Polycation Polyethylenimine Research Articles

Novel Anion Liposome-Encapsulated Antisense Oligonucleotide Restores Susceptibility of Methicillin-ResistantStaphylococcus aureusand Rescues Mice from Lethal Sepsis by TargetingmecA

Beta-lactam resistance in methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is caused by the production of an additional low-affinity penicillin-binding protein 2a, which is encoded by the mecA gene. The disruption of mecA may inhibit mecA expression and thereafter lead to the restoration of MRSA susceptibility to beta-lactams. In this study, we developed a novel anionic liposome for encapsulating and delivering the complexes of a specific anti-mecA phosphorothioate oligodeoxynucleotide (PS-ODN833) and polycation polyethylenimine (PEI). The efficiencies of liposome encapsulation of the complexes were around 79.7% +/- 2.7%. The liposomes showed sustained release of PS-ODN833 at 37 degrees C but very low levels of release at 4 degrees C and room temperature. The addition of the encapsulated anti-mecA PS-ODN833-PEI complex to cultures of MRSA strains caused 45, 76, 82, and 93% reductions in mecA expression, accompanied by the inhibition of MRSA growth on Mueller-Hinton agar containing oxacillin (6 microg/ml) in a concentration-dependent manner. The encapsulated-PS-ODN833 treatment also reduced the MICs of five of the most commonly used antibiotics for MRSA clinical isolates to values within the sensitivity range and rescued mice from MRSA-caused septic death by downregulating mecA. The survival rates of septic mice increased from 0% for the control group to 53% for the PS-ODN833-treated group. The results were associated with reductions of bacterial titers in the blood of surviving mice. The findings of the present study indicate that an antisense oligodeoxynucleotide targeted to mecA can significantly restore the susceptibility of MRSA to existing beta-lactam antibiotics, providing an apparently novel strategy for treating MRSA infections.

Influence of pH on the Structure of Multilayer Films Composed of Strong and Weak Polyelectrolytes

The sequential adsorption of oppositely charged polyelectrolytes called the "layer by layer" technique is a method for formation of ultrathin films with controlled thickness and interfacial properties. Composition of polyelectrolyte solutions, pH, and electrolyte concentration are important parameters governing formation of multilayer films. Since pH is the factor controlling charge of weak polyelectrolytes, the structure of multilayers should be sensitive to its value. In this paper we focused on formation of PE multilayer films composed from weak and strong polyelectrolytes. We used weak, branched polycation polyethyleneimine (PEI, 70 kDa) and strong polyanion poly-4-styrenesulfonate (PSS, 70 kDa) to form films by the layer-by-layer technique on the surface of silicon wafers under two deposition conditions: pH = 6 when PEI was strongly charged and pH = 10.5 when the charge density of PEI was low. Thicknesses of films were measured by single wavelength ellipsometry, and the results were confronted with ones concerning mass of the adsorbed films obtained by quartz crystal microbalance. We found that, depending on pH of the solutions, combination of weakly and strongly charged polyelectrolytes gave either linear or nonmonotonic increase of film thickness with a number of deposited PE layers. We observed a good correlation between multilayer film thickness and adsorbed mass. The atomic force microscopy images of surface topography of PEI/PSS films demonstrated large differences between films deposited at pH = 6 and 10.5. Additionally the cyclic voltamperometry was used to determine the differences in permeability of films formed at various pH conditions.

Oxygen tolerance of an implantable polymer/enzyme composite glutamate biosensor displaying polycation-enhanced substrate sensitivity

Biosensors were fabricated at neutral pH by sequentially depositing the polycation polyethyleneimine (PEI), the stereoselective enzyme L-glutamate oxidase (GluOx) and the permselective barrier poly-ortho-phenylenediamine (PPD) onto 125-microm diameter Pt wire electrodes (Pt/PEI/GluOx/PPD). These devices were calibrated amperometrically at 0.7 V versus SCE to determine the Michaelis-Menten parameters for enzyme substrate, l-glutamate (Glu) and co-substrate, dioxygen. The presence of PEI produced a 10-fold enhancement in the detection limit for Glu (approximately 20 nM) compared with the corresponding PEI-free configurations (Pt/GluOx/PPD), without undermining their fast response time (approximately 2 s). Most remarkable was the finding that, although some designs of PEI-containing biosensors showed a 10-fold increase in linear region sensitivity to Glu, their oxygen dependence remained low.

Glucose oxidase anode for biofuel cell based on direct electron transfer

This paper presents a new design concept of a glucose oxidase (GO x ) electrode as an anode for the biofuel cell based on direct electron transfer (DET) between the active site of an enzyme and the multi-walled carbon nanotube (MWNT)-modified electrode surface. Toray ® carbon paper (TP) with a porous three-dimensional network (78% porosity) was used as a matrix for selectively growing multi-walled carbon nanotubes. The incorporation of MWCNTs into TP was provided by the chemical vapor deposition technique after an electrochemical transition of cobalt metal seeds. This approach has the ability to efficiently promote DET reactions. The morphologies and electrochemical characteristics of the GO x modified electrodes were investigated by scanning electron microscopy, cyclic voltammetry, and potentiometric methods. The combination of poly-cation polyethylenimine (PEI) with negatively charged glucose oxidase provides formation of ∼100 nm thick films on the TP/MWCNT surface. The tetrabutylammonium bromide salt-treated Nafion ® was used as GO x binder and proton-conducting medium. The TP/MWCNT/PEI/GO x /Nafion ® modified electrode operates at 25 °C in 0.02 M phosphate buffer solution (pH 6.9) containing 0.1 M KCl in the presence of 20 mM glucose. The open circuit potential of GO x anode was between −0.38 V and −0.4 V vs. Ag/AgCl, which is closer to the redox potential of the FAD/FADH 2 cofactor in the enzyme itself. The GO x electrode has a potential to work in vivo by using endogenous substances, such as glucose and oxygen. Such a glucose anode allows for the development of a new generation of miniaturized membrane-less biofuel cells.

Development and in vitro validation of a targeted delivery vehicle for DNA vaccines

Usage of DNA vaccination has been limited by inefficient cellular expression of plasmid constructs used in DNA vaccines. We describe a novel system for enhancing delivery of DNA vaccine plasmids into cells and their nuclei. This delivery system uses recombinant reovirus type 3 σ1 attachment protein genetically modified with a nuclear localization sequence (σ1-NLS) as a targeting ligand. Purified σ1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with σ1 or σ1-NLS. In L929 fibroblasts loaded with σ1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified σ1. Transfection of L929 cells with σ1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using σ1-conjugated PEI. These results suggest that σ1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy.

Polymer−DNA Hybrid Nanoparticles Based on Folate−Polyethylenimine-block-poly(l-lactide)

The ability of amphiphilic block copolymers that consist of polyethylenimine (PEI) and poly(L-lactide) (PLLA) to modulate the delivery of plasmid DNA was evaluated. Folate-polyethylenimine-block-poly(l-lactide) (folate-PEI-PLLA) was synthesized by linking folic acid and PLLA to PEI diamine. Water-soluble polycation PEI provides gene-loading capability. Additionally, PEI is considered to exhibit high transfection efficiency and endosomal disrupting capacity. Hydrophobic PLLA that is incorporated into the gene delivery vector is believed to enhance the cell interactions and tissue permeability of the delivery system. Polymeric carrier containing folic acid is expected to be able to identify tumor surface receptors and transfect cells by receptor-mediated endocytosis. The results of agarose retardation assay indicated that the folate-PEI-PLLA began to form polyplexes at a polymer/DNA weight ratio (P/D) of over 10, whereas branched polyethylenimine (B-PEI) formed polyplexes with DNA at a ratio of above 1. The spherical particle morphology was supplemented with a particle size of approximately 100 nm at 10 P/D ratio. The results indicated that folate-PEI-PLLA with proper PEI/PLLA ratio effectively reduced cytotoxicity and maintained acceptable transfection efficiency. Low cytotoxicity of the folate-PEI-PLLA gives an advantage to high-dose administration.

Polyelectrolyte Nanoparticles Mediate Vascular Gene Delivery

The purpose is to develop a non-viral gene delivery system that meets the requirements of colloidal stability of DNA complexes expressed in terms of no particle aggregation under physiologic conditions. The system should be used to transfect cardiovascular tissues. We used a strategy based on the formation of polyelectrolyte nanoparticles by deposition of alternatively charged polyelectrolytes onto a DNA core. Polyelectrolytes were transfer RNA as well as the synthetic polyanion, polyvinyl sulfate (PVS), and the polycation polyethylenimine (PEI). The PEI/DNA complex formed the DNA core. We observed that the DNA is condensed by polycations and further packaged by association with a polyanion. These nanoparticles exhibited negative surface charge and low aggregation tendency. In vivo rat carotid artery experiments revealed high transfection efficiency, not only with the reporter gene but also with the gene encoding human urokinase plasminogen activator (Hu-uPA). Hu-uPA is one of the proteins involved in the recovery of the blood vessels after balloon catheter injury and therefore clinically relevant. A strategy for in vivo gene transfer is proposed that uses the incorporation of polyanions as RNA or PVS into PEI/DNA complexes in order to overcome colloidal instability and to generate a negative surface charge. The particles proved to be transfectionally active in vascular gene transfer.

Fine-Tuning the Wetting Behavior of Polyelectrolyte Films with Sodium Dodecyl Sulfate

Charge-alternating polyelectrolyte multilayers containing the anionic surfactant sodium dodecyl sulfate (SDS) were constructed by adsorption from aqueous solution. The polyelectrolyte films contained the polycation PEI (poly(ethylenimine)) and the polyanion PAZO (poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl, sodium salt]). SDS adsorption on the PEI/PAZO/PEI trilayer was studied using dynamic tensiometry and single wavelength ellipsometry. Advancing contact angle measurements of SDS adsorption onto PEI surfaces indicate that surfactant adsorption is complete within 1 min. The contact angle increases with surfactant concentration, from 30° (0 mM SDS) and plateaus to a constant value of 80° beyond 0.01 mM SDS. Constructing films from varying concentrations of PEI allowed the degree of PAZO interpenetration to be qualitatively measured. Contact angle and ellipsometric thickness studies indicate that PAZO interpenetrates 32 Å into the terminal PEI layer, above which the surface is composed entirely of PEI. Maximum SDS adsorption is observed above the interpenetrated region, consistent with maximum electrostatic interaction between the terminal PEI layer and SDS.

Non-viral vector-mediated uptake, distribution, and stability of chimeraplasts in human airway epithelial cells.

Chimeraplasty is a novel methodology that uses chimeric RNA/DNA oligonucleotides (chimeraplasts) to stimulate genomic DNA repair. Efficient uptake and nuclear localization of intact chimeraplasts are key parameters to achieve optimal correction of mutation defects into specific cell types. A 5'-end FITC-labeled 68-mer RNA/DNA oligonucleotide was complexed with the polycation polyethylenimine (PEI) and the cationic lipids Cytofectin and GenePorter. Flow cytometry was employed to evaluate chimeraplast uptake under different conditions. Intracellular chimeraplast distribution and co-localization with endocytosis markers were assessed by confocal microscopy. Relative quantification of chimeraplast metabolism was performed by denaturing PAGE and GeneScan(trade mark) analysis. In airway epithelial cells, optimized chimeraplast uptake reached near 100% efficiency with the carriers tested. However, chimeraplast nuclear localization could only be achieved using PEI or Cytofectin. Chimeraplast/GenePorter lipoplexes were retained in the cytoplasm. PEI polyplexes and Cytofectin lipoplexes displayed different uptake rates and internalization mechanisms. Chimeraplast/PEI polyplexes were internalized at least partially by fluid-phase endocytosis. In contrast, phagocytosis may have contributed to the internalization process of large-sized chimeraplast/Cytofectin lipoplexes. Moreover, significant chimeraplast degradation was detected 24 h after transfection with both PEI polyplexes and Cytofectin lipoplexes, although the latter seemed to confer a higher degree of protection against nuclease degradation. Both Cytofectin and PEI are efficient for chimeraplast nuclear uptake into airway epithelial cells. However, despite the distinct structures and trafficking pathways of the corresponding complexes, none of them could prevent nuclease-mediated metabolism of the chimeric oligonucleotides. These findings should be taken into account for future investigations of chimeraplast-mediated gene repair in airway epithelial cells.

Charge-Dependent Sidedness of Cytochrome P450 Forms Studied by Quartz Crystal Microbalance and Atomic Force Microscopy

Quartz crystal microbalance (QCM) resonance measurements were used to examine the surface charge characteristics of cytochrome P450 forms and the influence of charge on the docking of redox partners like cytochrome b5. The distal surface of cytochrome P450 (CYP)101 (pI = 4.5), relative to the heme, is fairly anionic, as is the proximal surface. The latter, however, also has two cationic clusters. A considerably greater extent of CYP101 binding was seen to the cationic, polyethylene-surfaced resonators. CYP2B4 (pI = 8.5) preferentially bound to the polyanionic, polystyrene sulfonate-surfaced resonators. Cytochrome b5 is an acidic protein that had a preferential binding to the poly(ethyleneimine (PEI)-surfaced resonators. When binding to CYP2B4-surfaced films, cytochrome b5 preferentially bound to those cytochrome P450 molecules that were adsorbed to cationic (PEI) films. It is suggested that adsorption of CYP2B4 to an anionic poly(styrenesulfonate) (PSS) surface is with cationic clusters that include the cytochrome b5 docking domain. This diminishes the extent of docking of the cytochrome b5. In contrast, when CYP2B4 is adsorbed to a cationic film the proximal surface with the cytochrome b5-docking site is available for cytochrome b5 binding. A film of the polycation PEI was adsorbed to the silver QCM surface. It formed polymer islands when viewed with atomic force microscopy. Polyanionic PSS was adsorbed intermittently with the PEI. By the third and fourth layer of polyions the polymer islands were essentially merged and protein adsorption as a fourth or fifth layer formed a nearly continuous film. CYP101 was seen to adsorb as globules with a molecular diameter of about 10 nm. CYP2B4 adsorbed to the polyionic films had a slightly elliptical globular shape, also with a molecular diameter of about 10 nm.

Xenogenization by tetanus toxoid loading into lymphoblastoid cell lines and primary human tumor cells mediated by polycations and liposomes

We explored the potential of the xenogenization concept as an adjuvant procedure in anti-tumor immunity. To mediate effective loading we used polyarginine (pArg) molecules of various degrees of polymerization, cationic liposomes, or chimeric molecules of transferrin (Tf) and the polycation polyethyleneimine (PEI). Tetanus toxoid (TT) was loaded onto primary human leukemia cells, culture adapted primary human neuroblastoma cells, and human lymphoblastoid cell lines (LCLs) with high efficiency by all procedures. Trypsin treatment of loaded cells provided evidence that only liposomes and Tf–PEI mediated internalization of TT. Lymphocytes primed with xenogenized LCLs and challenged with unmodified LCLs showed increased IFNγ secretion compared with lymphocytes primed with non-xenogenized LCLs.

Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery.

A simple and inexpensive method of condensing and linking plasmid DNA to carrier adenovirus particles is described. The synthetic polycation polyethylenimine is used to condense plasmid DNA into positively charged 100 nm complexes. These PEI-DNA complexes are then bound to adenovirus particles through charge interactions with negative domains on the viral hexon. The resulting transfection complexes deliver plasmid DNA to cells by the adenovirus infectious route without interference from virus gene expression because psoralen-inactivated virus is employed. The PEI-DNA-adenovirus complexes display DNA delivery comparable to more sophisticated DNA virus complexes employing streptavidin/biotin linkage, but require no special reagents and are much easier to prepare.

Displacement chromatography in biotechnological downstream processing

Displacement chromatography was used for the isolation and purification of recombinant antithrombin III (rh-AT III), an anticoagulant of high pharmaceutical interest, which is usually isolated from human blood plasma. A fully automated system was developed, where the preparative chromatographic unit is continuously monitored by fast analytical HPLC. High purities and high concentrations were achieved in a single step. Macroporous hydroxyapatite (HA, diameter 2 μm, pore size 1000 Å) was used to pack the displacement column. Adsorption isotherms were recorded for various proteins. The influence of the column length, the mobile phase composition and the conditioning of the adsorptive surface on the separation by displacement of both basic and acidic proteins was investigated. Much attention was paid to the design of a suitable protein displacer. Cytochrome c and the polycation PEI (polyethyleneimine) were used for the displacement of basic proteins. Better results were achieved for the cytochrome c. A number of polyacids, proteins and Ca 2+ complexing agents were investigated as putative displacers of acidic proteins from HA. While the polyacids, PA (polyacrylic acid, M r 6000) and PGA (polyglutaminic acid, M r 12 000), were unsuited to the task, the carboxylate cluster-carrying protein ß-casein was more successful. The best results were obtained with complexing agents such as the commercially available EGTA [ethylene glycol bis(ß-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, M r 380.4] and IDA-PEG (polyethylene glycol carrying iminodiacetic acid end-groups, M r peg 1000), synthesized for the purpose of protein displacement. Short reversed-phase columns packed with non-porous beads were used for the fast monitoring of the effluent of the displacement column. By working at 60°C the mobile phase flow-rate could be increased to an extent where the time required for a single analysis was reduced to 20 s. Including column regeneration, an analytical frequency of 2 min −1 could be maintained. As the flow-rates in the preparative column were chosen such (usually 100 μl/min), that a displacement including column regeneration required ca. 1 h, this analytical frequency is high enough to allow adequate monitoring and documentation of the purification and to permit automatic collection of the product-containing fractions.

Dose-dependent movement of cationic molecules across the glomerular wall.

Different concentrations of the polycation polyethyleneimine (PEI) were administered by single intravenous injections or by constant vascular perfusion to the kidneys of Sprague-Dawley rats. At a fixed time interval after administration of PEI, the kidneys were fixed and the distribution of PEI in the glomerular wall was evaluated by electron microscopy. At the lower concentrations (e.g., 0.005%), PEI bound only to the glomerular endothelial glycocalyx and preferentially to microvillous projections on this endothelium. At higher concentrations (e.g., 0.05%), PEI also bound to discrete anionic sites in the lamina rara interna (LRI) but was rarely seen in the lamina rara externa (LRE). As the concentration of PEI was further increased (e.g., 0.5%), PEI moved deeper into the glomerular basement membrane (GBM) and bound extensively to discrete anionic sites in the lamina rara externa. Although anionic sites in the LRI and LRE appeared nearly saturated following infusion of 0.5% PEI, this cationic molecule was rarely seen to cross filtration slits and pass into the urinary space. At still higher concentrations (e.g., 2%), however, PEI moved freely across the filtration slits, bound extensively to the glomerular epithelial glycocalyx, and induced a narrowing of the filtration slits. When PEI was mechanically perfused through the kidney vasculature for 3 minutes, PEI binding to the epithelial glycocalyx caused very extensive adherence of adjacent podocyte processes and the narrowing and loss of filtration slits. Also in these latter samples, discrete anionic sites in the LRE were no longer apparent and a dense band of PEI was seen under the foot processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of glomerular immune complex deposition by a circulating polycation.

It is known that polycations bind to and neutralize glomerular polyanions. Here we examine the effect of the polycation polyethyleneimine (PEI) on glomerular deposition of preformed immune complexes. Bovine serum albumin (BSA)-anti-BSA immune complexes made in 40 times antigen excess were administered following intravenous injection of PEI. Glomerular localization of immune deposits was assessed by quantitative immunofluorescence and electron microscopy and compared to controls receiving diluent without PEI followed by the same dose in immune complexes. In rats receiving PEI, deposits were localized within the glomerular basement membrane (GBM) of all peripheral capillary walls and in the mesangium. In controls, deposits localized exclusively within the mesangium in smaller amounts than after PEI. Thus, neutralization of glomerular polyanion by a circulating polycation enhances the deposition and alters the distribution of immune complexes in glomeruli.