (FPA, 20:5n-3) and arachidonic acid (AA, 20:4n-3)were obtained from the microalga Porphyridium cruentum by a three-stepprocess: fatty acid extraction by direct saponification of biomass,polyunsaturated fatty acid (PUFA) concentration by urea inclusion complexingand EPA isolation by high-performance liquid chromatography (HPLC). Twosolvents were tested for direct saponification of lipids in biomass. Themost efficient solvent, ethanol (96% v/v), extracted 75% ofthe fatty acids. PUFAs concentration by urea inclusion employed a urea/fattyacid ratio of 4:1 wt/wt at the crystallization temperatures of 4°C and28°C. Concentration factors were similar at both temperatures, but theEPA and AA recoveries were higher at 28°C (67.7% and 61.8%for the two acids, respectively). EPA and AA were purified from this PUFAconcentrate using analytical scale HPLC and the best results of thisseparation were scaled up to preparative level (4.7 i. d. × 30 cmcompression radial cartridge). A 94.3% pure EPA fraction and a81.4% pure AA fraction were obtained. Suitability of severalmicroalgae (Porphyridium cruentum, Phaeodactylum tricornutum and Isochrysisgalbana) and cod liver oil as sources of highly pure PUFAs, mainly EPA, wascompared.     

Isolation of Three Crystalline Proteins from Beef Liver After Partial Purification by Acetone Fractionation

The isolation of three proteins in crystalline form from ground beef liver is described. These proteins are FTBL protein (Arch. Biochem. Biophys. 188, 251–265 (1978), crotonase, and catalase. Crotonase is isolated by crystallization from a 32 acetone extract of the ground liver. FTBL protein and catalase can subsequently be isolated from the same extract. For optimal yield and ease of isolation, FTBL protein is isolated from a 46.5% acetone extract from which catalase can subsequently be crystallized by dialysis.

The isolation of FTBL protein as well as the isolation of catalase involves a preliminary fractional precipitation and solution before crystallization can be achieved. Isopropanol can be substituted for acetone in the isolation of the above three proteins and in the case of catalase, results in an exceptionally high yield.

Methods for the recrystallization of the proteins are presented and the role of organic solvents in recrystallization is discussed.     

Crystallization behavior of glucomannan

Five different glucomannan samples were recrystallized from dilute solution. Depending on the experimental conditions, the crystals obtained could be identified as corresponding to the mannan I (anhydrous precipitate of more or less regular lozenge-shaped crystals) or mannan II (hydrated gel-forming pseudo-fibrillar precipitate). High-molecular-weight material, low temperature of crystallization, or a polar crystallization medium favored the mannan II polymorph, whereas a low-molecular weight, a high temperature of crystallization, and a crystallization medium of low polarity yielded the mannan I polymorph. Since the base-plane unit-cell dimensions are fairly constant with respect to variation of glucose, it is likely that isomorphous replacement of mannose by glucose occurs in glucomannan crystallization; the data also indicate that perfection of the glucomannan crystals was reduced in specimens having a high glucose:mannose ratio. The oriented crystallization of glucomannan on cellulose microfibrils was also studied under conditions where the mannan I polymorph was obtained. This gave shish-kebab structures that were characterized.     

Factors controlling in vitro recrystallization of the Caulobacter crescentus paracrystalline S-layer.

The S-layer of Caulobacter is a two-dimensional paracrystalline array on the cell surface composed of a single protein, RsaA. We have established conditions for preparation of stable, soluble protein and then efficient in vitro recrystallization of the purified protein. Efficient recrystallization and long range order could not be obtained with pure protein only, though it was apparent that calcium was required for crystallization. Recrystallization was obtained when lipid vesicles were provided, but only when the vesicles contained the specific species of Caulobacter smooth lipopolysaccharide (SLPS) that previous studies implicated as a requirement for attaching the S-layer to the cell surface. The specific type of phospholipids did not appear critical; phospholipids rather different from those present in Caulobacter membranes or archaebacterial tetraether lipids worked equally well. The source of LPS was critical; rough and smooth variants of Salmonella typhimurium LPS as well as the rough form of Caulobacter LPS were ineffective. The requirement for calcium ions for recrystallization was further evaluated; strontium ions could substitute for calcium, and to a lesser extent, cobalt, barium, manganese and magnesium ions also stimulated crystallization. On the other hand, nickel and cadmium provided only weak crystallization stimulation, and zinc, copper, iron, aluminum ions, and the monovalent potassium, sodium, and lithium ions were ineffective. The recrystallization could also be reproduced with Langmuir-Blodgett lipid monolayers at an air-water interface. As with the vesicle experiments, this was only successful when SLPS was incorporated into the lipid mix. The best method for RsaA preparation, leading to apparently monomeric protein that was stable for many months, was an extraction with a low pH aqueous solution. We also achieved recrystallization, albeit at lower efficiency, using RsaA protein solubilized by 8 M urea, a method which allows retrieval of protein from inclusions, when expressed as heterologous protein in Escherichia coli or when retrieved as shed, precipitated protein from certain mutant caulobacters. In summary, the clarification of recrystallization methods has confirmed the requirement of SLPS as a surface attachment component and suggests that its presence in a membrane-like structure greatly stimulates the extent and quality of S-layer formation. The in vitro approach allowed the demonstration that specific ions are capable of participating in crystallization and now provides an assay for the crystallization potential of modified S-layer proteins, whether they were produced in or can be secreted by caulobacters.     

Involvement of Two Specific Causes of Cell Mortality in Freeze-Thaw Cycles with Freezing to −196°C

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to −196°C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than −70°C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

离子液体1-丁基-3-甲基咪唑氯代盐对溶菌酶结晶的影响

以亲水性离子液体1-丁基-3-甲基咪唑氯盐(BmimCl)为添加剂,研究离子液体对溶菌酶结晶的影响.分别考察了离子液体对溶菌酶晶体数量与尺寸、晶体形貌及蛋白质纯度的影响,并探讨了离子液体对结晶过程影响的作用机制.离子液体通过增大溶菌酶的溶解度和其自身低蒸气压两种途径,降低了溶菌酶在结晶过程中的过饱和度,更有利于晶体的成核和生长,得到更好的结果.如避免多晶态现象的发生,增大晶体的尺寸,降低溶菌酶样品纯度的要求.X-射线衍射分析表明,离子液体未改变晶体的晶型结构,但可提高晶体的衍射分辨率.     

Influence of the Dispersed Particulate in Chocolate on Cocoa Butter Microstructure and Fat Crystal Growth during Storage

The dispersed particulates present in chocolate are shown to influence the microstructural development and fat crystal growth of cocoa butter (CB) during storage. Atomic force microscopy of both chocolate and CB showed that surface crystal growth on both materials was similar during isothermal (25 °C) storage over 4 weeks. However, unique micron-scale amorphous mounds also appeared on the surface of chocolate. With time, these regions increased in number and diameter and eventually solidified into clustered crystalline masses. X-Ray diffraction, solid fat content, and whiteness index measurements substantiated the observed crystal growth, with gradual increases in the proportion of the form VI polymorph, solid fat, and whiteness over time. Overall, these findings suggest that typical chocolate refining and tempering protocols result in a heterogeneously distributed particulate network that has a substantial impact on the morphology and crystallization pathway of the fat phase.     

Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum

The effects of temperature, solvents, and cultural conditions on the fermentative physiology of an ethanol-tolerant (56 g/liter at 60°C) and parent strain of Clostridium thermohydrosulfuricum were compared. An ethanol-tolerant mutant was selected by successive transfer of the parent strain into media with progressively higher ethanol concentrations. Physiological differences noted in the mutant included enhanced growth, tolerance to various solvents, and alterations in the substrate range and the fermentation end product ratio. Ethanol tolerance was temperature dependent in the mutant but not in the parent strain. The mutant grew with ethanol concentrations up to 8.0% (wt/vol) at 45°C, but only up to 3.3% (wt/vol) at 68°C. Low ethanol concentration (0.2 to 1.6% [wt/vol]) progressively inhibited the parent strain to where glucose was not fermented at 2.0% (wt/vol) ethanol. Both strains grew and produced alcohols on glucose complex medium at 60°C in the presence of either 5% methanol or acetone, and these solvents when added at low concentration stimulated fermentative metabolism. The mutant produced ethanol at high concentrations and displayed an ethanol/glucose ratio (mole/mole) of 1.0 in media where initial ethanol concentrations were ≤4.0% (wt/vol), whereas when ethanol concentration was changed from 0.1% to 1.6% (wt/vol), the ethanol/glucose ratio for the parent strain changed from 1.6 to 0.6. These data indicate that C. thermohydrosulfuricum strains are tolerant of solvents and that low ethanol tolerance is not a result of disruption of membrane fluidity or glycolytic enzyme activity.     

Involvement of two specific causes of cell mortality in freeze-thaw cycles with freezing to -196 degrees C

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to -196 degrees C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than -70 degrees C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.