Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.) Copyright 1999 John Wiley & Sons, Inc.     

Mitochondrial morphology and function impaired by dimethyl sulfoxide and dimethyl Formamide

In this work, the effects of two non-ionic, non-hydroxyl organic solvents, dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) on the morphology and function of isolated rat hepatic mitochondria were investigated and compared. Mitochondrial ultrastructures impaired by DMSO and DMF were clearly observed by transmission electron microscopy. Spectroscopic and polarographic results demonstrated that organic solvents induced mitochondrial swelling, enhanced the permeation to H⁺/K⁺, collapsed the potential inner mitochondrial membrane (IMM), and increased the IMM fluidity. Moreover, with organic solvents addition, the outer mitochondrial membrane (OMM) was broken, accompanied with the release of Cytochrome c, which could activate cell apoptosis signaling pathway. The role of DMSO and DMF in enhancing permeation or transient water pore formation in the mitochondrial phospholipid bilayer might be the main reason for the mitochondrial morphology and function impaired. Mitochondrial dysfunctions induced by the two organic solvents were dose-dependent, but the extents varied. Ethanol (EtOH) showed the highest potential damage on the mitochondrial morphology and functions, followed by DMF and DMSO.     

Surface functionalized electrospun biodegradable nanofibers for immobilization of bioactive molecules

A blend mixture of biodegradable poly(epsilon-caprolactone) (PCL) and poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol)-NH(2) (PLGA-b-PEG-NH(2)) block copolymer was electrospun to produce surface functionalized nanofibers. The resulting nanofibrous mesh with primary amine groups on the surface was applied for immobilization of biologically active molecules using lysozyme as a model enzyme. Lysozyme was immobilized via covalent conjugation by using a homobifunctional coupling agent. The nanofibrous mesh could immobilize a far greater amount of lysozyme on the surface with concomitantly increased activity, primarily due to its larger surface area, compared to that of the solvent casting film. It was also found that the enzyme immobilization process slightly altered thermal and pH-dependent catalytic activity profiles compared to those of native lysozyme. The results demonstrated the surface functionalized electrospun nanofibrous mesh could be used as a promising material for immobilizing a wide range of bioactive molecules.     

Application of electrospun poly(ethylene terephthalate) nanofiber mat to apple juice clarification

Electrospinning was used to produce self-supporting nanofibrous poly(ethylene terephthalate) membranes with good mechanical properties and straightforward handling. The application of this type of membranes in apple juice clarification process was investigated. Processing characteristics and quality parameters of apple juice were analyzed in order to compare the proposed method to traditional clarification techniques. In general, the apple juice obtained from electrospun nanofiber membrane filtration revealed physico-chemical characteristics comparable to those from juice clarified by ultrafiltration or by conventional clarification using filtering aids. Nevertheless, the new process showed a high flux performance and revealed to be much faster, simple and more economical than the traditional processes. This work demonstrated the filtration potential of an electrospun PET membrane thus introducing a new concept of clarification and opening new approaches for the juice processing industry or even for other food industry fields.     

Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions

Further utilization of chitosan nanofibrous membranes that are electrospun from chitosan solutions in trifluoroacetic acid (TFA) with or without dichloromethane (DCM) as the modifying cosolvent is limited by the loss of the fibrous structure as soon as the membranes are in contact with neutral or weak basic aqueous solutions due to complete dissolution of the membranes. Dissolution occurs as a result of the high solubility in these aqueous media of -NH(3)(+)CF(3)COO(-) salt residues that are formed when chitosan is dissolved in TFA. Traditional neutralization with a NaOH aqueous solution only maintained partial fibrous structure. Much improvement in the neutralization method was achieved with the saturated Na(2)CO(3) aqueous solution with an excess amount of Na(2)CO(3)(s) in the solution. We showed that electrospun chitosan nanofibrous membranes, after neutralization in the Na(2)CO(3) aqueous solution, could maintain its fibrous structure even after continuous submersion in phosphate buffer saline (pH = 7.4) or distilled water for 12 weeks.     

Chitosan-modified poly(acrylonitrile-co-acrylic acid) nanofibrous membranes for the immobilization of concanavalin A

Lectin affinity membranes have been receiving much attention for the separation and detection of various glycoconjugates. In this work, we present a simple and efficient method for the preparation of lectin affinity nanofibrous membranes. Chitosan-modified poly(acrylonitrile-co-acrylic acid) (PANCAA) nanofibrous membranes were first prepared by a coupling reaction between the primary amino groups of chitosan and the carboxyl groups of PANCAA electrospun membranes. Surface characterizations by attenuated total reflectance Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscopy (XPS) and field-emission scanning electron microscopy (FESEM) confirm the chemical and morphological changes of the studied nanofibrous membranes. Fluorescence-labeled concanavalin A (FL-Con A) was then immobilized on these membranes via noncovalent binding. Analyses by fluorescence spectrophotometer (FS) and confocal laser scanning microscopy (CLSM) reveal that the immobilization of Con A onto the modified nanofibrous membranes has been successfully achieved on the basis of the electrostatic interaction and the specific recognition between Con A and chitosan. The results show that the amount of adsorbed FL-Con A increases dramatically with the increasing coupling degree of chitosan (CDC) on the nanofibrous membrane. Moreover, Con A immobilized on the chitosan-modified nanofibrous membranes (CMNMs) can remain relatively stable at pH 5.3. Therefore, it is believed that this work may provide a new kind of material for affinity application.     

Surface modification of nanofibrous poly(acrylonitrile-co-acrylic acid) membrane with biomacromolecules for lipase immobilization

In this work, poly(acrylonitrile-co-acrylic acid) (PANCAA) was electrospun into nanofibers with a mean diameter of 180 nm. To create a biofriendly microenvironment for enzyme immobilization, collagen or protein hydrolysate from egg skin (ES) was respectively tethered on the prepared nanofibrous membranes in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiamine (EDC)/N-hydroxyl succinimide (NHS). Confocal laser scanning microscopy (CLSM) was used to verify the surface modification and protein density on the nanofibrous membranes. Lipase from Candida rugosa was then immobilized on the protein-modified nanofibrous membranes by covalent binding using glutaraldehyde (GA) as coupling agent, and on the nascent PANCAA nanofibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized enzyme were assayed. It was found that different pre-tethered biomacromolecules had distinct effects on the immobilized enzyme. The activity retention of the immobilized lipase on ES hydrolysate-modified nanofibrous membrane increased from 15.0% to 20.4% compared with that on the nascent one, while it was enhanced up to more than quadrupled (activity retention of 61.7%) on the collagen-modified nanofibrous membrane. The kinetic parameter, K_m and V_max, were also determined for the free and immobilized lipases. Furthermore, the stabilities of the immobilized lipases were obviously improved compared with the free one.     

Wound-dressing materials with antibacterial activity from electrospun polyurethane-dextran nanofiber mats containing ciprofloxacin HCl

Dextran is a versatile biomacromolecule for preparing electrospun nanofibrous membranes by blending with either water-soluble bioactive agents or hydrophobic biodegradable polymers for biomedical applications. In this study, an antibacterial electrospun scaffold was prepared by electrospinning of a solution composed of dextran, polyurethane (PU) and ciprofloxacin HCl (CipHCl) drug. The obtained nanofiber mats have good morphology. The mats were characterized by various analytical techniques. The interaction parameters between fibroblasts and the PU-dextran and PU-dextran-drug scaffolds such as viability, proliferation, and attachment were investigated. The results indicated that the cells interacted favorably with the scaffolds especially the drug-containing one. Moreover, the composite mat showed good bactericidal activity against both of Gram-positive and Gram-negative bacteria. Overall, our results conclude that the introduced scaffold might be an ideal biomaterial for wound dressing applications.     

Fabrication of gelatin/calcium phosphate composite nanofibrous membranes by biomimetic mineralization

Based on the principles of biomimetic mineralization, biocomposite nanofibrous membranes were fabricated by the growth of CaP crystals on electrospun gelatin nanofibers to mimic both the physical architecture and chemical composition of natural bone ECM. Plenty more CaP crystals formed on the nanofibrous membrane containing Ca(2+) ion precursors, in which these crystals were also observed on the inner side of membrane. The release rate of Ca(2+) ion precursors from the nanofibrous membrane was slower than that of PO(4)(3-) ion precursors, suggesting the existence of more strong intermolecular interaction between gelatin and Ca(2+) ions. ATR-FTIR and XRD results clearly revealed the formation of CaP crystals mixed with apatite and CaCO(3), or apatite and TCP on the membranes. The Ca/P molar ratio of crystals obtained from the XPS data was 2.03 and 1.60, which depended on the mineralization conditions. Higher amount of CaP crystals significantly accelerated the deposit rate of bone-like apatite on the surface of composite membrane, meaning to the improved in vivo bone bioactivity.     

溶剂稳定性蛋白酶产生菌的筛选和鉴定

自油污土样等样品中分离获得一株具有产胞外溶剂稳定性蛋白酶的耐有机溶剂极端微生物,经BIOLOG系统鉴定及16S rDNA序列(GenBank,EF105377)分析,该菌株为Bacillus licheniformis YP1。该菌株能耐受中浓度盐、强碱性环境(pH12)及多种不同浓度的有机溶剂,但对多种抗生素敏感。在YP1产蛋白酶发酵过程中添加各种有机溶剂结果表明,丙酮虽抑制了菌体生物量的生长却促进了单位菌体的蛋白酶分泌,而长链烷醇如辛醇、十二醇等能强烈抑制该蛋白酶的分泌。该菌株所产蛋白酶经11种50%(V/V)有机溶剂处理后均能保留高活力。该溶剂稳定性蛋白酶在有机相生物催化等领域具有良好的应用前景。     

Entrapment of l-tryptophan producing Escherichia coli in different matrices: activity of immobilized cells

Cells of Escherichia coli induced for l-tryptophan synthase [l-serine hydro-lyase (adding indole-glycerol-phosphate), EC 4.2.1.20] have been assayed in DMF and DMSO aqueous solvents as reaction medium. Up to 20% DMF/water, cells retained 90% of their tryptophan synthase activity. Concentrations of 20 mM indole, which did not inhibit this reactivity, could be reached with 5% DMF/water. Four matrices were compared for cell immobilization: polyacrylamide, foam particles of bovine seum albumin, alginate and κ-carrageenan. The best activity was retained with the latter matrix, and the preparations thus obtained allowed high productivity of l-tryptophan. Various systems of production of l-tryptophan with κ-carrageenan and DMF/water were studied.     

Enzymatic transesterification of purine nucleoside having a low solubility in organic medium

Enzymatic transesterification of guanosine having low solubility against organic solvent was examined. For the transesterification between guanosine and divinyl adipate catalyzed by alkaline protease from Bacillus (Bioprase), DMSO was added to DMF to increase the solublility of the nucleoside, and the conversion rate of guanosine to the vinyl guanosine ester was less than 30%. To overcome the reversible inactivation of enzyme by hydrophilic organic solvents, the reaction was carried out with 10% (v/v) water. The transesterification reaction was effectively catalyzed in DMF/DMSO in the presence of water and the conversion rate increased ca. 70% after 7 d reaction. The result shows that the water effect of Bioprase would be a useful method for the synthesis of low solublility nucleoside esters.     

On protein solubility in organic solvent

Solubility of a model protein, hen egg-white lysozyme, was investigated in a wide range of neat nonaqueous solvents and binary mixtures thereof. All solvents that are protic, very hydrophilic, and polar readily dissolve more than 10 mg/mL of lysozyme (lyophilized from aqueous solution of pH 6.0). Only a marginal correlation was found between the lysozyme solubility in a non-aqueous solvent and the letter's dielectric constant or Hildebrand solubility parameter, and no correlation was observed with the dipole moment. Lysozyme dissolved in dimethyl sulfoxide (DMSO) could be precipitated by adding protein nondissolving co-solvents, although the enzyme had a tendency to form supersaturated solutions in such mixtures. The solubility of lysozyme, both in an individual solvent (1,5-pentanediol) and in binary solvent mixtures (DMSO/acetonitrile), markedly increased when the pH of the enzyme aqueous solution prior to lyophilization was moved away from the proteins's isoelectric point. (c) 1994 John Wiley & Sons, Inc.     

The mechanism of the stabilization of the hexagonal II (HII) phase in phosphatidylethanolamine membranes in the presence of low concentrations of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO), a water-miscible organic solvent, has been used as a cryoprotectant for cells. It is known that DMSO stabilizes the HII phase of phosphatidylethanolamine (PE) membranes rather than the Lalpha phase, while most other water-miscible organic solvents such as acetone and ethanol destabilize the HII phase. To elucidate the mechanism for this stabilizing effect of DMSO on the HII phase, we have investigated its effects on the structures and physical properties of PE membranes. X-ray diffraction data indicated that dipalmitoleoylphosphatidylethanolamine (DPOPE) membranes in H2O at 20 degrees C were in the Lalpha phase and that an Lalpha to HII phase transition occurred at X=0.060 (mole fraction of DMSO) in water/DMSO mixtures. As the DMSO concentration increased, the basis vector length of the dioleoylphosphatidylethanolamine (DOPE)/ 16 wt% tetradecane membrane and also of the DPOPE/ 16 wt% tetradecane membrane in the HII phase decreased, suggesting that the spontaneous curvature of these membranes increased. We have also investigated the effects of DMSO on the physical properties of the PE membranes, and compared them with those of acetone. As the DMSO concentration increased, the excimer to monomer fluorescence intensities of pyrene-phosphatidylcholine in the PE membranes decreased, indicating that the membrane fluidity decreased, and also the generalized polarization value of the Laurdan fluorescent probe in the DPOPE membrane increased, indicating that the polarity of the membrane interface decreased. On the other hand, acetone had the opposite effects to DMSO. The interaction free energy between the membrane surface segments and solvent increased with an increase in DMSO concentration. It decreased the amount of solvent in the membrane interface, inducing an increase in the spontaneous curvature. This can reasonably explain the effects of DMSO on the phase stability and the physical properties of the membranes.     

Electrospinning of carboxymethyl chitin/poly(vinyl alcohol) nanofibrous scaffolds for tissue engineering applications

A novel fibrous membrane of carboxymethyl chitin (CMC)/poly(vinyl alcohol) (PVA) blend was successfully prepared by electrospinning technique. The concentration of CMC (7%) with PVA (8%) was optimized, blended in different ratios (0–100%) and electrospun to get nanofibers. Fibers were made water insoluble by chemical followed by thermal cross-linking. In vitro mineralization studies identified the ability of formation of hydroxyapatite deposits on the nanofibrous surfaces. Cytotoxicity of the nanofibrous scaffold was evaluated using human mesenchymal stem cells (hMSCs) by the MTT assays. The cell viability was not altered when these nanofibrous scaffolds were pre-washed with phosphate buffer containing saline (PBS) before seeding the cells. The SEM images also revealed that cells were able to attach and spread in the nanofibrous scaffolds. Thus our results indicate that the nanofibrous CMC/PVA scaffold supports cell adhesion/attachment and proliferation and hence this scaffold will be a promising candidate for tissue engineering applications.     

电纺技术在生物医学中的应用进展

电纺技术已经成为结合多组分化合物与织造技术的关键工具,可改变电纺丝材料的化学、物理和生物特性,使其与不同的应用环境相适应。通过电纺技术制作的功能化纳米电纺丝材料,在组织工程、创伤敷料、酶的固定化和药物（基因）载体等生物医学方面得到了广泛的应用。新型的电纺技术可以进一步优化纳米电纺丝的特性,如同轴电纺、二相电纺技术;电纺丝膜的修饰也为调控电纺丝的各向异性和多孔性提供了有效的方法。该文将概述功能化电纺丝的纺织技术及修饰方法在生物医学领域的研究与应用进展。     

Ion transport through dimethyl sulfoxide (DMSO) induced transient water pores in cell membranes

It is well known that dimethyl sulphoxide (DMSO) increases membrane permeability, which makes it widely used as a vehicle to facilitate drug delivery across biological membranes. However, the mechanism of how DMSO increases membrane permeability has not been well understood. Recently, molecular dynamics simulations have demonstrated that DMSO can induce water pores in biological membranes, but no direct experimental evidence is so far available to prove the simulation result. Using FluxOR Tl? influx assay and intracellular Ca2? imaging technique, we studied the effect of DMSO on Tl? and Ca2? permeation across cell membranes. Upon application of DMSO on CHO-K1 cell line, Tl? influx was transiently increased in a dose-dependent manner. The increase in Tl? permeability induced by DMSO was not changed in the presence of blockers for K? channel and Na?-K? ATPase, suggesting that Tl? permeates through transient water pores induced by DMSO to enter into the cell. In addition, Ca2? permeability was significantly increased upon application of DMSO, indicating that the transient water pores induced by DMSO were non-selective pores. Furthermore, similar results could be obtained from RAW264.7 macrophage cell line. Therefore, this study provided experimental evidence to support the prediction that DMSO can induce transient water pores in cell membranes, which in turn facilitates the transport of active substances across membranes.     

Electrospun water-soluble carboxyethyl chitosan/poly(vinyl alcohol) nanofibrous membrane as potential wound dressing for skin regeneration

Biocompatible carboxyethyl chitosan/poly(vinyl alcohol) (CECS/PVA) nanofibers were successfully prepared by electrospinning of aqueous CECS/PVA solution. The composite nanofibrous membranes were subjected to detailed analysis by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). SEM images showed that the morphology and diameter of the nanofibers were mainly affected by the weight ratio of CECS/PVA. XRD and DSC demonstrated that there was strong intermolecular hydrogen bonding between the molecules of CECS and PVA. The crystalline microstructure of the electrospun fibers was not well developed. The potential use of the CECS/PVA electrospun fiber mats as scaffolding materials for skin regeneration was evaluated in vitro using mouse fibroblasts (L929) as reference cell lines. Indirect cytotoxicity assessment of the fiber mats indicated that the CECS/PVA electrospun mat was nontoxic to the L929 cell. Cell culture results showed that fibrous mats were good in promoting the cell attachment and proliferation. This novel electrospun matrix would be used as potential wound dressing for skin regeneration.     

Process and economic evaluation for monoclonal antibody purification using a membrane-only process

In recent years, the market for therapeutic monoclonal antibodies (mAb) has grown exponentially, and with this there has been a desire to reduce the costs associated with production and purification of these high-value biological products. A typical mAb purification process involves three adsorption/chromatography steps [protein A, ion exchange (IEX), and hydrophobic interaction (HIC)], along with ultrafiltration, nanofiltration, and microfiltration. With the development of membrane adsorption/chromatography as a viable alternative to traditional pack bed systems, the opportunity exists to complete the entire downstream purification process using only membrane operations. In this study, the process simulation tool SuperPro Designer was used to evaluate the application of recently developed ultra-high capacity electrospun nanofibrous adsorption membranes as a replacement for conventional chromatographic media in the downstream mAb production process. The simulation showed that nanofibrous adsorption membranes in place of the three packed bed chromatography steps reduced the required volume of protein A, IEX, and HIC adsorptive medium by 25, 80, and 80%, respectively. In addition, the membrane-only process reduced the downstream processing time by 50%, decreased the number of labor hours associated with the purification steps by 40%, generated 40% less aqueous waste, and reduced the overall downstream process operating expenses per unit product by 23%. There were also significant savings in facility construction costs and the price of fixed equipment required for separations. With these savings not only is the membrane-only process economically competitive with the traditional packed bed operations, but it offers the possibility of moving toward more disposable process.     

Diffusion of sucrose and dextran through agar gel membranes

Mass transfer limitations severely impede the performance of bioreactions involving large molecules by gel-entrapped microorganisms. This paper describes a quantitative investigation of such diffusional limitations in agar gel membranes. Sucrose and commercial dextran fractions with (weight-average) molecular weights ranging from 10,000 to 2,000,000 Da were used as standard diffusants. For all tested solutes but sucrose, the values of the agar/water partition coefficients highlighted steric hindrance at the entrance of the membrane pores. The effective diffusivity of sucrose in agar was similar to that in water. All dextran fractions, however, displayed restricted diffusion in the agar membranes. Their effective diffusivities were a decreasing function of the agar content of the gel membrane (0.5, 1.0, or 1.5% w/v). The effective diffusivity in a given membrane decreased as the molecular weight of the diffusing molecule increased. T500 (ucbar|M_w = 470,000 Da) and ucbar|M_w = 1,950,000 Da) fractions were unable to diffuse through 1.0 or 1.5% agar membranes. The diffusion data did not agree with the classical (Renkin) model for a hard sphere diffusing through a cylindrical pore. These results are discussed in terms of gel and diffusant characteristics.