Enhanced stability of core-surface cross-linked micelles fabricated from amphiphilic brush copolymers

"Stealth" nanoparticles made from polymer micelles have been widely explored as drug carriers for targeted drug delivery. High stability (i.e., low critical micelle concentration (CMC)) is required for their intravenous applications. Herein, we present a "core-surface cross-linking" concept to greatly enhance nanoparticle's stability: amphiphilic brush copolymers form core-surface cross-linked micelles (nanoparticles) (SCNs). The amphiphilic brush copolymers consisted of hydrophobic poly(epsilon-caprolactone) (PCL) and hydrophilic poly(ethylene glycol) (PEG) or poly(2-(N,N-dimethylamino)ethyl methacrylate) (PDMA) chains were synthesized by macromonomer copolymerization method and used to demonstrate this concept. The resulting SCNs were about 100 times more stable than micelles from corresponding amphiphilic block copolymers. The size and surface properties of the SCNs could be easily tailored by the copolymer's compositions.     

Bursal anti-steroidogenic peptide (BASP): modulation of mitogen-stimulated bursal-lymphocyte DNA synthesis

The present study examined the effects of bursal anti-steroidogenic peptide (BASP) on mitogen-induced DNA synthesis in bursa-derived B-lymphocytes in short-term culture. Partially purified extracts of chicken bursa of Fabricius tissue, containing BASP, significantly (P < 0.05) reduced DNA synthesis in bursal-lymphocytes exposed to increasing concentrations of phorbol 12,13-dibutyrate (PDB). Following these initial observations, BASP, further purified from bursal extracts using sequential rpHPLC fractionation, was observed to reduce (P < 0.05) both B-lymphocyte PDB-stimulated DNA synthesis and ovarian granulosa cell progesterone biosynthesis with bioactivity observed at similar retention times in each assay, suggesting that each bioactivity may be due to the same or similar molecules. A similar BASP-enriched fraction was not effective in altering basal levels of DNA synthesis in chick embryonic kidney cells. Subsequently, BASP was further purified by several sequential chromatographic methods including: C-18 rpHPLC (preparative rpHPLC followed by a semi-preparative rpHPLC column), cation exchange chromatography, molecular sieve HPLC chromatography, and SDS-PAGE. Biologically active material was observed at approximately 29 or 34 kDa. Protein concentration was determined and bioactivity was evaluated. Anti-proliferative effects of this partially purified BASP on bursal-lymphocytes was observed at concentrations as low as 1.6 micrograms ml-1, with complete suppression of mitogen-stimulated DNA synthesis observed at approximately 25 micrograms ml-1. This partially purified BASP was also efficacious for attenuation of ovarian granulosa cell progesterone biosynthesis at concentrations as low as 0.4 microgram ml-1, with complete suppression of gonadotrophin-stimulated progesterone biosynthesis observed at approximately 0.8 microgram ml-1. While BASP is efficacious for attenuation of both granulosa cell steroidogenesis and bursal-lymphocyte proliferation, these data suggest that BASP is much more potent with regard to anti-steroidogenic activity.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

Ferrous iron uptake by rat duodenal brush border membrane vesicles: Effects of dietary iron level and competing minerals (Zn, Mn, and Ca)

We sought to confirm a recent report that Fe⁺² uptake into rat brush-border membrane vesicles is markedly increased by short-term consumption of iron-deficient diet, with no additional enhancement as the animal becomes functionally iron-deficient with continuing dietary Fe deprivation. In addition, we investigated whether previously observed in vivo absorption interactions of iron, zinc, and manganese occur in the brush border membrane vesicles uptake process, and whether short-term or long-term consumption of an iron-deficient diet affects the interaction at the uptake level. We did not observe any differences in Fe⁺² uptake between normal and iron-deficient brush border membrane vesicles, even when the iron status contrast was intensified by feeding a high iron versus iron-deficient diet for 3 weeks. Equimolar Zn⁺² and Mn⁺² decreased Fe⁺² uptake by 29 to 50% and 11 to 39%, respectively. Iron deficiency did not alter these effects. Equimolar Fe⁺² decreased Zn⁺² uptake by 13 to 22%. Calcium, included as a negative control, did not affect Fe⁺² uptake. Thus, some competition between Fe⁺² and similar divalent cations does occur at the level of the brush border membrane; the exact nature of this competition remains to be determined.     

Synthesis and characterization of core-shell star copolymers for in vivo PET imaging applications

The synthesis of core-shell star copolymers via living free radical polymerization provides a convenient route to three-dimensional nanostructures having a poly(ethylene glycol) outer shell, a hydrophilic inner shell bearing reactive functional groups, and a central hydrophobic core. By starting with well-defined linear diblock copolymers, the thickness of each layer, overall size/molecular weight, and the number of internal reactive functional groups can be controlled accurately, permitting detailed structure/performance information to be obtained. Functionalization of these polymeric nanoparticles with a DOTA-ligand capable of chelating radioactive (64)Cu nuclei enabled the biodistribution and in vivo positron emission tomography (PET) imaging of these materials to be studied and correlated directly to the initial structure. Results indicate that nanoparticles with increasing PEG shell thickness show increased blood circulation and low accumulation in excretory organs, suggesting application as in vivo carriers for imaging, targeting, and therapeutic groups.     

A zeolite as a tool for successful refolding of PEGylated proteins and their reassembly with tertiary structures

In the present study, we demonstrated zeolites' potential contribution to establish a method for preparing successfully refolded and reassembled PEGylated protein nanoparticles without the use of protein denaturants through the proteins' reassembly process. At first, the PEGylated nanoparticles are disassembled into identical PEGylated protein subunits by means of protein denaturants, and then the denatured subunits are adsorbed to zeolites. After the complete removal of denaturants, high-molecular-weight poly(ethylene glycol) (PEG) molecules are added to a solution where the zeolites suspend. Consequently, the PEGylated proteins are gradually reassembled into nanoparticles because the subunits are desorbed from the zeolites by the steric hindrance of the added PEG molecules. The present study reveals that PEGylated encapsulin was reassembled and hollow encapsulin nanoparticles were obtained. The results clearly demonstrate the usefulness of zeolites as a tool for the successful refolding of PEGylated proteins and their reassembly with tertiary structures.     

Entropic interaction chromatography: separating proteins on the basis of size using end-grafted polymer brushes

Partitioning of a macromolecule into the interfacial volume occupied by a grafted polymer brush decreases the configurational entropy (DeltaSbrush(c)) of the terminally attached linear polymer chains due to a loss of free volume. Self-consistent field theory (SCF) calculations are used to show that DeltaSbrush(c) is a strong function of both the size (MWp) of the partitioning macromolecule and the depth of penetration into the brush volume. We further demonstrate that the strong dependence of DeltaSbrush(c) on MWp provides a novel and powerful platform, which we call entropic interaction chromatography (EIC), for efficiently separating mixtures of proteins on the basis of size. Two EIC columns, differing primarily in polymer grafting density, were prepared by growing a brush of poly(methoxyethyl acrylamide) chains on the surface of a wide-pore (1,000-A pores, 64-microm diameter rigid beads) resin (Toyopearl AF-650M) bearing surface aldehyde groups. Semipreparative 0.1-L columns packed with either EIC resin provide reduced-plate heights of 2 or less for efficient separation of globular protein mixtures over at least three molecular-weight decades. Protein partitioning within these wide-pore EIC columns is shown to be effectively modeled as a thermodynamically controlled process, allowing partition coefficients (K(P)) and elution chromatograms to be accurately predicted using a column model that combines SCF calculation of K(P) values with an equilibrium-dispersion type model of solute transport through the column. This model is used to explore the dependence of column separation efficiency on brush properties, predicting that optimal separation of proteins over a broad MWp range is achieved at low to moderate grafting densities and intermediate chain lengths.     

Peptide-polymer biotherapeutic synthesis on novel cross-linked beads with "spatially tunable" and "isolated" functional sites

Solid phase synthesis of polymer biotherapeutics using conventional polymers suffers from many limitations such as low synthetic yield and purity. The conventional polymers prepared by either pre- or post-functionalization strategies have no control over the point of functionalization. Hence we report a novel cross-linked polymer in which the functional groups are spatially tuned to predefined distance with optimal site isolation. This has been achieved by the design and synthesis of a tetra functional PEG, 3,3'-(PEG)bis(1-(4-vinylphenoxy)propan-2-ol) (bis(VPP)PEG). It has been incorporated at cross-linking of 1-12%, into a polystyrene network by free radical suspension polymerization. In this polymer, the distance between hydroxyl functional groups has been spatially tuned in a predefined manner by varying the length of the cross-linker backbone from ethylene glycol to PEG1000 Da and the loading capacity could be varied from 0.1 to 0.9 mmol/g. The polymer has been characterized by SEM, FTIR, and 13C NMR. The polymer exhibits excellent swelling behavior and high chemical stability. The synthetic efficiency of the polymer was demonstrated by the successful synthesis of three structural classes of PEGylated antimicrobial peptide biotherapeutics and the difficult ACP (65-74) fragment. Thus the "spatially defined" and "site isolated" synthesis within the new polymer offers a novel strategy for synthesis of difficult peptide-polymer bioconjugates. The bioassay studies shows that PEGylation of AMPs significantly reduces their hemolytic potential but the retainment of antibacterial property was dependent both on the peptide sequence and the size of PEG used.     

Isolation and characterization of Brush border fragments from mosquito mesenterons

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca²⁺ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.     

Intestinal mucosal lipid peroxidation and absorptive function In Salmonella typhimurium mediated intestinal infection

S. typhimurium infection is associated with neutrophil infiltration within the intestinal mucosa. Neutrophil activation provides a major source of reactive oxygen species (ROS). The mucosal pathology of S. typhimurium infection may be in part due to the excessive production of these reactive species. This study was carried out to investigate if ROS play a role in mediating the changes in the structural components and functional properties of brush border membrane (BBM) in rats during S. typhimurium infection. This was done by determining the changes in the BBM extent of lipid peroxidation and absorptive function. A significant increase in the extent of lipid peroxidation of BBM during S. typhimurium infection was observed as judged by malondialdehyde (MDA) and conjugated diene formation and depletion of -tocopherol and protein associated thiol groups. A significant decrease in the BBMV (brush border membrane vesicle) transport of amino acids was also observed. However there was no change in the transport of D-glucose. The decrease in amino acid transport further led to a significant decrease in the enterocyte level of protein synthesis. Exposure of BBMV to a free radical donor, cumene hydroperoxide, also led to an increase in the extent of lipid peroxidation and a decrease in the amino acid transport. Possibly ROS might play a significant role in mediating the mucosal damage during S. typhimurium infection.     

A photoactivatable probe for the Na+/H+ exchanger cross-links a 66-kDa renal brush border membrane protein

Earlier studies on LLC-PK1 cells have demonstrated two pharmacologically distinct Na+/H+ exchangers in renal epithelia. In addition, the cDNA clone for the human Na+/H+ antiporter which is growth factor activatable has been isolated and expressed (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280). We report here the synthesis of an amiloride analogue that can be photoactivated and labeled with 125I. This analogue covalently cross-links a 66-kDa protein of bovine renal brush border membranes. A rabbit polyclonal antibody that was directed against a 20-amino acid peptide of the cytoplasmic domain of its human Na+/H+ antiporter also gives a positive Western against 66-kDa protein of bovine brush border membranes. Thus, the photoactive probe may be helpful in the isolation and purification of the brush border Na+/H+ exchanger.     

Rapid synthesis of silver nanoparticles using culture supernatants of Enterobacteria: A novel biological approach

The development of reliable processes for the synthesis of silver nanomaterials is an important aspect of current nanotechnology research. Reports on the cell-associated biosynthesis of silver nanoparticles using microorganisms have been published, but these methods of synthesis are rather slow. In this paper, we report on the rapid synthesis of metallic nanoparticles of silver using the reduction of aqueous Ag+ ion using the culture supernatants of Klebsiella pneumonia, Escherichia coli, and Enterobacter cloacae (Enterobacteriacae). The synthetic process was quite fast and silver nanoparticles were formed within 5 min of silver ion coming in contact with the cell filtrate. Through a limited screening process involving a number of common microorganisms, we observed that the culture supernatants of different bacteria from Enterobacteriacae were potential candidates for the rapid synthesis of silver nanoparticles; further, we revealed that this method of synthesis requires far less time than previously published biological methods. Our investigation also showed that piperitone can partially inhibit the reduction of Ag⁺ to metallic silver nanoparticles by Enterobacteriacae.     

Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles

Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.     

Polysaccharide-poly(ethylene glycol) star copolymer as a scaffold for the production of bioactive hydrogels

The production of polysaccharide-derivatized surfaces, polymers, and biomaterials has been shown to be a useful strategy for mediating the biological properties of materials, owing to the importance of polysaccharides for the sequestration and protection of bioactive proteins in vivo. We have therefore sought to combine the benefits of polysaccharide derivatization of polymers with unique opportunities to use these polymers for the production of bioactive, noncovalently assembled hydrogels. Accordingly, we report the synthesis of a heparin-modified poly(ethylene glycol) (PEG) star copolymer that can be used in the assembly of bioactive hydrogel networks via multiple strategies and that is also competent for the delivery of bioactive growth factors. A heparin-decorated polymer, synthesized by the reaction of thiol end-terminated four-arm star PEG (M(n) = 10 000) with maleimide functionalized low molecular weight heparin (LMWH, M(r) = 3000), has been characterized via (1)H NMR spectroscopy and size-exclusion chromatography; results indicate attachment of the LMWH with at least 73% efficiency. Both covalently and noncovalently assembled hydrogels can be produced from the PEG-LMWH conjugate. Viscoelastic noncovalently assembled hydrogels have been formed on the basis of the interaction of the PEG-LMWH with a PEG polymer bearing multiple heparin-binding peptide motifs. The binding and release of therapeutically important proteins from the assembled hydrogels have also been demonstrated via immunochemical assays, which demonstrate the slow release of basic fibroblast growth factor (bFGF) as a function of matrix erosion. The combination of these results suggests the opportunities for producing polymer-polysaccharide conjugates that can assemble into novel hydrogel networks on the basis of peptide-saccharide interactions and for employing these materials in delivery applications.     

Natural N-terminal fragments of brain abundant myristoylated protein BASP1

BASP1 (also known as CAP-23 and NAP-22) is a novel myristoylated calmodulin-binding protein, abundant in nerve terminals. It is considered as a signal protein participating in neurite outgrowth and synaptic plasticity. BASP1 is also present in significant amounts in kidney, testis, and lymphoid tissues. In this study, we show that BASP1 is accompanied by at least six BASP1 immunologically related proteins (BIRPs), which are present in all animal species studied (rat, bovine, human, chicken). BIRPs have lower molecular masses than that of BASP1. Similarly to BASP1, they are myristoylated. Peptide mapping and partial sequencing have shown that BIRPs represent a set of BASP1 N-terminal fragments devoid of C-terminal parts of different length. In a definite species, the same set of BASP1 fragments is present in both brain and other tissues. The sum amount of the fragments is about 50% of the BASP1 amount in a tissue. Obligatory accompanying of BASP1 by a set of specific fragments indicates that these fragments are of physiological significance.     

Self-assembly and photo-cross-linking of eight-armed PEG-PTMC star block copolymers

Eight-armed poly(ethylene glycol)-poly(trimethylene carbonate) star block copolymers (PEG-(PTMC)(8)) linked by a carbamate group between the PEG core and the PTMC blocks were synthesized by the metal-free, HCl-catalyzed ring-opening polymerization of trimethylene carbonate using an amine-terminated eight-armed star PEG in dichloromethane. Although dye solubilization experiments, nuclear magnetic resonance spectroscopy, and dynamic light scattering clearly indicated the presence of aggregates in aqueous dispersions of the copolymers, no physical gelation was observed up to high concentrations. PEG-(PTMC(9))(8) was end-group-functionalized using acryloyl chloride and photopolymerized in the presence of Irgacure 2959. When dilute aqueous dispersions of PEG-(PTMC(9))(8)-Acr were UV irradiated, chemically cross-linked PEG-PTMC nanoparticles were obtained, whereas irradiation of more concentrated PEG-(PTMC(9))(8)-Acr dispersions resulted in the formation of photo-cross-linked hydrogels. Their good mechanical properties and high stability against hydrolytic degradation make photo-cross-linked PEG-PTMC hydrogels interesting for biomedical applications such as matrices for tissue engineering and controlled drug delivery systems.     

Spectroscopic investigation of collagen scaffolds impregnated with AgNPs coated by PEG/TX-100 mixed systems

Silver nanoparticles have received attention as novel antimicrobial agents due to their high surface area to volume ratio and the unique chemical and physical properties. In order to study the effects of capping agents on silver nanoparticles (AgNPs), the nanoparticles were synthesized via chemical reduction method using different concentrations (0.3 mM, 0.6 mM and 0.9 mM) of polyethylene glycol (PEG) and Triton X-100 (TX). Also, AgNPs capped by the combinations of both PEG and TX were synthesized. These coated AgNPs were incorporated into collagen, lyophilized to form scaffolds and characterized by FTIR and FT-Raman spectroscopy. Results on mechanical property of all the scaffolds displayed no significant difference in the percentage elongation at break. However, the maximum percentage of 46.67% was observed with the combinations (0.9 mM PEG+0.9 mM TX). This implies that the combinations of surfactants increase the elasticity, which is useful for biomedical applications, e.g., Heart-valve preparations.