Polycation Polyethylenimine Research Articles

Electron Transfer of Cellobiose Dehydrogenase in Polyethyleneimine Films

AbstractCellobiose dehydrogenase (CDH) is applied as a bioelectrocatalyst in biosensors because its mobile cytochrome domain is capable of direct electron transfer. This study investigates the electron transfer mechanism of CDH molecules embedded in the polycation polyethyleneimine (PEI), which has been reported as a current‐boosting component of CDH‐based biosensors. By immobilizing different concentrations of CDH and its isolated cytochrome domain in PEI films, we found that increasing concentrations of cytochrome enhanced the film conductivity (up to 251±8 mS cm−1) through improved electron transfer between the protein redox centers. The increased electrical conductivity of the film contacts CDH molecules at a greater distance from the electrode. The cross‐linker poly(ethylene glycol) diglycidyl ether improves the packing and contacting of the cytochrome domains, whereas glutaraldehyde reduces the current obtained. Deglycosylation of CDH enhances the conductivity of enzyme‐polymer films by up to 34 %, implying a higher number of productive electron‐hopping events between cytochrome domains due to enhanced mobility or reduced shielding. By balancing negative charges on the CDH surface at neutral and alkaline pH, PEI increases the interdomain electron transfer and the electrical film conductivity. The resulting increased current output is relevant for in vivo bioanalytical applications.

High-Performance and Stable Two-Dimensional MXene-Polyethyleneimine Composite Lamellar Membranes for Molecular Separation.

Two-dimensional (2D) materials are candidates for use in advanced molecular separation and water treatment. Among them, MXenes are cutting-edge two-dimensional (2D) materials with favorable properties such as high hydrophilicity, adjustable interlayer spacing, high mechanical strength, and structural stability. Therefore, they can be used to construct advanced lamellar membranes to ensure enhanced separation performance of modified membranes. Here, we prepared novel stable lamellar membranes through electrostatic attraction between polycation polyethyleneimine (PEI) and a negatively charged MXene, with hydrogen bond formation between their functional groups. By changing the pH of the suspension, the interlayer d-spacing of the prepared membrane could be altered to achieve precise molecular separation and ultrahigh organic solvent penetration. Furthermore, inserting PEI into the interlayer d-spacing of the membrane did not hinder the passage of water molecules. The prepared pH = 2-MXene-PEI membrane for dyes larger than 1.5 nm exhibited a rejection rate of greater than 96%, and the pH = 10-MXene-PEI membrane had a rejection rate of greater than 96% for dyes larger than 1.6 nm. In addition, the optimized MXene-PEI membranes showed channel stability. In this work, high-performance, stable, 2D MXene-PEI membranes with tunable nanochannels were developed. These membranes have great potential for use in precise molecular separation applications.

Physicochemical characterization and targeting performance of triphenylphosphonium nano-polyplexes

Mitochondrial gene therapy can be seen as a promising tool and a revolutionary approach towards mitochondrial diseases arising from mitochondrial DNA mutations. To pursue this goal, the mitochondria-targeted polycation polyethylenimine (PEI)/triphenylphosphonium (TPP) conjugate has been used to complex and condense both GFP or ND1 encoded plasmid DNA (pDNA) aiming at the formation of suitable vectors for mitochondrial gene therapy. The properties of pDNA based TPP-polyplexes have been addressed and appear to be valuable for gene delivery applications. In vitro studies using fluorescent pDNA demonstrated the internalization of the developed carriers and mitochondria targeting. Evidences of gene expression into mitochondria are also reported. Following this achievement, significant levels of both mitochondrial recoded GFP and ND1 proteins were quantified. This work represents a great step towards effective mitochondria-targeting gene delivery and functional protein expression, providing a significant contribution in mitochondrial gene therapy field.

Crown ether containing polyelectrolyte multilayer membranes for lithium recovery

Achieving solute selectivity has always been a goal of membrane development studies. The continuing growth of global consumption of scarce metals by different industries has put a strain on traditional sources of these species. Achieving cation selectivity in membranes, especially among monovalent cations, is a major step in introducing alternative sources for scarce metals such as lithium.Polyelectrolyte multilayer membranes (PEMMs) are a novel class of membranes, offering great potentials in monovalent/bivalent ion selectivity. On the other hand, crown ethers are a well-studied family of macrocyclic ligands capable of forming stable complexes with cations. In the current study, for the first time, we report on a PEMM nanofiltration membrane with crown ether moieties embedded in its structure for the goal of achieving monovalent salt selectivity. The crown ether 15-crown-5 was successfully incorporated in the polycation polyethylenimine (PEI), which was then used as the polycation in PEMM formation through layer by layer deposition. Both the synthesized polymer and the polyelectrolyte multilayer (PEM) were characterized and the performance of the resulting membrane was studied. It was determined that crown ether containing polymer forms more stable complexes with lithium than potassium. This was explained by the limitation put on 2:1 potassium-crownether complexes by steric hindrance from polymer chain. The manufactured membranes showed Li/K selectivity for a period of around 90 min, after which the crown ethers became saturated and selectivity was lost. The modified membranes became non selective after this point, but possessed high salt rejection potential.

Permeability of the Plasma Membrane for Propidium Iodide and Destruction of Cell Nuclei in the Epidermis of Pea Leaves: The Effect of Polyelectrolytes and Detergents

Damage to the plasma membrane (PM) of cells in pea leaf epidermis determined by its permeability to propidium iodide (PI), which binds to DNA of cell nuclei, and programmed cell death (PCD) detected by the destruction of cell nuclei were investigated. PM of the epidermal cells in the isolated epidermis was permeable to PI (it stained their nuclei). PM of the guard cells did not allow PI to pass through. KCN, an inducer of PCD, caused the destruction of both epidermal and guard cell nuclei. KCN-induced destruction of guard cell nuclei was accompanied by the penetration of PI into the cells. The polycation chitosan at a concentration of 0.1 mg/mL caused the destruction of the epidermal cell nuclei, but the permeability of the guard cell PM for PI staining their nuclei was induced at a concentration of 1 mg/mL. Other polycations (cytochrome c, polylysine, polyethylenimine, and protamine) also caused staining of the guard cell nuclei by PI. Polyanions (polyacrylic acid, dextran, and heparin) initiated the destruction of cell nuclei, which was accompanied by the penetration of PI into cells. Detergents Triton X-100 and lauryldimethylamine-N-oxide produced the permeability of the guard cell PM for PI and prevented the destruction of the nuclei that was induced by KCN. Treatment of the epidermis with Triton X-100 (for 2 h with its subsequent washing) increased the destruction of the guard cell nuclei that was caused by KCN. Polycations polyethyleneimine and protamine were prevented, while chitosan, cytochrome c, and polylysine, on the contrary, enhanced KCN-induced destruction of the guard cell nuclei. The obtained data shows that the destruction of cell nuclei upon induction of cell death with KCN or polyanions is accompanied by damage to PM (producing its permeability for PI). Damage to PM caused by detergents or polycations prior to cell treatment by KCN can prevent or, on the contrary, intensify the destruction of cell nuclei.

Gradient nanoporous phenolics as substrates for high-flux nanofiltration membranes by layer-by-layer assembly of polyelectrolytes

Abstract Thin film composite (TFC) membranes represent a highly promising platform for efficient nanofiltration (NF) processes. However, the improvement in permeance is impeded by the substrates with low permeances. Herein, highly permeable gradient phenolic membranes with tight selectivity are used as substrates to prepare TFC membranes with high permeances by the layer-by-layer assembly method. The negatively charged phenolic substrates are alternately assembled with polycation polyethylenimine (PEI) and polyanion poly(acrylic acid) (PAA) as a result of electrostatic interactions, forming thin and compact PEI/PAA layers tightly attached to the substrate surface. Benefiting from the high permeances and tight surface pores of the gradient nanoporous structures of the substrates, the produced PEI/PAA membranes exhibit a permeance up to 506 L ∙ m−2 ∙ h−1 ∙ MPa−1, which is ~ 2–10 times higher than that of other membranes with similar rejections. The PEI/PAA membranes are capable of retaining > 96.1% of negatively charged dyes following the mechanism of electrostatic repulsion. We demonstrate that the membranes can also separate positively and neutrally charged dyes from water via other mechanisms. This work opens a new avenue for the design and preparation of high-flux NF membranes, which is also applicable to enhance the permeance of other TFC membranes.

Adsorption of polyelectrolytes onto the oppositely charged surface of tubular J-aggregates of a cyanine dye

The adsorption of three different polycations at the negatively charged surface of tubular J-aggregates of the amphiphilic cyanine dye 3,3′-bis(2-sulfopropyl)-5,5′,6,6′-tetrachloro-1,1′-dioctylbenzimidacarbocyanine (C8S3) is investigated by means of cryogenic electron microscopy and optical absorption spectroscopy. All three polycations could be adsorbed at the tubular aggregates without flocculation or precipitation when added in molar amounts of monomers sufficiently smaller than that of the dye molecules. It is found that preferably, a minority of aggregates is coated by the polycations while a majority of aggregates is left uncoated. For the coated aggregates, the adsorption leads to charge reversal of the aggregate surface as supported by zeta potential measurements. The morphology of the coating differs significantly for the three polycations: The branched polycation polyethylenimine (PEI) attaches to the tubular aggregate by hit-and-stick adsorption of the coiled state in solution forming irregular clot-like coatings; the flexible and weakly cationic poly (allylamine hydrochloride) (PAH) forms a more homogeneous coating but destroys the integrity of the dye aggregate; the more hydrophobic and strong polycation poly (diallyldimethylammonium chloride) (PDADMAC) forms a thin and homogeneous layer, supposedly by wrapping around the tubular aggregate. For the latter growth of a second double layer of dyes is observed for the aggregates. The different morphologies of the coating layers are explained by the details of the chemical structure of the polycations. The possible adsorption of polyelectrolytes at these amphiphilic tubular structures, stabilized by means of hydrophobic forces, is far from obvious and demonstrates an applicable route to the build-up of more complex nanostructures in solution by means of a self-assembly process.

A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro.

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.

Edge activators and a polycationic polymer enhance the formulation of porous voriconazole nanoagglomerate for the use as a dry powder inhaler

Purpose: Voriconazole has both low aqueous solubility and stability. We hypothesize that designing voriconazole in the form of a nano powder inhaler at a geometric diameter within 1–5 μm will enhance its stability and solubility. Therefore, we prepared nanoagglomerates of voriconazole which will collapse in the lungs to reform the nanoparticles.Method: The nanoparticles were formulated using both stearic acid and sodium deoxycholate as edge activators. Osmogenic polycation polyethyleneimine (PEI) was used to form agglomerates of controllable size.Results: Voriconazole nanoparticles and agglomerates showed a significant higher cumulative drug release than the pure powder (p < 0.05) with R2 = 0.95. Small-sized particles were formed (353 nm), while their zeta potential was −30.7 mV. The agglomerates were 2.7 μm in size and their zeta potential was −20.9 mV. The formation of porous agglomerates was confirmed using a transmission electron microscope. Cascade impactor was used to evaluate the aerodynamic properties of the nanoparticles and the agglomerates. The aerodynamic characterization of the nanoparticles and the agglomerates resulted in a significant smaller mass median aerodynamic diameter (MMAD) (p < 0.05) and higher fine particle dose (FPD) (p < 0.01), fine particle fraction (FPF) (p < 0.01), and total emitted dose (TED) (p < 0.01) than the pure powder.Conclusion: The results suggest that using the combination of edge activators and diluted polycationic polymer solution provides porous voriconazole nanoagglomerates in a respirable range, which is proved successful in enhancing both the deposition and the dissolution of water insoluble-drugs in the lung.

Optoacoustic imaging enabled biodistribution study of cationic polymeric biodegradable nanoparticles.

Nanosized contrast agents for molecular imaging have attracted widespread interest for diagnostic applications with high resolution in medicine. However, many solid nanoparticles exhibit a great potential to induce toxicity, hindering their use for clinical applications. On the other hand, near-infrared (NIR) dyes have also been used for extensive biological applications, but show some limitations due to their poor aqueous stability, tendency to aggregation and rapid elimination from the body. An alternative proposed in this work to overcome these limitations is the use of NIR dye-loaded nanoparticles. Here we introduce nanoparticles constructed with poly(D,L-lactide-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer widely used for biomedical applications, attached to the polycation polyethyleneimine (PEI) to obtain positively charged nanoparticles. The in vivo biodistribution of the cationic PEI-PLGA nanoparticles was investigated after administration through three different routes (intravenous, intraperitoneal and subcutaneous) using multispectral optoacoustic tomography (MSOT). The prepared nanoparticles exhibited good colloidal stability and adequate optical properties for optoacoustic imaging. The in vivo biodistribution assays indicated a strong accumulation of the particles in the liver and spleen, and retention in these organs for at least 24 h. Therefore, these nanoparticles could find promising applications in MSOT due to a sharp and characteristic optoacoustic spectrum and high optoacoustic signal generation, and become a promising building block for theranostic strategies.

Use of the polycation polyethyleneimine to improve the physical properties of alginate–hyaluronic acid hydrogel during fabrication of tissue repair scaffolds

Recently alginate-based tissue repair scaffolds fabricated using 3D printing techniques have been extensively examined for use in tissue engineering applications. However, their physical and mechanical properties are unfavorable for many tissue engineering applications because these properties are poorly controlled during the fabrication process. Some improvement of alginate gel properties can be realized by addition of hyaluronic acid (HA), and this may also improve the ability of cells to interact with the gel. Here, we report improvement of the physical properties of alginate–HA gel scaffolds by the addition of the polycation polyethyleneimine (PEI) during the fabrication process in order to stabilize alginate molecular structure through the formation of a polyelectrolyte complex. We find that PEI has a significant beneficial influence on alginate–HA scaffold physical properties, including a reduction in the degree of gel swelling, a reduction in scaffold degradation rate, and an increase in the Young’s modulus of the gel. Further study shows that fabrication of alginate–HA gels with PEI increases the encapsulation efficiency of bovine serum albumin, a model protein, and reduces the subsequent initial protein release rate. However, it was also found that survival of Schwann cells or ATDC-5 chondrogenic cells encapsulated during the scaffold fabrication process was modestly reduced with increasing PEI concentration. This study illustrates that the use of PEI during scaffold fabrication by plotting can provide an effective means to control alginate-based scaffold properties for tissue engineering applications, but that the many effects of PEI must be balanced for optimal outcomes in different situations.

Polyion multilayers with precise surface charge control for antifouling.

We report on a molecular fabrication approach to precisely control surface ζ potentials of polymeric thin layers constructed by electrostatic layer-by-layer (LbL) assembly methods. The protocol established allows us to achieve surface isoelectric points (IEP) in the pH range of 6-10. Poly(acrylic acid) (PAA, a weak polyanion) and poly(diallyldimethylammonium chloride) (PDADMAC, a strong polycation) were chosen to build up the bulk films. The weak polycation polyethylenimine (PEI) was applied as a top layer. A unique feature of this approach is that the chemical composition of the top layer is not affected by the manipulation of the ζ potential of the films. Surface charge tuning is achieved by controlling the degree of ionization of the weak polyelectrolytes at various pH values and subsequent manipulation of the amount of polyelectrolyte deposited in the penultimate and last layers, respectively. Following assembly and characterization, the films were used as candidates for antifouling surfaces. The fouling behavior of barnacle cyprids and bacteria on the LbL films with similar hydrophilicity and roughness but different surface charge densities were studied. We found that more cyprids of Amphibalanus amphitrite settled on the negatively charged LbL film compared to the neutral or positively charged LbL film. In bacterial adhesion tests employing Pseudomonas, Escherichia coli, and Staphylococcus aureus, more bacteria were observed on the positively charged LbL film compared with the neutral and negatively charged LbL films, possibly as a result of the negative potential of the bacterial cell wall. The procedures proposed allow one to adjust surface isoelectric points of LbL architectures to achieve optimal antifouling performance of a given material taking into account specific pH values of the environment and the character of the fouler.

Bioplotting Alginate/Hyaluronic Acid Hydrogel Scaffolds with Structural Integrity and Preserved Schwann Cell Viability

Bioplotting is an emerging freeform scaffold fabrication technique useful for creating artificial tissue scaffolds containing living cells. Simultaneous maintenance of scaffold structural integrity and cell viability is a challenging task. In this article, we present strategies developed to bioplot alginate-based three-dimensional tissue scaffolds containing hyaluronic acid and living Schwann cells for potential use in peripheral nerve tissue engineering. The fabrication platform, upon which the scaffold is created, was coated with the polycation polyethylenimine to immobilize the first layer of the scaffold on the platform. Each layer was then dispensed into a bath containing calcium chloride to cross-link the alginate, polyvinyl alcohol, and low concentrations of polyethylenimine to improve the structural integrity of the resulting scaffolds while retaining biocompatibility. The present study illustrated that with these strategies, porous alginate/hyaluronic acid scaffolds can be fabricated with good structural integrity and long-term cell viability. Plotting method and speed were also important factors determining the printability of single-layered scaffolds. The methods developed and the results obtained from this study form the basis for a general bioplotting method by which more sophisticated alginate/hyaluronic acid scaffolds can be fabricated with the inclusion of living cells into desired structures.

Polyethyleneimine as a promoter layer for the immobilization of cellobiose dehydrogenase from Myriococcum thermophilum on graphite electrodes.

Cellobiose dehydrogenase (CDH) is a promising enzyme for the construction of biofuel cell anodes and biosensors capable of oxidizing aldoses as cellobiose as well as lactose and glucose and with the ability to connect to an electrode through a direct electron transfer mechanism. In the present study, we point out the beneficial effect of a premodification of spectrographic graphite electrodes with the polycation polyethyleneimine (PEI) prior to adsorption of CDH from Myriococcum thermophilum (MtCDH). The application of PEI shifts the pH optimum of the response of the MtCDH modified electrode from pH 5.5 to 8. The catalytic currents to lactose were increased up to 140 times, and the K(M)(app) values were increased up to 9 times. The previously investigated, beneficial effect of divalent cations on the activity of CDH was also present for graphite/PEI/MtCDH electrodes but was less pronounced. Polarization curves revealed a second unexpected catalytic wave for graphite/PEI/MtCDH electrodes especially pronounced at pH 8. Square wave voltammetric studies revealed the presence of an unknown redox functionality present at 192 mV vs Ag|AgCl (0.1 M KCl) at pH 8, probably originating from an oxidized adenosine derivative. Adenosine is a structural part of the flavin adenine dinucleotide (FAD) cofactor of the dehydrogenase domain of CDH. It is suggested that for some enzyme molecules FAD leaks out from the active site, adsorbs onto graphite, and is oxidized on the electrode surface into a product able to mediate the electron transfer between CDH and the electrode. PEI is suggested and discussed to act in several manners by (a) increasing the surface loading of the enzyme, (b) possibly increasing the electron transfer rate between CDH and the electrode, and (c) facilitating the creation or immobilization of redox active adenosine derivatives able to additionally mediate the electron transfer between CDH and the electrode.

Nucleic Acid Carrier Systems Based on Polyethylenimine Conjugates for the Treatment of Metastatic Tumors

Nucleic acid (NA) based drugs offer the potential of highly selective treatments for malignant diseases. They act as an initially inactive pro-drug being activated at the intended site of action, either by translation into a protein in case of plasmid DNA or through expression shutdown by interfering specifically with messenger RNA (RNAi technology). In case of already metastasized cancer, systemic treatment via the blood stream is often inevitable to reach the lesion. This makes it necessary to protect NAs from enzymatic degradation, but also to target them to the tumor with appropriate ligands. Polycationic molecules can provide such functions by condensing NAs into virus sized particles by virtue of charge interaction with the negatively charged phosphate backbone of NAs. Here we review the application of NA carrier systems based on the polycation polyethylenimine (PEI), where peptide based ligands are attached to the polycation via heterobifunctional polyethylene glycol linker molecules. Conjugate synthesis, in vitro testing and in vivo application in subcutaneous and disseminated cancer models in rodents are discussed.

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

Transient gene expression in mammalian cells is a valuable alternative to stable cell lines for the rapid production of large amounts of recombinant proteins. While the establishment of stable cell lines takes 2–6months, milligram amounts of protein can be obtained within a week following transfection. The polycation polyethylenimine (PEI) is one of the most utilized reagents for small- to large-scale transfections as it is simple to use and, when combined with optimized expression vectors and cell lines, provides high transfection efficiency and titers. As with most transfection reagents, PEI-mediated transfection involves the formation of nanoparticles (polyplexes) which are obtained by its mixing with plasmid DNA. A short incubation period that allows polyplexes to reach their optimal size is performed prior to their addition to the culture. As the quality of polyplexes directly impacts transfection efficiency and productivity, their formation complicates scalability and automation of the process, especially when performed in large-scale bioreactors or small-scale high-throughput formats. To avoid variations in transfection efficiency and productivity that arise from polyplexes formation step, we have optimized the conditions for their creation directly in the culture by the consecutive addition of DNA and PEI. This simplified approach is directly transferable from suspension cultures grown in 6-well plates to shaker flasks and 5-L WAVE bioreactors. As it minimizes the number of steps and does not require an incubation period for polyplex formation, it is also suitable for automation using static cultures in 96-well plates. This “direct” transfection method thus provides a robust platform for both high-throughput expression and large-scale production of recombinant proteins.

Kinetically Controlled Cellular Interactions of Polymer−Polymer and Polymer−Liposome Nanohybrid Systems

Although bioactive polymers such as cationic polymers have demonstrated potential as drug carriers and nonviral gene delivery vectors, high toxicity and uncontrolled, instantaneous cellular interactions of those vectors have hindered the successful implementation In Vivo. Fine control over the cellular interactions of a potential drug/gene delivery vector would be thus desirable. Herein, we have designed nanohybrid systems (100-150 nm in diameter) that combine the polycations with protective outer layers consisting of biodegradable polymeric nanoparticles (NPs) or liposomes. A commonly used polycation polyethylenimine (PEI) was employed after conjugation with rhodamine (RITC). The PEI-RITC conjugates were then encapsulated into (i) polymeric NPs made of either poly(lactide-co-glycolide) (PLGA) or poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA); or (ii) PEGylated liposomes, resulting in three nanohybrid systems. Through the nanohybridization, both cellular uptake and cytotoxicity of the nanohybrids were kinetically controlled. The cytotoxicity assay using MCF-7 cells revealed that liposome-based nanohybrids exhibited the least toxicity, followed by PEG-PLGA- and PLGA-based NPs after 24 h incubation. The different kinetics of cellular uptake was also observed, the liposome-based systems being the fastest and PLGA-based systems being the slowest. The results present a potential delivery platform with enhanced control over its biological interaction kinetics and passive targeting capability through size control.

Removable Nanocoatings for siRNA Polyplexes

To assist in overcoming the inherent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(l-lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (M(w)), the hydrodynamic radius (R(h)), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.

OprMas a new target for reversion of multidrug resistance inPseudomonas aeruginosaby antisense phosphorothioate oligodeoxynucleotides

Multidrug-resistant Pseudomonas aeruginosa (MDR-PA) is one of the leading Gram-negative organisms associated with nosocomial infections. The increasing frequency of MDR-PA has represented a huge challenge in conventional antibacterial therapy. The loss of effectiveness of commonly used antibiotics calls for the immediate need to develop an alternative strategy for combating MDR-PA infections. The multiantibiotic resistance of MDR-PA is largely attributable to the production of multidrug efflux pumps, MexAB-OprM. OprM forms the antibiotic-ejecting duct and plays a crucial role in exporting incoming chemotherapeutic agents across the membranes. Disruption of the OprM expression may inhibit the function of multidrug efflux pumps and lead to restoration of MDR-PA susceptibility to antibiotics. In this study, we developed a novel anion liposome for encapsulating and delivering specific anti-oprM phosphorothioate oligodeoxynucleotide (PS-ODN617) and polycation polyethylenimine (PEI) complexes. The additions of the encapsulated anti-oprM PS-ODN617/PEI to MDR-PA isolates caused a significant reduction of oprM expression and inhibition of MDR-PA growth in the presence of piperacillin in a concentration-dependent manner. The encapsulated PS-ODN617 treatment also reduced minimal inhibitory concentrations of five most commonly used antibiotics to the sensitive margin values on MDR-PA clinical isolates, respectively. The results of present study firstly indicate that PS-ODN targeted to oprM can significantly restore the susceptibility of MDR-PA to existing antibiotics, which appears to be a novel strategy for treating MDR-PA infections.

Self-Assembly of SiO&lt;SUB&gt;2&lt;/SUB&gt;/Gd-DTPA-Polyethylenimine Nanocomposites as Magnetic Resonance Imaging Probes

Controlled self-assembly of organic/inorganic magnetic hybrid materials have important applications in magnetic resonance imaging (MRI). In this study, a widely used polycation polyethylenimine was conjugated with gadopentetic acid (Gd-DTPA) as a gadolinium bearing polyelectrolyte (Gd-DTPA-PEI). Next, multilayers of Gd-DTPA-PEI were coated on silica nanoparticles through layer-by-layer (LbL) self-assembly with polyanions as monitored by dynamic light scattering, zeta-potential, and scanning electron microscopy. The thickness of the multilayer film was estimated from quartz crystal microbalance based on counting frequency change of each adsorbed layer. The magnetic relaxation of SiO2/(Gd-DTPA-PEl/polyanion), core-shell nanocomposite was tested at 1.5 T magnetic field in a clinical MRI scanner, and a 3-fold increase in T1 relaxivity to 15.1 Gd mM(-1)s(-1) was noticed comparing to Gd-DTPA small molecules. Dextran sulfate was coated as the outermost layer on the nanocomposite for better biocompatibility as verified by in vitro cytotoxicity studies. This formulation provides good signal intensity enhancement of mouse liver in vivo with only 1/25 dose of clinical standard at 30 and 60 minutes after intravenous injection. This sensitive imaging probe with unique core-shell structures may find broad applications in cellular and molecular imaging.