Interaction of Polymer Aggregates Based on Stearoyl-poly-N-vinylpyrrolidone with Blood Components

Stearoyl-poly-N-vinylpyrrolidone (PVP-stear) of various molecular weights (M(n) = 1500-5500) self-assemble in aqueous medium. Particles prepared from PVP-stear were characterized in terms of shape and size distribution, and the mechanical stability of the particles was studied. The interaction of PVP-stear and its aggregates with blood components was investigated. Aggregates formed by the polymers with M(n) = 1500-3500 in the presence of human serum are stable. The direct lytic action of PVP-stear preparations was studied using sheep and human erythrocytes. The influence of PVP-stear aggregates on the activation of complement system both on classical and alternative pathways was examined. The aggregates prepared from PVP-stear of various molecular weights had no effect on the activation of the complement system.     

Virus attachment to transparent exopolymeric particles along trophic gradients in the southwestern lagoon of New Caledonia

Viruses on organic aggregates such as transparent exopolymeric particles (TEP) are not well investigated. The number of TEP-attached viruses was assessed along trophic gradients in the southwestern lagoon of New Caledonia by determining the fraction of viruses removed after magnetic isolation of TEP. In order to isolate TEP magnetically, TEP were formed in the presence of magnetic beads from submicrometer precursors collected along the trophic gradients. The mixed aggregates of TEP-beads-viruses were removed from solution with a magnetic field. The percentage of viruses associated with newly formed TEP averaged 8% (range, 3 to 13%) for most of the stations but was higher (ca. 30%) in one bay characterized by the low renewal rate of its water mass. The number of viruses (N) attached to TEP varied as a function of TEP size (d [in micrometers]) according to the formulas N = 100d(1.60) and N = 230d(1.75), respectively, for TEP occurring in water masses with short (i.e., <40 days) and long (i.e., >40 days) residence times. These two relationships imply that viral abundance decreases with TEP size, and they indicate that water residence time influences viral density and virus-bacterium interactions within aggregates. Our data suggest that the fraction of viruses attached to TEP is highest in areas characterized by a low renewal rate of the water mass and can constitute at times a significant fraction of total virus abundance. Due to the small distance between viruses and hosts on TEP, these particles may be hot spots for viral infection.     

Isoelectric precipitation of soybean protein using carbon dioxide as a volatile acid

A novel process is presented for the isoelectric precipitation of soy protein, using carbon dioxide as a volatile acid. By contacting a soy meal extract with pressurized carbon dioxide, the solution pH was decreased to the isoelectric region of the soy proteins. Complete precipitation of the precipitable soy proteins could be achieved for protein concentrations up to 40 g/l at pressures less than 50 bar. Isoelectric precipitation with a volatile acid enabled accurate control of the solution pH by pressure and eliminated the local pH overshoot, usual in conventional precipitation techniques. The advantage of the improved precipitation control was reflected by the morphology of the precipitate particles. Protein aggregates formed by CO₂ were perfectly spherical whereas protein precipitated by sulfuric acid had an irregular morphology. The influence of process variables to control particle size is discussed.     

Synthesis and aggregates of cellulose-based hydrophobically associating polymer

A novel cellulose-based hydrophobically associating water-soluble polymer-cellulose octaonate sulfate (COS) was synthesized in this paper. The basic physico-chemical properties such as surface tension and the critical aggregation concentration (cac) were measured by the conventional Wilhelmy plate method. The obtained cac value was compared with environmental scanning electron microscopy (ESEM) and Rheology data. All these results indicated that cac of this amphiphilic polymer was between the range 0.04 and 0.2 wt% and the corresponding surface tension was around 55 mN/m. The conformation of aggregates and size of particles in aqueous solution were carefully investigated by ESEM and dynamic laser scattering (DLS) measurements. When the concentration is around 0.04 wt%, loose aggregates are formed; around 0.5 wt%, network structure formed. DLS results indicated that average size of particle was increased from 54.7 nm to 73 nm and finally to 168.1 nm with the increase of concentration from 0.04% to 0.1% and even to 0.2%. These results suggested that almost all of micelles in aqueous solution aggregated at the experimental concentration range 0.04–0.5 wt%. Rheological properties of this polymer were similar to hydrophobically associating polymers’ (HMP). As the shear rate increased, the solution passed through a shear-thickening region before exhibiting a sharp decrease in viscosity, eventually exhibited Newtonian behavior.     

Effect of nucleocapsid on multimerization of gypsy structural protein Gag

The structural protein (Gag) of the gypsy Drosophila retrovirus lacks matrix, but contains capsid and nucleocapsid domains. The Gag forms virus-like particles in a bacterial cell; besides, its capsid alone is able to form aggregates. However, aggregates assembled from the capsid were variable in size and displayed much less organization than particles formed by the whole Gag. The nucleocapsid exerts influence on the organization and structure of particles, and this function is directed by sequence of amino acid residues at its N-terminus (a nucleocapsid proximal part). The particle assembling occurs in the presence of any RNAs or single stranded DNA oligonucleotides.     

Effect of nucleocapsid on multimerization of <Emphasis Type="Italic">gypsy</Emphasis> structural protein GAG

The structural protein (Gag) of Drosophila retrovirus gypsy contains capsid and nucleocapsid domains. Gag forms virus-like particles in a bacterial cell; furthermore, its capsid alone is able to form aggregates. However, aggregates assembled from the capsid vary in size and are less organized than particles formed by a full-length Gag. The nucleocapsid determines the organization and structure of the particles, which is ensured by the amino acid residues at its N-terminal (a nucleocapsid proximal part). The assembly of the particle occurs in the presence of any RNAs or single-stranded DNA oligonucleotides.     

Self-organized Polymer Aggregates with a Biomimetic Hierarchical Structure and its Superhydrophobic Effect

Nature always gives us inspirations to fabricate functional materials by mimicking the structure design of biomaterials. In this article, we report that polymeric aggregates with morphology similar to the papilla on lotus leaf can be self-organized in the polymer solution by adding 16 wt% water into 5 mg/ml polycarbonate solution in N, N′-dimethylformamide. The hierarchically structured aggregates at micro- and nano-scale alone show superhydrophobic effect without the need of modification with low surface energy compound. Small amount of liquid can be wrapped by the aggregates to form the so-called liquid marble. Influence of the amount of water added into the solution on the morphology of resultant polymer aggregates was investigated. By using the hierarchical aggregates as the surface building blocks, superhydrophobic coating with a static water contact angle larger than 160° and sliding angle less than 5° (for a water drop of 5 μl) was formed. Other solutions, like acid, basic and blood plasma are also repelled on the coating.     

Formation of flower- or cake-shaped stereocomplex particles from the stereo multiblock copoly(rac-lactide)s

Flower- or cake-shaped particles with uniform particle size ranging from nanometers to micrometers were prepared from the stereo multiblock copoly(rac-lactide)s (smb-PLAs) by precipitating the polymer from its solution in methylene chloride/ethanol via three different methods: slowly lowering the solution temperature, slowly evaporating the solvent, and slowly adding a nonsolvent. Under the same condition, sheet-shaped crystals in 10 mum size but not particles were obtained from the pure PLLA with almost the same molecular weight. Electron diffraction and WAXD data demonstrated that the stereocomplex particles belonged to the monoclinic system. All three methods resulted in particles with identical morphology and almost the same particle size. At a given stereoregularity of 88%, as the molecular weight of the polymer increased from 8700 to 23,200 Da, the crystallinity decreased, the particle morphology changed from flower-shaped to cake-shaped, and the diameter and height of the particles increased from 0.8 and 0.45 to 3.6 microm and 2.0 microm, respectively. The initial concentration of the polymer solution influenced the particle size slightly but affected the morphology markedly. On the basis of the above experimental observations, it was proposed that the smb-PLA particles of flower- or cake-shape were formed in four steps: (1) complexation in solution of the smb-PLA chains; (2) particle nucleation; (3) particle growth in the width direction; and (4) particle growth in the height direction. The curvature of the paired smb-PLA chains and the inner stress governed the particle size, and the interconnection between the neighboring particles determined the layered structure and the package density of the particles formed.     

Reversible meso-scale smart polymer--protein particles of controlled sizes

Functionalized beads and particles in the size range of tens to hundreds of nanometers (nano- to meso-scale) are finding increased applications in the bioanalytical field. We show here that conjugates of streptavidin and the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm), synthesized with low polydispersities by reversible addition--fragmentation chain transfer (RAFT) polymerization, rapidly formed mesoscale polymer--protein particles above the lower critical solution temperature (LCST). The average hydrodynamic diameters of these particles could be controlled between 250 nm to 900 nm by the choice of conjugate concentration and polymer molecular weight, and/or through control of the rate of temperature change. Once formed, the biohybrid particles were found to be stable for >16 h at the controlled size, unlike the free PNIPAAm which continued to aggregate and grow over time into very large and polydisperse aggregates. The reversibility between the smart polymer--protein particles and the free polymer--protein conjugates opens potential uses in traditional diagnostic formats and in microfluidic formats where the differential diffusive and physical properties might be exploited for separations, analyte concentration, and signal generation.     

Virus Attachment to Transparent Exopolymeric Particles along Trophic Gradients in the Southwestern Lagoon of New Caledonia

Viruses on organic aggregates such as transparent exopolymeric particles (TEP) are not well investigated. The number of TEP-attached viruses was assessed along trophic gradients in the southwestern lagoon of New Caledonia by determining the fraction of viruses removed after magnetic isolation of TEP. In order to isolate TEP magnetically, TEP were formed in the presence of magnetic beads from submicrometer precursors collected along the trophic gradients. The mixed aggregates of TEP-beads-viruses were removed from solution with a magnetic field. The percentage of viruses associated with newly formed TEP averaged 8% (range, 3 to 13%) for most of the stations but was higher (ca. 30%) in one bay characterized by the low renewal rate of its water mass. The number of viruses (N) attached to TEP varied as a function of TEP size (d [in micrometers]) according to the formulas N = 100d^1.60 and N = 230d^1.75, respectively, for TEP occurring in water masses with short (i.e., <40 days) and long (i.e., >40 days) residence times. These two relationships imply that viral abundance decreases with TEP size, and they indicate that water residence time influences viral density and virus-bacterium interactions within aggregates. Our data suggest that the fraction of viruses attached to TEP is highest in areas characterized by a low renewal rate of the water mass and can constitute at times a significant fraction of total virus abundance. Due to the small distance between viruses and hosts on TEP, these particles may be hot spots for viral infection.     

Protamine assembled in multilayers on colloidal particles can be exchanged and released

Biocomposite thin films assembled on colloidal particles by means of layer-by-layer adsorption have been suggested as drug carriers and diagnostic devices. Protamine (PRM)/dextransulfate (DXS) and protamine/bovine serum albumine (BSA) multilayers were fabricated on colloidal silica and subsequently investigated by means of fluorescence activated cell sorting (FACS) and microelectrophoresis. Fluorescein labeled polyelectrolytes were embedded at different positions in the multilayers as a marker for layer growth. FACS showed that PRM and DXS formed regular growing stable multilayers, yet adsorbed PRM can be nevertheless exchanged with PRM in solution during layer formation and also after the multilayer formation has been completed. Up to 90% of the PRM pool was available for exchange. PRM together with BSA as demonstrated by SFM did not form multilayers under the applied conditions although the zeta-potential, commonly used as an indicator for stepwise adsorption, observed characteristic alternations. The capability of bound PRM to exchange with PRM in solution is attributed to its relatively small size. The demonstrated exchange may have importance in designing multilayers with smart release features. Furthermore, FACS proved to be a rather suitable means to quantify the aggregation behavior during coating and washing. Singulets, doublets, triplets, and aggregates of higher order could be clearly resolved. The aggregation of particles coated with PRM/DXS layers was higher than that of silica particles coated with PAH/PSS layers. In the first case about 50% of all recorded events are attributed to aggregats, while the PAH/PSS coating produced only about 10% aggregates.     

The self-assembly of collagen molecules

The aggregation of native acid-soluble collagen (N-ASC) and of pronase-treated acid soluble collagen (P-ASC) was examined in solution under conditions which varied from those of minimum collagen-collagen interaction to those leading to incipient fiber formation. Molecular weights and weight distributions were determined in the analytical ultracentrifuge using the Yphantis high speed sedimentation equilibrium and Aarchiblad approach-to-equilibrim techniques. The aggregation was pH and ionic strength dependent in each case. Under conditions of minimum aggregation (low pH, low ionic strength), N-ASC showed the presence of permant aggregates. At higher pH and ionic strength, a higher fraction of aggregate was formed but these were of the same charcter and molecular weight as the permanent aggregates. The aggregates were of a single molecular size, with a weight of 1.5 × 10⁶ daltons, compared with a monomer collagen weight of 3.1 × 10⁵ daltons. The P-ASC formed aggregates also but to a much lower extent and the maximum aggregate size corresponded to dimers in molecular weight. These data show the major importance of molecular end-regions in collagen aggregation to form native type fibers and, by virtue of the discrete size of the N-ASC aggregates, support the microfibrillar hypothesis for the assembly of collagen fibrills.     

Investigation of processing parameters of spray freezing into liquid to prepare polyethylene glycol polymeric particles for drug delivery

The objective of this study was to investigate the influence of processing parameters on the morphology, porosity, and crystallinity of polymeric polyethylene glycol (PEG) microparticles by spray freezing into liquid (SFL), a new particle engineering technology. Processing parameters investigated were the viscosity and flow rate of the polymer solution, nozzle diameter, spray time, pressure, temperature, and flow rate of the cryogenic liquid. By varying the processing parameters and feed composition, atomization and heat transfer mechanisms were modified resulting in particles of different size distribution, shape, morphology, density, porosity, and crystallinity. Median particle diameter (M50) varied from 25 μm to 600 μm. Particle shape was spherical or elongated with highly irregular surfaces. Granule density was between 0.5 and 1.5 g/mL. In addition to producing particles of pure polymer, drug particles were encapsulted in polymeric microparticles. The encapsulation efficiency of albuterol sulfate was 96.0% with a drug loading of 2.4%, indicating that SFL is useful for producing polymeric microparticles for drug delivery applications. It was determined that the physicochemical characteristics of model polymeric microparticles composed of PEG could be modified for use as a drug delivery carrier.     

Self-aggregates of oligoarginine-conjugated poly(amino acid) derivatives as a carrier for intracellular drug delivery

N_ω-2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (N_ω-Pbf)-protected oligoarginine was directly conjugated to poly(amino acid) derivatives modified with a long alkyl chain. The final concentration of conjugated peptides was easily controlled by the feed ratio of oligoarginine to polymer backbone and a final soluble polymeric system was obtained by the deprotection of N_ω-Pbf groups. The polymeric conjugates formed stable self-aggregates of size range of 8–40 nm in aqueous solution and effectively internalized into HeLa cells by adsorptive endocytosis.Revisions requested 8 April 2005; Revisions received 6 May 2005     

Preparation and evaluation of polyvinyl alcohol-co-oleylvinyl ether derivatives as tumor-specific cytotoxic systems

A series of poly(vinyl alcohol) amphiphilic derivatives have been prepared to obtain polymeric aggregates in aqueous phase holding thermodynamic instability. The aim was to evaluate their ability to interact with tumor cells eliciting selective cytotoxicity. The poly(vinyl alcohol) derivatives were prepared by partial substitution of poly(vinyl alcohol) (MW 10 kDa) with both oleyl chains and poly(ethylene glycol) monoethyl ethers (PEGMEE) of different molecular weights. The substitution degree was 1.5% for the oleyl chains and 1% for the PEGMEE chains (moles of substituent per 100 mol of hydroxyvinyl monomer). The polyvinyl derivatives obtained easily dissolved in water. Dynamic and static light scattering measurements on the polymer aqueous solutions indicated the formation of polymeric aggregates characterized by low polydispersity (0.232-0.299) and mean size (218-382 nm) in the range suitable for intravenous administration. Moreover, they were characterized by different packing densities and thermodynamic instabilities driving the polymers to interact with hydrophobic membranes. Among the analyzed polymers, the poly(vinyl alcohol)-co-oleylvinyl ether substituted with triethylene glycol monoethyl ether (P10(4)) provided in solution the highest affinity for hydrophobic membranes. P10(4), moreover, was the most cytotoxic toward the tumor cell lines analyzed (neuroblastoma: SH-SY5Y, IMR-32, HTLA-230. melanoma: MZ2-MEL, RPMI7932.), while it did not appreciably alter the viability of the normal resting lymphocytes. The peculiar behavior of the P10(4) aggregates has been correlated to their high thermodynamic instability in solution due to the high packing density that triggers the polymeric aggregates to interact with hydrophobic membranes such as the tumor cell membranes, thus eliciting cytotoxicity.     

The role of helper lipids in cationic liposome-mediated gene transfer.

In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a "helper lipid" to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton-directed membrane motion, after their respective DNA:liposome complexes attached to cell surfaces by electrostatic means. Formation of granules on the cell surface facilitated and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.     

Study of antiproteinase activity of acylated derivatives of Bowman-Birk soybean proteinase inhibitor

The effect of acylation of Bowman–Birk soybean proteinase inhibitor (BBI) by derivatives of various unsaturated fatty acids on inhibition of trypsin, -chymotrypsin, and human leukocyte elastase was investigated. Inhibition (K _i) and kinetic (k _ass, k _diss) constants of interaction between proteases and acylated BBI derivatives were determined. For mono-, di-, and triacylated BBI derivatives, insertion of two oleic residues into the BBI molecule was demonstrated to be more potent for exhibiting antiproteinase activity.     

Evidence for the formation of start aggregates as an initial stage of protein aggregation

The kinetics of thermal aggregation of glycogen phosphorylase b and glyceraldehyde 3-phosphate dehydrogenase from rabbit skeletal muscles were studied using dynamic light scattering. Use of high concentrations of the enzymes (1-3 mg/ml) provided a simultaneous registration of the native enzyme forms and protein aggregates. It was shown that initially registered aggregates (start aggregates) were large-sized particles. The hydrodynamic radius of the start aggregates was about 100 nm. The intermediate states between the native enzyme forms and start aggregates were not detected. The initial increase in the light scattering intensity is connected with accumulation of the start aggregates, the size of the latter remaining unchanged. From a certain moment in time aggregates of higher order, formed as a result of sticking of the start aggregates, make a major contribution to the enhancement of the light scattering intensity.     

Anti-fibrillogenic properties of phthalocyanines: Effect of the out-of-plane ligands

The axially-coordinated phthalocyanines were previously reported as agents possessing strong anti-fibrillogenic properties. In the presented study we used the atomic force microscopy to investigate the intermediates and the products of insulin aggregation reaction formed in the presence of Zr and Hf phthalocyanine complexes that contain out-of-plane ligands of different size and nature. It is shown that while phthalocyanine-free insulin generated mostly amyloid fibrils with a diameter of 2–8 nm and a length of up to 5 μm, the presence of phthalocyanines with spatial bulky ligands (PcZrDbm₂) leads to the redirection of the fibrillization reaction to the formation of the spherical oligomer aggregates with a diameter of 4–12 nm. At the same time the phthalocyanine complex PcHfCl₂ having the small-volume ligands induces the formation of large size insulin aggregates with a height of about 100 nm that are supposed to be amorphous species. The study of the aggregation intermediates showed the certain similarity of the reaction passing for phthalocyanine-free insulin and insulin in the presence of PcZrDbm₂. The large-size amorphous species were observed at the beginning of reaction, later they dissociated, leading to the formation and growth of the smaller size particles. The amyloid-sensitive cyanine dye 7519 demonstrates the strong fluorescent response both in the presence of fibrils and spherical oligomers, while it is non-sensitive to amorphous aggregates.     

Soybean Bowman–Birk Inhibitor Conjugates with Clinical Dextran. Synthesis and Antiproteolytic Activity

Conjugates of the classical soybean Bowman-Birk inhibitor (BBI) with clinical dextran were synthesized. Clinical dextran was preliminarily oxidized with periodate to dialdehydedextran (DAD). The effect of the degree of oxidation of DAD on coupling of the inhibitor was evaluated. The binding of the protein was shown to increase with increasing degree of DAD oxidation (5, 10, 20%). Total coupling of the inhibitor occurred when the degree of oxidation of the dextran was 20%. The BBI-DAD (20%) conjugate contained 13% protein with BBI/DAD molar ratio 1 : 1. The conjugates retained the ability to inhibit trypsin (Ki = 0.2-0.3 nM) and alpha-chymotrypsin (Ki = 15-30 nM). Thus, the coupling of BBI with the polymeric carrier caused practically no decrease in the antiproteolytic activity of the inhibitor.