The effect of cosolvents on the formulation of nanoparticles from low-molecular-weight poly(l)lactide

The aim of this study was to formulate nanoparticles from poly(l)lactide by a modified nanoprecipitation method. The main focus was to study the effect of cosolvent selection on the shape, size, formation efficiency, degree of crystallinity, x-ray diffraction (XRD) reflection pattern, and zeta potential value of the particles. Low-molecular-weight (2000 g/mol) poly(l)lactide was used as a polymer, and sodium cromoglycate was used as a drug. Acetone, ethanol, and methanol were selected as cosolvents. Optimal nanoparticles were achieved with ethanol as a cosolvent, and the formation efficiency of the particles was also higher with ethanol as compared with acetone or methanol. The particles formulated by ethanol and acetone appeared round and smooth, while with methanol they were slightly angular. When the volume of the inner phase was decreased during the nanoprecipitation process, the mean particle size was also decreased with all the solvents, but the particles were more prone to aggregate. The XRD reflection pattern and the degree of crystallinity were more dependent on the amount of the solvents in the inner phase than on the properties of the individual cosolvents. The zeta potential values of all the particle batches were slightly negative, which partially explains the increased tendency toward particle aggregation.     

Cosolvent effects on gel-entrapped oxidoreductase: the glucose oxidase model

An intrinsic problem often involved in biotransformations carried out by immobilized cells is the poor solubility of substrate and product in water. Increase in hydrophobic substrate availability to such gel-entrapped cells may be attained by the replacement of a fraction of the aqueous medium by water-miscible solvents (cosolvents). The introduction of cosolvents results in increased solubility, but may simultaneously affect enzymic activity and stability. Recently, criteria and guidelines for cosolvent selection on the basis of its effect on intracellular enzyme stability were reported (Freeman, A., and Lilly, M.D. (1987) Appl. Microbiol. Biotechnol. 25, 495-501). In order to understand the impact of the preferable or unsuitable cosolvents on enzyme kinetics and stability, the effects of 1-5 M concentrations of a series of cosolvents (e.g., ethylene glycol, dimethylsulfoxide, N,N-dimethylformamide, ethanol) on a well-characterized, highly specific enzyme model (glucose oxidase) were investigated. The presence of 1-5 M of the cosolvents studied imposed 10-50% reduction in Vmax of the enzyme, but Km was only mildly affected (+/- 25%). This inhibition was attributed to cosolvent effect on small, reversible, conformational changes in the enzyme native structure. Determination of the rate constant of thermal inactivation (at 55 degrees C) of glucose oxidase, in the presence of cosolvents, was employed for the quantitative evaluation of cosolvent effect on enzyme stability. A clear pattern of cosolvent preference in respect to its denaturing effect was obtained, which was identical to the pattern previously observed in a study of oxidoreductases operating from within a whole cell. In both cases diols (e.g., ethylene glycol) were found to be the preferable group of cosolvents. Our results indicate that a soluble enzyme and an intracellular enzyme operating from a whole cell are affected by cosolvents via the same mechanism.     

Modeling and optimization of methanol as a cosolvent in Amoxicillin synthesis and its advantage over ethylene glycol

The production of semi-synthetic beta-lactam antibiotics such as Amoxicillin may be performed enzymatically using penicillin acylase under mild conditions. However, the thermodynamically favored hydrolysis of the antibiotic product and the acyl donor substrate needs to be minimized to use the kinetically controlled route. The addition of cosolvents such as ethylene glycol and methanol (the two best solvents identified so far for semi-synthetic beta-lactam antibiotics) can achieve this to some degree, but these additives also produce enzyme inhibition and deactivation. In this study, we compared ethylene glycol and methanol under various substrate conditions. Methanol gave a better synthesis to hydrolysis ratio, although its deactivating effects adversely affected production at lower cosolvent concentrations than ethylene glycol. This effect and its dependence on substrate concentration was further modeled and optimized. A few targets of optimization such as Amoxicllin level, the synthesis to hydrolysis ratio, or a combination, were employed. While maximum levels of Amoxicillin synthesis were achievable only at high substrate concentrations, improvements derived from cosolvents were most significant at low substrate concentrations.     

Efficient approach to design stable water-dispersible nanoparticles of hydrophobic cellulose esters

Commercially prepared cellulose acetate, cellulose acetate propionate, -butyrate, and -phthalate as well as cellulose acetates prepared in the laboratory scale with varying degree of substitution (DS) self-assemble into regular nanoparticles, ranging in size from 86 to 368 nm, by using two different techniques of nanoprecipitation. Dialysis of polymers dissolved in N,N-dimethylacetamide results in the formation of regular nanospheres whereas the preparation in acetone by successive adding of water leads to bean-shaped particles in the nanoscale. One criterion for nanoprecipitation is the existence of dilute polymer solutions. Furthermore, the formation of nanoparticles strongly depends on DS and distribution of the substituents. Concerning this issue, quantitative (13)C NMR spectroscopy was applied for detailed structure characterization of selected cellulose acetates. The stability of the nanoparticle suspensions in the physiological pH range was observed by zeta potential measurements.     

Nanoparticles of beta-cyclodextrin esters obtained by self-assembling of biotransesterified beta-cyclodextrins

The synthesis of decanoate beta-cyclodextrin esters (beta-CDd) and hexanoate beta-cyclodextrin esters (beta-CDh) was biocatalyzed by thermolysin from native beta-cyclodextrin (beta-CD) and vinyl hexanoate or vinyl decanoate used as acyl donors. The products were chemically characterized by infrared, NMR, and mass spectrometry. Both beta-CDd and beta-CDh esters were identified as a mixture of beta-CD preferentially substituted on the C2 position by the corresponding acyl chain. The degree of substitution varied from 2 to 7 for beta-CDd and from 4 to 8 for beta-CDh. The ability of beta-CD esters to self-organize into nanoparticles was tested using a nanoprecipitation technique in various solvents. The mean size diameter and polydispersity measured by quasi-elastic light scattering were dramatically affected by the nature of solvent (acetone, ethanol, or tetrahydrofuran) used in the nanoprecipitation technique. When directly observed using cryo-transmission electron microscopy, beta-CDh appeared as uniformly dense nanospheres, whereas beta-CDd exhibited a multilamellar onion-like organization. A structural model was rationalized for the beta-CDd nanoparticles.     

Micronization of Organic Materials by Crystallization with Compressed Gases

A pilot plant is presented, which has been built to prepare fine particles by the P recipitation with a C ompressed Fluid A ntisolvent (PCA process). This technique offers interesting applications for products, which are produced in small amounts, as certain pharmaceuticals or energetic materials. In this contribution the micronization of paracetamol and tartaric acid is presented. Liquid solutions of tartaric acid in acetone, ethanol and methanol/ethanol mixtures have been sprayed into supercritical carbon dioxide used as antisolvent. The mean particle size of the precipitated powder can be manipulated by changing the precipitation pressure and solvent type however the precipitation temperature has no significant influence on the particle size. Paracetamol is micronized from acetone, methanol and DMF and morphologies from needles to spheres were found depending on the solvent. The particle size was in the submicron range.     

葡萄籽中原花青素提取方法优化处理

溶剂提取法是提取葡萄籽原花青素的常用方法 ,然而 ,在不同提取条件下 ,提取效果并不一致。利用水、甲醇、乙醇、丙酮及它们的水溶液提取葡萄籽中的原花青素 ,而后用铁盐催化比色法测原花青素含量 ,考察了提取剂的浓度、粉碎度对提取的影响。在优化处理的基础上 ,获得了有效的提取条件 :葡萄籽粉碎度 10 0目 ,提取剂 70 %甲醇水溶液。     

Rosin microparticles as drug carriers: Influence of various solvents on the formation of particles and sustained-release of indomethacin

The aim of this study was to formulate a sustained release system for indomethacin (IND) with rosin gum obtained from a pine tree. Rosin microparticles were prepared by a dispersion and dialysis method without the addition of surfactant. In order to investigate the influence of solvents on the formation of colloidal microparitcles, various solvents like ethanol, DMF, DMAc, and acetone were used. The rosin microparticles containing IND were characterized by X-ray diffractometry (XRD) and differential scanning calorimetry (DSC). The morphologies of rosin microparticles observed by scanning electron microscopy (SEM) were spherical. The solvents used to dissolve rosin significantly affected the drug content and drug release rate of IND. The release behaviors of IND from the rosin microparticles were dependent on the drug content and size of the particles. Rosin microparticles with a higher drug content and of a larger particle size had a slower drug release rate. Also, the IND release rate from the rosin microparticles could be regulated by the rosin content in the microparticles. From these results, rosin microparticles have the potential of being used as a sustained release system of IND.     

真眼点藻类色素的提取与测定方法

分别采用甲醇、乙醇和丙酮3种有机溶剂提取7种真眼点藻的色素,比较3种有机溶剂提取色素的效果,测定3种有机溶剂色素提取液的吸收光谱,利用分光光度法计算藻的叶绿素a和类胡萝卜素的含量,并比较甲醇和乙醇色素提取液在A₄₇₀和A₆₆₆的最大吸收峰。结果表明:使用乙醇比甲醇和90%丙酮操作更简便、快捷并且毒害低。3种有机溶剂色素提取液的叶绿素a和类胡萝卜素的含量均无显著性差异(P>0.05),提取率基本一致。色素在3种有机溶剂中的吸收光谱相似,甲醇和乙醇的色素提取液在A₄₇₀和A₆₆₆的最大吸收峰并无显著性差异(P>0.05)。乙醇色素提取液可使用Lichtenthaler的公式计算色素含量。     

Water-induced precipitation of cholesterol dissolved in organic solvents in the absence and presence of surfactants and salts

The precipitation of cholesterol dissolved in organic solvents, viz. methanol, ethanol, n-propanol, isopropanol, acetone and 1,4-dioxane, by the addition of water has been studied. The effects of the solvents towards the precipitation follow the order: methanol greater than ethanol greater than acetone greater than dioxane greater than n-propanol greater than iso-propanol, the solvent dioxane however exhibits a change in the order at higher concentration. Additives like Triton X-100, sodium cholate, sodium deoxycholate, sodium dehydro cholate, sodium salicylate and sodium chloride have some protective action against precipitation, the maximum protection being that of Triton X-100. The additives have shown better protective action in propanols and dioxane than in methanol, ethanol and acetone. Analysis of solvent composition and dielectric constant has revealed specific solvent effects on the water-induced precipitation of cholesterol. Thermodynamic analysis of the precipitation phenomenon and the unique role of solvent structure on cholesterol precipitation has been discussed.     

Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis

The efficiency of pigment extraction forms the crux of the spectrophotometric analysis of chlorophyll a. The alcoholic solvents, methanol and ethanol, proved to be superior to acetone and acetone with DMSO. Homogenisation and sonication did not improve the extraction in the alcoholic solvents. Boiling at 100°C had an adverse effect whereas complete extraction of the pigments was obtained at the solvents boiling point and allowing the samples to stand for 24 h in the dark.     

The influence of organic solvents on estimates of genotoxicity and antigenotoxicity in the SOS chromotest

In this work, the toxicity and genotoxicity of organic solvents (acetone, carbon tetrachloride, dichloromethane, dimethylsulfoxide, ethanol, ether and methanol) were studied using the SOS chromotest. The influence of these solvents on the direct genotoxicity induced by the mutagens mitomycin C (MMC) and 4-nitroquinoline-1-oxide (4-NQO) were also investigated. None of the solvents were genotoxic in Escherichia coli PQ37. However, based on the inhibition of protein synthesis assessed by constitutive alkaline phosphatase activity, some solvents (carbon tetrachloride, dimethylsulfoxide, ethanol and ether) were toxic and incompatible with the SOS chromotest. Solvents that were neither toxic nor genotoxic to E. coli (acetone, dichloromethane and methanol) significantly reduced the genotoxicity of MMC and 4-NQO. When these solvents were used to dissolve vitamin E they increased the antigenotoxic activity of this compound, possibly through additive or synergistic effects. The relevance of these results is discussed in relation to antigenotoxic studies. These data indicate the need for careful selection of an appropriate diluent for the SOS chromotest since some solvents can modulate genotoxicity and antigenotoxicity.     

Compatibility of organic solvents with the Microscreen prophage-induction assay: solvent--mutagen interactions

The following solvents did not induce prophage lambda in the Escherichia coli WP2s(lambda) Microscreen assay: acetone, benzene, chloroform, ethanol, n-hexane, isopropanol, methanol, toluene, and a mixture of the three isomers of xylene. Dimethyl sulfoxide was genotoxic in the presence and absence of S9, and methylene chloride was weakly genotoxic in the presence of S9. The genotoxic potencies of 2-aminoanthracene and 2-nitrofluorene were reduced when dissolved in DMSO or methanol compared to their potencies when dissolved in acetone.     

Development and structure of a novel barrier membrane composed of drug-loaded poly(lactic-co-glycolic acid) particles for guided bone regeneration

A novel barrier membrane composed of poly(lactic-co-glycolic acid) particles loaded with dexamethasone (DEX) as a bioactive molecule was produced via a modified nanoprecipitation method without any mixing. The particle membranes had a bilayer structure: one side was smooth and had a compact surface that was connected to larger particles, while the opposite side was rough, porous and connected to smaller particles. Additionally, a cross-section of the particle membrane had a porous structure with nano and micro sized irregular pores. Process optimization revealed that NaCl concentration in the water phase, with acetone as solvent and water as a non-solvent, played critical roles in determining the properties of the particle membranes, such as DEX encapsulation efficiency, thickness and surface morphologies of the particle membranes. A novel barrier membrane containing DEX using polymer particle drug capture technology has been successfully developed.     

Influence of Drug Brittleness,Nanomilling Time,and Freeze-Drying on the Crystallinity of Poorly Water-Soluble Drugs and Its Implications for Solubility Enhancement

The aim of this study was to assess whether wet bead milling of dexamethasone and tacrolimus suspensions leads to a lower degree of crystallinity of nanocrystals, and if the degree of crystallinity affects the drug solubility, in addition to particle size. Powder X-ray diffraction (XRD) was used to determine the degree of crystallinity of the particles, which decreased during milling until reaching a plateau: the particles had ～79% degree of crystallinity after 5 h milling. Different milling times were required for the two drugs in order to reach their plateaux, 2 h for dexamethasone and 3 h for tacrolimus. These results could be explained with the brittleness of the drugs. Dexamethasone was more brittle than tacrolimus, with an apparent elastic modulus of 16 GPa compared to ～12 GPa of tacrolimus. Freeze-drying the nanosuspensions resulted in a reduction in the degree of crystallinity to ～35% for dexamethasone and to ～45% for tacrolimus in comparison to non-freeze-dried particles. Solubility studies were performed with a Sirius® inForm based on in situ UV/VIS spectroscopy. The reduced degree of crystallinity of nanocrystals after milling was responsible, in addition to the nanoparticle size, for the solubility increase. Indeed, while the smallest particle size (394 nm for dexamethasone and 240 nm for tacrolimus) did not always result in the highest increase in solubility (factor of 1.04 for dexamethasone and 1.3 with tacrolimus), the smallest degree of crystallinity was always characteristic of the maximum solubility obtained (factor of 1.15 for dexamethasone and 1.7 for tacrolimus).     

Synthesis of lovastatin with immobilized Candida rugosa lipase in organic solvents: Effects of reaction conditions on initial rates

Lipase from Candida rugosa immobilized on a nylon support has been used to synthesize lovastatin, a drug which lowers serum cholesterol levels, by the regioselective acylation of a diol lactone precursor with 2-methylbutyric acid in mixtures of organic solvents. Analogs of lovastatin having a different side chain were also obtained through this method by reacting the diol substrate with different carboxylic acids. The selection of reaction conditions that maximize the initial reaction rate is investigated. Since the diol substrate has very low solubility in non-polar solvents, reaction solvents consisting of mixtures of hexane with a different, more polar cosolvent are considered. For each of the cosolvent mixtures studied, the reaction rate is maximum for an intermediate percentage of cosolvent in hexane. With total concentrations of the diol lactone in the range 6.25-12.5 mM, maximum initial rates correspond approximately to those cosolvent concentrations that permit a complete solubilization of the substrate. At higher cosolvent concentrations, lower rates are obtained. When considering the same dissolved substrate concentration, the reaction rate was found to increase with increasing values of logP(mix) and decreasing values of the dielectric constant, when varying the composition of a binary solvent mixture. However, when comparing different cosolvents, no general trend with respect to these properties was observed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56:671-680, 1997.     

Role of protein-solvent interactions in refolding: effects of cosolvent additives on the renaturation of porcine pancreatic elastase at various pHs

The effects of cosolvent additives on the refolding of porcine pancreatic elastase were studied by comparing the enzymatic activity and the conformation of the enzyme renatured at various pHs with those of the native elastase under the same cosolvent and pH conditions. A lag period was observed before reaching the steady state of the hydrolysis of an amide substrate, and the lag period measured with the refolding enzyme was longer than that measured with the native elastase. Depending on the cosolvent studied (acetonitrile, dimethylsulfoxide, glycerol, methanol) there was or was not a dramatic increase in the duration of the lag period measured with the refolding enzyme, but not in the case of the native elastase. These results and additional kinetic data on inactivation of the enzyme demonstrated that dimethylsulfoxide, glycerol, and methanol enhance the stability of the intermediates able to refold into the native form, contrary to acetonitrile. In neither the case of the native enzyme nor that of the renatured enzyme, did the cosolvents modify the pK(app) of ionization of the amino acids that control enzymatic activity, indicating that they did not penetrate the core of the refolded elastase. Conversely, they shifted toward a more alkaline pH the structural transition of the native elastase, and the amplitude of the shift was comparable to that observed in bulk water with elastase whose Ser 195 has been acylated, suggesting that cosolvents stabilized the structure of the folded molecule by increasing its packing.     

Acetone preservation: a practical technique for molecular analysis

In attempts to establish a convenient and reliable method for field collection and archival preservation of insects and their endosymbiotic microorganisms for molecular analysis, acetone, ethanol, and other organic solvents were tested for DNA preservability of the pea aphid Acyrthosiphon pisum and its intracellular symbiotic bacterium Buchnera sp. After 6 months' storage, not only the band of high-molecular-size DNA but also the bands of rRNA were well preserved in acetone, ethanol, 2-propanol, diethyl ether and ethyl acetate. Polymerase chain reaction (PCR) assays confirmed that the DNA of both the insects and their symbionts was well preserved in these solvents. In contrast, methanol and chloroform showed poor DNA preservability. When water-containing series of acetone and ethanol were examined for DNA preservability, acetone was apparently more robust against water contamination than ethanol. Considering that most biological materials contain high amounts of water, acetone may be a more recommendable preservative for DNA analysis than ethanol which has been widely used for this purpose. The DNA of various insects could be preserved in acetone at room temperature in good condition for several years. In addition to the DNA of the host insects, the DNA of their endosymbionts, including Buchnera and other mycetocyte symbionts, Wolbachia, and gut bacteria, was amplified by PCR after several years of acetone storage. The RNA and protein of the pea aphid and its endosymbiont were also preserved for several years in acetone. After 2 years' storage in acetone, proteins of A. pisum could be analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and the endosymbiotic bacteria were successfully detected by immunohistochemistry and in situ hybridization on the tissue sections.     

烟草废料中绿原酸的提取工艺研究

讨论了从烟草废料中提取绿原酸的优势和甲醇、乙醇、丙酮、水等不同溶剂经超声波辅助提取绿原酸的效果：研究结果表明,用体积分数40％的甲醇得到的浸提液中,绿原酸质量浓度为2．11mg／mL,比以水为溶剂时高出近50％.不同浓度的甲醇溶液中,体积分数50％的甲醇提取绿原酸的浓度最高。对树脂的吸附动力学分析表明,大孔树脂CN-101对烟草浸提液中绿原酸的吸附遵循Freundlich等温方程,吸附和解析分离所得的绿原酸收率为87．6％.在超声辅助条件下,利用甲醇等有机溶剂提取烟草中的绿原酸,进而用大孔树脂进行吸附分离的方法可行。