Core-shell structured PEO-chitosan nanofibers by coaxial electrospinning

Core-shell structured PEO-chitosan nanofibers have been produced using a coaxial electrospinning setup. PEO and chitosan solutions, both in an aqueous acetic acid solvent, were used as the inner (core) and outer (shell) layer, respectively. Uniform-sized defect-free nanofibers of 150-190 nm diameter were produced. In addition, hollow nanofibers could be obtained subsequent to PEO washing of the membranes. The core-shell nanostructure and existence of chitosan on the shell layer were confirmed by TEM images obtained before and after washing the PEO content with water. The presence of chitosan on the surface of the composite nanofibers was further supported by XPS studies. The chitosan and PEO compositions in the nanofibrous mats were determined by TGA analysis, which were similar to their ratio in the feed solutions. The local compositional homogeneity of the membranes and the efficiency of the washing step to remove PEO were also verified by FTIR. In addition, DSC and XRD were used to characterize the crystalline structure and morphology of the co-electrospun nonwoven mats. The prepared coaxial nanofibers (hollow and solid) have several potential applications due to the presence of chitosan on their outer surfaces.     

Carbon nanotube reinforced Bombyx mori silk nanofibers by the electrospinning process

Nanocomposite fibers of Bombyx mori silk and single wall carbon nanotubes (SWNT) were produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun into nanofibers. The morphology, structure, and mechanical properties of the electrospun nanofibers were examined by field emission environmental scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and microtensile testing. TEM of the reinforced fibers shows that the single wall carbon nanotubes are embedded in the fibers. The mechanical properties of the SWNT reinforced fiber show an increase in Young's modulus up to 460% in comparison with the un-reinforced aligned fiber, but at the expense of the strength and strain to failure.     

ATP-dependent import of a lumenal protein by isolated thylakoid vesicles

The 33 kd protein of the photosynthetic oxygen-evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non-chloroplast proteins and precursors of stromal proteins are not imported.     

Counter-current distribution of sonicated inside-out thylakoid vesicles

Summary Inside-out thylakoid vesicles were isolated from spinach chloroplasts, and fragmented by sonication. Different fragments were separated by counter-current distribution and analyzed for chlorophyll and P700. The inside-out vesicles had a chlorophyll a/b ratio of 2.2–2.4 (original chloroplasts 2.8–3.0). After further fragmentation of the inside-out vesicles by sonication and separation by countercurrent distribution three populations of vesicles were obtained having chlorophyll a/b ratios of 1.7, 1.9 and 2.5 respectively. The P-700 was depleted in fractions with lower chlorophyll a/b ratio and was nearly absent in the fraction having a chlorophyll a/b ratio of 1.7 (chlorophyll/P700 > 4500 mol/mol). That PSII membrane vesicles, with such a low chlorophyll a/b ratio and lacking PSI, can be prepared by a non-detergent method provides strong support for the notion that PSI and PSII are segregated along the thylakoid membrane.A plot of P700 per chlorophyll against chlorophyll b/(a+b) fits a straight line connecting the pure PSI membrane (chlorophyll a/b = 6; P700/chlorophyll = 5.6 mmol/mol) with the pure PSII membrane (chlorophyll a/b = 1.7; P700 = 0). These two membranes can be considered as separate phases of a two-dimensional phase system. Models for the thylakoid membrane are discussed.Abbreviations PSI Photosystem I - PSII Photosystem II - PEG Polyethylene Glycol - P700 Reaction Center of PSI     

Protonmotive force and photophosphorylation in single swollen thylakoid vesicles

Swollen vesicles generally 40 micron in diameter were prepared from spinach chloroplasts. These vesicles appear to originate from thylakoids. The present study reports results obtained with individual vesicles using micromanipulative procedures. The electric potential across the membrane was measured with microelectrodes and the pH of the internal space was calculated from the fluorescence of the pH indicator pyranine. The individual vesicles photophosphorylate as measured with luciferin-luciferase. Impalement with microelectrodes did not affect the ability of individual vesicles to photophosphorylate. However, there was no significant membrane potential either with continuous illumination or light flashes. In contrast, we found a delta pH of 3.7 under photophosphorylative conditions and the incubation with the appropriate buffers blocked photophosphorylation presumably by preventing formation of a pH gradient. We propose that, in these vesicles, the membrane potential plays no role in photophosphorylation, whereas a pH gradient is obligatory.     

Composition of photosynthetic pigments in thylakoid membrane vesicles from spinach

Thylakoid membranes from spinach were fragmented mechanically and separated into vesicles originating from grana and stroma-exposed lamellae (Andreasson et al. (1988) Biochim Biophys Acta 936: 339–350). The grana vesicles were further fragmented and separated into smaller vesicles originating from different parts of the grana (Svensson and Albertsson (1989) Photosynth Res 20: 249–259). All vesicles so obtained were analyzed with respect to chlorophyll and carotenoid composition by reverse phase HPLC. For all fractions the following relations (mole/mole) were found: 1 carotenoid per 4 chlorophyll (a+b), 2 lutein per 5 chlorophyll b and 5 violaxanthin per 100 chlorophyll (a + b). The contents of lutein and neoxanthin were each linearly related to chlorophyll b and -carotene was linearly related to chlorophyll a.     

Preparation, characterization and biocompatibility of electrospinning heparin-modified silk fibroin nanofibers

In this study, the electrospun silk fibroin nanofibrous scaffolds were modified with heparin by grafting after plasma treatment and blending electrospinning. Morphology, microstructure, chemical composition and grafting efficiency of the heparin-modified silk fibroin nanofibrous scaffolds were characterized to evaluate the effect of modification by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectrometer (XPS). The results showed that the heparin was successfully introduced to the silk fibroin nanofibrous scaffolds by both the two kinds of modification, and there was a hydrogen bonding between the silk fibroin and heparin. Moreover, the hydrophilicity, O-containing groups and negative charge density of the heparin-modified scaffolds were enhanced. In vitro coagulation time tests showed that the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) of the heparin-modified scaffolds were much higher than those of the pure silk fibroin scaffolds. L929 fibroblasts and EVCs spread and proliferated better on the heparin-modified scaffolds than on the pure silk fibroin scaffolds. Macrophages, neutrophils and lymphocytes were not observed in the heparin-modified scaffolds, which indicated that the modified scaffolds could induce minor inflammation in vivo. The results indicated that the electrospun heparin-modified silk fibroin nanofibrous scaffolds could be considered as ideal candidates for tissue engineering scaffolds.     

Modified coaxial electrospinning for the preparation of high-quality ketoprofen-loaded cellulose acetate nanofibers

This study investigates the use of a modified coaxial electrospinning process in the production of drug-loaded cellulose acetate (CA) nanofibers. With CA employed as a filament-forming matrix and ketoprofen (KET) as an active pharmaceutical ingredient, modified coaxial processes using sheath fluids comprising only mixed solvents were undertaken. With a sheath-to-core flow rate ratio of 0.2:1, the nanofibers prepared from the coaxial process had a smaller average diameter, narrower size distribution, more uniform structures, and smoother surface morphologies than those generated from single fluid electrospinning. In addition, the coaxial fibers provided a better zero-order drug release profile. The use of a sheath solvent means that the core jet is subjected to electrical drawing for a longer period, facilitating homogeneous core jet solidification and retarding the formation of wrinkles on the surface of the nanofibers. This modified coaxial electrospinning protocol allows the systematic fabrication of functional polymer nanofibers with improved quality.     

Direct measurement of k channels in thylakoid membranes by incorporation of vesicles into planar lipid bilayers

Light-driven electron transfer reactions cause the active accumulation of protons inside thylakoids, yet at steady state the electrical potential difference across the thylakoid membrane is very small; therefore, there must be a flux of other ions to balance the charge that would otherwise be built up by the net movement of H⁺. This paper presents direct measurements of ion movements through channels in the thylakoid membrane. These were made possible by fusing thylakoid vesicles from spinach (Spinacia oleracea L.) into planar lipid bilayers, using techniques developed originally to study sarcoplasmic reticulum. No Mg²⁺ current was found, but voltage-dependent channels have been characterized, these being somewhat selective for K⁺ over Cl⁻. The data are consistent with a role for these channels in charge balance during light-driven H⁺ movements.     

Fabrication of silk sericin nanofibers from a silk sericin-hope cocoon with electrospinning method

In this study, silk sericin nanofibers from sericin hope-silkworm, whose cocoons consist almost exclusively of sericin were successfully prepared by electrospinning method. Scanning electron microscopy (SEM) was used to observe the morphology of the fibers. The effect of spinning conditions, including the concentration of sericin cocoon solution, acceleration voltage, spinning distance and flow rate on the fiber morphologies and the size distribution of sericin nanofibers were examined. The structure and physical properties were also observed by Fourier transform infrared (FT-IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TG). The optimum conditions for producing finely thinner fibrous sericin nanofibers without beads were the concentration of sericin solution above 6-8 wt%, acceleration voltage ranging from 25 to 32 kV, spinning distance above 9 cm, and flow rate above 0.06 cm min(-1). The mean diameter of as spun sericin fibers varied from 114 to 430 nm at the different spinning conditions. In the as-spun fibers, silk sericin was present in a random coil conformation, while after methanol treatment, the molecular structure of silk sericin was transformed into a β-sheet containing structure. Sericin hope nanofiber demonstrated thermal degradation at lower temperature than the sericin hope cocoon, which probably due to the randomly coiled rich structure of the sericin hope nanofiber.     

Immobilization of phospholipid vesicles and protein-lipid vesicles containing red cell membrane proteins on octyl derivatives of large-pore gels

For improved immobilization of phospholipid vesicles and protein-lipid vesicles (cf. Sandberg, M., Lundahl, P., Greijer, E. and Belew, M. (1987) Biochim. Biophys. Acta 924, 185-192) and for chromatographic experiments with vesicles containing membrane protein, we have prepared octyl sulfide derivatives of the large-pore gels Sephacryl S-1000 and Sepharose 2B with ligand concentrations up to 14 and 5 mumol/ml gel, respectively. The Sephacryl derivatives allowed higher flow rates, gave higher rates of adsorption and showed equally high or higher capacities than the Sepharose adsorbents. 'Small', 'medium' and 'large' vesicles of radii approx. 20, 50 and 100 nm showed distribution coefficients on Sephacryl S-1000 of 0.7, 0.5 and 0.05, respectively and could be immobilized on octyl sulfide-Sephacryl S-1000 in amounts corresponding to 110, 40 and 20 mumol of phospholipids per ml gel, respectively. 'Small' vesicles became absorbed onto this gel at a rate of 1.5 mumol of phospholipids per min per ml gel until 60 mumol of phospholipids had become immobilized, whereas the initial adsorption rate was about 0.4 mumol.min-1.ml-1 on octyl sulfide-Sepharose 4B (see reference above) and on octyl sulfide-Sepharose 2B. Lower ligand concentrations gave lower capacities for 'small' vesicles. When vesicles entrapping calcein were immobilized on octyl sulfide-Sephacryl S-1000 some calcein was released during the adsorption process. For 'small' and 'medium' vesicles, respectively, the leakage was 75 and 25% at a ligand concentration of 14 mumol/ml but only 3 and 2% at 5 mumol/ml. The internal volumes of immobilized 'small' and 'medium' vesicles were estimated at 0.97 and 2.9 microliters per mumol of phospholipid by determination of entrapped calcein, which could indicate vesicle radii 20 and 50 nm, respectively. The total volumes of immobilized 'medium' lipid vesicles and 'medium' protein-lipid vesicles containing integral membrane proteins from human red cells, were estimated at 2.9 and 2.0 microliters/mumol, respectively, by chromatography of D- and L-[14C]glucose and calcein on the octyl sulfide-Sephacryl S-1000 column before and after immobilization. These volumes are roughly consistent with the internal volume of the vesicles. A zone of D-glucose eluted 90 microliters later than a zone of L-glucose on a 4- or 5-ml column of octyl sulfide-Sephacryl S-1000 with immobilized 'medium' protein-lipid vesicles containing the glucose transporter from human red cells, probably since part of the internal vesicle volume was accessible to the D-glucose but not to the L-glucose. This indicates that the glucose transporter was active in the immobilized vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Poly(vinyl alcohol) nanofibers by electrospinning as a protein delivery system and the retardation of enzyme release by additional polymer coatings

Protein-loaded (bovine serum albumin (BSA) or luciferase) poly(vinyl alcohol) (PVA) nanofibers were obtained by electrospinning. Poly(p-xylylene) (PPX, also coined as parylene) coated PVA/BSA nanofibers were prepared by chemical vapor deposition (CVD). The release of BSA from PVA nanofibers under physiological conditions was monitored by absorption spectroscopy. Burst release of BSA was noted with uncoated PVA nanofibers. In contrast, PPX-coated nanofibers exhibited a significantly retarded release of BSA depending on the coating thickness of PPX (ranging from 40 to 300 nm). Luciferase was used here as model enzyme, which after electrospinning retained its enzyme activity. This preservation of enzyme activity and the continuous release of the intact enzyme from the immersed fibers meets a fundamental prerequisite for the application of enzymes or other sensitive agents released from electrospun nanofibers under physiological conditions.     

Novel multilayered lipid vesicles: comparison of physical characteristics of multilamellar liposomes and stable plurilamellar vesicles

The preparation of a new kind of multilayered liposome, called a stable plurilamellar vesicle (SPLV), is described. Although SPLVs and classical multilamellar vesicles (MLVs) are made of the same materials and appear overtly similar in the electron microscope, the two types of vesicles differ as determined by stability, entrapment efficiency, electron spin resonance (ESR), NMR, X-ray diffraction, and biological effects. It is demonstrated that, contrary to what has been assumed, classical MLVs exclude solutes during their formation and, thus, are under a state of osmotic compression. By contrast, the SPLV process produces liposomes that are not compressed. The effects of osmotic compression are discussed. It is suggested that the state of osmotic stress is an important variable that distinguishes various types of liposomes and that has significant physical and biological consequences.     

Preparation of poly(ε-caprolactone)/poly(trimethylene carbonate) blend nanofibers by electrospinning

Poly(ε-caprolactone) (PCL)/poly(trimethylene carbonate) (PTMC) blend nanofibers have been prepared for the first time using an electrospinning process. The mixed dichloromethane (DCM) and N,N-dimethylformamide (DMF) (75/25, v/v) was found to be the most suitable solvent for electrospinning. Various blends of PCL/PTMC solutions were investigated for the formation of nano-scale fibers and it was found that the average diameter of the fibers was reduced and the morphology became finer when PTMC content was increased. FT-IR and DSC analysis indicated that the molecular interactions between PCL and PTMC were weak and they were phase-separated in the fibers. Due to the biocompatible properties of PCL and PTMC, the spun nanofibers developed here could have applications in the biomedical field.     

A calorimetric examinations of stable and fusing lipid bilayer vesicles

Mixed lipid samples containing dimyristoylglycerophosphocholine and small amounts of myristic acid were examined calorimetrically. Examination of multilamellar and small vesicle samples indicated that upon heating small vesicles combine to form more extended structures. An exothermic peak (at 19.5°C) can be associated with the structural transformation. The enthalpy for this process, which may be interpreted as vesicle-vesicle fusion, is found to be approx. -2 kcal/mol.     

Transverse heterogeneity in lipid fluidity in spinach thylakoids,photosystem II preparations,and thylakoid galactolipid vesicles

We have used three doxyl stearic acid spin labels to study the transverse hetero-geneity in lipid fluidity in thylakoids, photosystem II (PS II) preparations, and thylakoid galactolipid vesicles. This comparative study shows that spin labels incorporated into the membrane of the PS II preparation experience far more immobilization than do the same spin labels incorporated into either thylakoids or vesicles prepared from the polar lipids extracted from thylakoids. The spin label immobilization found in the PS II preparation is manifest even near the center of the bilayer, where lipid mobility is normally at its maximum. Analysis of the lipid content of the PS II preparation, relative to chlorophyll, suggests that the PS II preparation may be lipid depleted. This lipid depletion could explain the results presented. However, electron microscopy [Dunahay et al. (1984) Biochim. Biophys. Acta 764:179–193] has not indicated that major delipidation has occurred, and so it remains possible that the immobilization found in the PS II preparation is due primarily to the normal (but close) juxtaposition of adjacent PS II complexes and the cooperative immobilization of their surrounding lipids. Based on the results presented, we conclude that highly mobile lipids are not required for oxygen evolution, the primary photochemistry or the secondary reduction of exogenously added quinones. Unfortunately, the relationship between the plastoquinone pool and the fluidity of the membrane in the PS II preparation remains ambiguous.Abbreviations PS II photosystem II - SDSA 5-doxylstearic acid - 12DSA 12-doxylstearic acid - 16DSA 16-doxylstearic acid - 7N14 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl - chromium oxalate potassium trioxalatochromiate - EPR electron paramagnetic resonance - Chl chlorophyll - MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol     

Incorporation of PEG-lipids in arsonoliposomes results in formation of highly stable arsenic-containing vesicles

We investigated the effect of pegylation on the physical stability, morphology and membrane integrity of arsonoliposomes. Arsonoliposomes composed of distearoylglycerophosphocholine (DSPC), cholesterol (Chol) and the palmitoyl side chain arsonolipid (with concentrations ranging from 0 mol% [DSPC/Chol vesicles] to 53 mol% of total lipid) containing either 4 or 8 mol% DPPE-PEG2000 or DSPE-PEG2000, were prepared by sonication. Arsonoliposome membrane integrity was evaluated by measuring the retention of encapsulated calcein in vesicles (during incubation in buffer or fetal calf serum [FCS]) while physical stability was evaluated by measuring vesicle dispersion turbidity (during incubation in water or CaCl(2)). Vesicle morphology was studied by cryo-electron microscopy. Experimental results show that: (i) PEG-lipids are incorporated in arsonoliposomes (as confirmed by the vesicle zeta potential modulation), (ii) pegylation of arsonoliposomes prevents their aggregation and fusion in the presence of calcium ions and (iii) when 8 mol% of PEG-DSPE is incorporated in arsonoliposomes based on their arsonolipid content, two groups of pegylated vesicles are formed: low content arsonoliposomes (<20 mol% arsonolipid) which are highly leaky and high content arsonoliposomes (>27 mol% arsonolipid) which are highly stable (70% calcein retention after 24h incubation in fetal calf serum [FCS]). In addition to high membrane integrity, the high content pegylated arsonoliposomes are morphologically perfect round-shaped vesicles without the sharp edges typically observed with non-pegylated DSPC-containing arsonoliposomes.     

EDTA-induced release of manganese and proteins from inside-out thylakoid vesicles and the inhibition of oxygen evolution

Washing of inside-out, but not right-way-round, pea chloroplast thylakoid vesicles with 2 mM EDTA inhibits O2 evolution. Artificial electron donor/acceptor studies indicate that the site of inhibition is on the oxidising side of photosystem two (PS2), a conclusion reinforced by chlorophyll fluorescence measurements. Evidence is presented that the EDTA inhibition of O2 evolution is linked partly to the removal of one Mn atom per PS2 reaction centre and partly to the removal of extrinsic membrane proteins having apparent molecular weights between 58 and 70 kdaltons.     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.