Aggregation of phospholipid vesicles by water-soluble polymers.

Water-soluble polymers such as dextran and polyethylene glycol are known to induce aggregation and size growth of phospholipid vesicles. The present study addresses the dependence of these processes on vesicle size and concentration, polymer molecular weight, temperature, and compartmentalization of the vesicles and polymers, using static and dynamic light scattering. Increasing the molecular weight of the polymers resulted in a reduction of the concentration of polymer needed for induction of aggregation of small unilamellar vesicles. The aggregation was fully reversible (by dilution), within a few seconds, up to a polymer concentration of at least 20 wt %. At relatively low phosphatidylcholine (PC) concentrations (up to approximately 1 mM), increasing the PC concentration resulted in faster kinetics of aggregation and reduced the threshold concentration of polymer required for rapid aggregation (CA). At higher PC concentrations, CA was only slightly dependent on the concentration of PC and was approximately equal to the overlapping concentration of the polymer (C*). The extent of aggregation was similar at 37 and 4 degrees C. Aggregation of large unilamellar vesicles required a lower polymer concentration, probably because aggregation occurs in a secondary minimum (without surface contact). In contrast to experiments in which the polymers were added directly to the vesicles, dialysis of the vesicles against polymer-containing solutions did not induce aggregation. Based on this result, it appears that exclusion of polymer from the hydration sphere of vesicles and the consequent depletion of polymer molecules from clusters of aggregated vesicles play the central role in the induction of reversible vesicle aggregation. The results of all the other experiments are consistent with this conclusion.     

Self-assembled pH-responsive hydrogels composed of the RATEA16 peptide

Peptide RATEA16 spontaneously self-assembled into higher-order nanofiber hydrogels with extremely high water content (>99.5% (wt/vol)) under physiological condition. The hydrogels could undergo pH-reversible transitions from viscous solution to elastic hydrogel and to precipitate. The supramolecular self-assembly and the three phase transitions are driven by hydrophobic interactions, intermolecular hydrogen bonds, and a combination of attractive or repulsive electrostatic interactions. These hydrogels are rich in beta-sheet nanofibers, as demonstrated by CD and FTIR data. Rheological measurements reveal that the viscoelasticity of the material can be tuned by environmental pH and peptide concentration. The storage modulus of the hydrogels increases with increasing peptide concentration, and the self-assembled hydrogels are able to recover from mechanical breakdowns. AFM images show that the elasticity is attributed to a network nanostructure consisting of fibrous self-assemblies. The hydrogels are promising for a variety of possible biomedical applications, including drug delivery.     

Regulation of mitogenesis by water-soluble phospholipid intermediates.

Many recent observations implicate choline and ethanolamine kinases as well as phosphatidylcholine-specific phospholipase C in the regulation of mitogenesis and carcinogenesis. For example, human cancers generally contain high concentrations of phosphoethanolamine and phosphocholine, and in different cell lines various growth factors, cytokines, oncogenes and chemical carcinogens were all shown to stimulate the formation of phosphocholine and phosphoethanolamine. In addition, other reports have appeared showing that both extracellular and intracellular phosphocholine as well as ethanolamine and its derivatives can regulate cell growth. This area of research has clearly arrived at a stage when it becomes important to examine critically the feasibility of water-soluble phospholipid intermediates serving as potential regulators of cell growth in vivo. Accordingly, the goal of this review is to summarise available information relating to the formation and mitogenic actions of intracellular and extracellular phosphocholine as well as ethanolamine and its derivatives.     

Enzyme purification with aqueous two-phase systems: comparison between systems composed of pure polymers and systems composed of crude polymers

The main drawback when using aqueous two-phase systems for macromolecule purification is the high cost of most polymers used. The purification of an enzyme, alcohol dehydrogenase, from a crude extract of Saccharomyces cerevisiae was tested in systems composed of poly(ethylene glycol) and a crude hydroxypropyl starch or Reppal PES 100, a purified fraction of hydroxypropyl starch. Purification factors measured for the enzyme were very similar in both systems (between 0.8 and 1.4 for both systems in the upper phase). However, systems composed of Reppal PES present a greater recovery of enzyme, between 77% and 100% versus 60% and 100%, while systems composed of crude hydroxypropyl starch exhibit a larger Δlog K for the tested ligand, 1.26 versus 0.81.     

Controlled release mechanisms of spontaneously forming unilamellar vesicles

Spontaneously forming small unilamellar vesicles (SULVs) are easy to prepare and show great promise for use in delivering therapeutic payloads. We report of SULVs made up of the ternary phospholipid mixture, dimyristoyl-phosphatidylcholine (DMPC), dihexanoyl-phosphatidylcholine (DHPC) and dimyristoyl-phosphatidylglycerol (DMPG), which have been characterized by small angle neutron scattering (SANS). These low-polydispersity (0.14-0.19) SULVs range in size (i.e., radius) from 110 to 215 A and are capable of entrapping, and subsequently releasing, hydrophilic molecules (e.g., fluorescent dyes and quenchers) in a controlled fashion over two different temperature ranges. The low-temperature release mechanism involves the SULVs transforming into discoidal micelles, with an onset temperature (T(o)) of ~32 degrees C, while the high-temperature release mechanism is more gradual, presumably the result of defects formed through the continuous dissolution of DHPC into solution. Both of these mechanisms differ from other, previously reported thermosensitive liposomes.     

Controlled release mechanisms of spontaneously forming unilamellar vesicles

Spontaneously forming small unilamellar vesicles (SULVs) are easy to prepare and show great promise for use in delivering therapeutic payloads. We report of SULVs made up of the ternary phospholipid mixture, dimyristoyl-phosphatidylcholine (DMPC), dihexanoyl-phosphatidylcholine (DHPC) and dimyristoyl-phosphatidylglycerol (DMPG), which have been characterized by small angle neutron scattering (SANS). These low-polydispersity (0.14-0.19) SULVs range in size (i.e., radius) from 110 to 215 Å and are capable of entrapping, and subsequently releasing, hydrophilic molecules (e.g., fluorescent dyes and quenchers) in a controlled fashion over two different temperature ranges. The low-temperature release mechanism involves the SULVs transforming into discoidal micelles, with an onset temperature (T_o) of ~ 32 °C, while the high-temperature release mechanism is more gradual, presumably the result of defects formed through the continuous dissolution of DHPC into solution. Both of these mechanisms differ from other, previously reported thermosensitive liposomes.     

Novel living cell sheet harvest system composed of thermoreversible methylcellulose hydrogels

In this study, a novel yet simple method, using a thermoreversible hydrogel system coated on tissue culture polystyrene (TCPS) dishes, was developed for harvesting living cell sheets. The hydrogel system was prepared by simply pouring aqueous methylcellulose (MC) solutions blended with distinct salts on TCPS dishes at 20 degrees C. For the applications to cell culture, only those aqueous MC compositions that may form a gel at 37 degrees C were chosen for the study. It was found that the hydrogel coating composed of 8% MC blended with 10 g/L PBS (phosphate buffered saline) (the MC/PBS hydrogel, with a gelation temperature of approximately 25 degrees C) stayed intact throughout the entire course of cell culture. To improve cell attachments, the MC/PBS hydrogel at 37 degrees C was evenly spread with a neutral aqueous collagen at 4 degrees C. The spread aqueous collagen gradually reconstituted with time and thus formed a thin layer of collagen (the MC/PBS/collagen hydrogel). After cells reached confluence, a continuous monolayer cell sheet formed on the surface of the MC/PBS/collagen hydrogel. When the grown cell sheet was placed outside of the incubator at 20 degrees C, it detached gradually from the surface of the thermoreversible hydrogel spontaneously, without treating with any enzymes. The results obtained in the MTT assay demonstrated that the cells cultured on the surface of the MC/PBS/collagen hydrogel had an even better activity than those cultured on an uncoated TCPS dish. After harvesting the detached cell sheet, the remaining viscous hydrogel system is reusable. Additionally, the developed hydrogel system can be used for culturing a multilayer cell sheet. The obtained living cell sheets may be used for tissue reconstructions.     

Highly stable vesicles composed of a new chain-terminus acetylenic photopolymeric phospholipid

Stability, ease of production, and storage convenience were addressed for polymerized vesicles composed of 1,2-bis(trideca-12-ynoyl)-sn-glycero-3-phosphocholine. The following vesicle properties were investigated before and after polymerization: size, shape, lamellarity, dispersity, degree of polymerization, membrane fluidity, and structural stability. A fairly monodisperse, unilamellar sub-micron vesicle suspension undergoes nearly complete polymerization of the chain-terminus acetylenic to polyacetylenic conversion as monitored by Fourier transform infrared spectroscopy. (1)H nuclear magnetic resonance spectroscopy and thin layer chromatography provide additional evidence for extensive lipid polymerization. Using differential scanning calorimetry, a gel/liquid transition was not observed for either polymerized or non-polymerized vesicles within the temperature range of 5-65 degrees C. These polymerized vesicles remained structurally stable and suspended for months at room temperature. However, vesicle size did decrease with increasing degree of polymerization. Polymerized vesicles remained spherical but decreased in size by 15% when subjected to 52 wt.% aqueous ethanol and did not change significantly in size and dispersity after a freeze-dry/resuspend cycle.     

Complex formation of protein with different water-soluble synthetic polymers

The formation of protein-polymer complexes was studied in an aqueous system using dynamic light scattering (DLS) and static light scattering (SLS) as the main experimental tools. Human serum albumin (HSA) was used as a protein and complexed with four representative water-soluble polymers: poly(N-isopropylacrylamide) (PNIPA), poly(ethylene glycol) (PEG), poly(vinyl pyrrolidone) (PVP), and poly(vinyl alcohol) (PVA). The first three molecular weights were within 420,000-540,000 and the last one was 270,000. The complexation was performed at 25 degrees C in 0.01 M NaCl solution adjusted to pH 3 with HCl as a function of mixing ratio (rm; molar ratio of polymer to HSA). From SLS experiments, we determined the molecular weight of the resulting complexes, from the value of which the number (nb) of bound proteins per polymer was estimated. It was found that each polymer forms an intrapolymer complex over a wide range of rm (1.2 > or = rm > or = 0.01). Then, a marked decrease in nb with increasing rm was found. Over the whole rm range, the HSA-PNIPA complex exhibited a large nb value, as compared with the other three complexes whose nb values at the same rm were close to one another. Both the hydrodynamic radius (Rh) by DLS and the radius of gyration (Rg) by SLS for the complexes of PNIPA, PVP, and PVA decreased and then reached a constant value as nb decreased with increasing rm. In the PEG system, however, there were a few changes in Rh and Rg with nb. The Rg/Rh ratio, as an indication of chain expansion, was found to increase with decreasing nb in the PNIPA system. The complexes of PVA and PVP displayed a similar tendency, although the magnitude of the increasing trend was smaller than that of the PNIPA complex. In contrast, the Rg/Rh ratio of the PEG complex hardly varied depending on nb. These results were discussed in connection with the differences of physicochemical properties among four water-soluble polymers.     

Effects of diacylglycerol on the structure and phase behaviour of non-bilayer forming phospholipid

The phase behaviour of mixed aqueous dispersions of the monomethyl derivative of dioleoylphosphatidylethanolamine and dipalmitoylglycerol has been characterised by X-ray diffraction, differential scanning calorimetry and freeze-fracture electron microscopy for mixtures containing dipalmitoylglycerol in the concentration range 0-20 mol%. Dispersions prepared at temperatures where the phospholipid exhibits a liquid-crystalline lamellar phase show that dipalmitoylglycerol is completely phase separated into aggregates of stable crystal phase (beta'-phase). Heating mixed dispersions results in transformation of lamellar into hexagonal-II structure commencing at approximately 45 degrees C. This temperature coincides with a disappearance of beta'-phase of DPG which becomes incorporated into hexagonal-II phase. The pure phospholipid is transformed upon cooling from hexagonal-II into characteristic cubic phases; the formation of cubic phase is prevented by the presence of dipalmitoylglycerol and mixed dispersions initially form a lamellar liquid-crystalline phase in which the lipids are phase separated. The X-ray and thermal data suggest that relatively small domains of metastable crystal phase (alpha-phase) of DPG form initially on cooling and these subsequently coalesce and transform to beta'-phase.     

Interaction of water-soluble form of apoproteolipid of bovine brain myelin with phospholipid vesicles

The water-soluble form of apoproteolipid (APL) from bovine brain myelin was found to bind with phosphatidylcholine (PC)/phosphatidylethanolamine (PE) (6:4) vesicles below pH 5. The protein bound to vesicles was photoactively labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I)TID) and was digested with trypsin. A [125I]TID-labeled fragment with an apparent molecular weight of approximately 2,500 was extracted. An APL fragment with an identical Mr value was also obtained from the tryptic digest of APL/vesicle complex without prior labeling with [125I]TID. Determination of amino acid composition and the identification of the N-terminal amino acid residue of this unlabeled fragment showed that this protected segment covers the amino acid residues from Met-205 to Lys-228. In another experiment, the [125I]TID-labeled APL obtained from the above experiment without the proteolysis step was extracted and reconstituted into PC vesicles. Subsequent tryptic digestion of the exposed segment and comparison of the elution profile of the extracted polypeptides on a Sephadex LH-60 column with the published profile of these polypeptides indicated that the membrane-inserted segment of the water-soluble form of APL when bound to vesicles is the C-terminal region of this apoprotein within the amino acid residues between Met-205 and Lys-268.     

Biodegradable polymers from renewable sources: rheological characterization of hemicellulose-based hydrogels

Hemicellulose-based hydrogels were prepared by radical polymerization of 2-hydroxyethyl methacrylate or poly(ethylene glycol) dimethacrylate with oligomeric hydrosoluble hemicellulose modified with well-defined amounts of methacrylic functions. The polymerization reaction was carried out in water at 40 degrees C using a redox initiator system. The hydrogels were in general elastic, soft, and easily swellable in water. Their viscoelastic properties were determined by oscillatory shear measurements on 2 mm thick hydrogels under a slight compression to avoid slip, over the frequency range 10(-1) to 10(2). The rheological characterization indicated that the elastic response of the hydrogels was stronger than the viscous response, leading to the conclusion that the hydrogel systems displayed a predominantly solid-like behavior. The curves showed an increase in shear storage modulus with increasing cross-linking density. The nature of the synthetic comonomer in the hemicellulose-based hydrogels also influenced the shear storage modulus. Comparison of hemicellulose-based hydrogels with pure poly(2-hydroxyethyl methacrylate) hydrogels showed that their behaviors were rather similar, demonstrating that the synthetic procedure made it possible to prepare hemicellulose-based hydrogels with properties similar to those of pure poly(2-hydroxyethyl methacrylate) hydrogels.     

Dissociation of aggregated ferroheme complexes and protoporphyrine IX by water-soluble polymers

THe ferroheme · pyridine complex, ferroheme and protoporphyrin IX form the aggregates by the hydrophobic interaction in aqueous solutions. We found by spectrophotometric and fluorometric measurements that the aggregates dissociated into the monomers by the addition of water-soluble polymers, such as, poly(ethyleneoxide), poly(vinylalcohol), poly(vinylpyrrolidone) and poly(styrene sulfonate). The dissociation by the polymers proceeded as their hydrophobicities increased. The aggregated ferroheme was effectively dissociated by the copolymers of 4-vinylpyridine which were water-soluble polymer-ligands.     

Characterization of Miscanthus cell wall polymers

Efficient utilization of lignocellulosic Miscanthus biomass for the production of biochemicals, such as ethanol, is challenging due to its recalcitrance, which is influenced by the individual plant cell wall polymers and their interactions. Lignocellulosic biomass composition differs depending on several factors, such as plant age, harvest date, organ type, and genotype. Here, four selected Miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus, Miscanthus × giganteus, Miscanthus sinensis × Miscanthus sacchariflorus hybrid) were grown and harvested, separated into stems and leaves, and characterized for their non‐starch polysaccharide composition and structures, lignin contents and structures, and hydroxycinnamate profiles (monomers and ferulic acid dehydrodimers). Polysaccharides of all genotypes are mainly composed of cellulose and low‐substituted arabinoxylans. Ratios of hemicelluloses to cellulose were comparable, with the exception of Miscanthus sinensis that showed a higher hemicellulose/cellulose ratio. Lignin contents of Miscanthus stems were higher than those of Miscanthus leaves. Considering the same organs, the four genotypes did not differ in their Klason lignin contents, but Miscanthus × giganteus showed the highest acetylbromide soluble lignin content. Lignin polymers isolated from stems varied in their S/G ratios and linkage type distributions across genotypes. p‐Coumaric acid was the most abundant ester‐bound hydroxycinnamte monomer in all samples. Ferulic acid dehydrodimers were analyzed as cell wall cross‐links, with 8‐5‐coupled diferulic acid being the main dimer, followed by 8‐O‐4‐, and 5‐5‐diferulic acid. Contents of p‐coumaric acid, ferulic acid, and ferulic acid dimers varied depending on genotype and organ type. The largest amount of cell wall cross‐links was analyzed for Miscanthus sinensis.     

The optical activity and circular dichroic spectra of diacetylenic phospholipid polymers

Phospholipids (phosphatidylcholines) which contain a diacetylene group in a single acyl chain and within both acyl chains have been synthesized. Upon irradiation with ultraviolet light, both types of lipid crosslink via the diacetylene groups to produce coloured polymers. The colour arises form the conjugated double and triple bonds which make up the polymer backbone. These phospholipid polymers can exhibit optical activity, as shown by their circular dichroic spectra. The optical activity is thought to stem from asymmetric packing of the polydiacetylene chains, a packing of one particular screw sense being favoured by the chiral glycerol moiety of the lipid. The presence of an intrinsic membrane protein within the liposome structure affects the CD spectra of polymer produced by irradiation.