Nonviral gene delivery to the lung with copolymer-protected and transferrin-modified polyethylenimine

Polyethylenimine (PEI) has been shown to efficiently mediate topical gene transfer to the lungs after either direct intratracheal instillation or nebulisation. Recently, the protection of polyplexes with novel copolymers of poly(ethylene glycol) (PEG) via electrostatic interaction has been reported. In this study, such coated PEI polyplexes were investigated for their stability and interaction with human plasma and bronchoalveolar lavage fluid (BALF). Further, their potential for gene delivery to the mouse lungs in vivo was examined. Plasma protein and mucin adsorption was effectively inhibited when polyplexes were coated with the novel copolymers. Gene transfer efficiency of the coated PEI polyplexes decreased as compared with uncoated PEI polyplexes when administered intratracheally to the lung. The higher the molecular weight of the copolymerized PEG was, the stronger the observed gene transfer reduction. Gene transfer decreased presumably due to reduced interaction of the coated gene vectors with the cell surface. To circumvent this problem, transferrin was combined with PEI/DNA polyplexes for specific binding to the cell surface. In this case, gene transfer efficiency decreased. Gene transfer of the copolymer-protected and transferrin-modified gene vectors increased as compared with the copolymer-protected gene vectors alone but did not reach the level of uncoated gene vectors. These data show that copolymers could be used to effectively shield polyplexes from interaction with components of the airway surface liquid (ASL). Increased gene delivery was found upon transferrin modification of the coated PEI polyplexes suggesting a targeting effect.     

Adsorption of polyethyleneimine on silver nanoparticles and its interaction with a plasmid DNA: a surface-enhanced Raman scattering study

Raman spectroscopy is applied in this work to study the adsorption of poly(ethyleneimine) (PEI) on Ag nanoparticles obtained by reduction with citrate, as well as to the study of the interaction between PEI and a plasmid. The surface-enhanced Raman spectroscopy (SERS) affords important information about the interaction and orientation of the polymer on the particles. In particular we have found that this polymer interacts with the surface through their amino groups in an interaction which also involves a change in the protonation state of amino groups as well as an increase of the chain order. This interaction implies a charge-transfer effect as deduced from the strong resonant effect in Raman spectra obtained at different excitation wavelengths. The complex formed by PEI and a plasmid, obtained by encoding the HBV (hepatitis B virus) genome inside the EcoRI restriction site of pGEM vector, was also studied by SERS. The interaction between both polymers leads to a conformational change affecting both macromolecules that can be detected by Raman at different excitation wavelengths. PEI undergoes a change to a more disordered structure as well as an increase of the number of protonated amino groups. The plasmid undergoes a structural change from A-DNA structure to B-DNA, along with a change in the superhelicity resulting in a more lineal structure when the plasmid interacts with PEI.     

A molecular dynamics study on the thermal rectification effect at the solid–liquid interfaces between the face-centred cubic (FCC) of gold (Au) with the surfaces of (100), (110) and (111) crystal planes facing the liquid methane (CH4)

A molecular dynamics study on the solid–liquid (S-L) interfaces for solid wall of gold having the face-centred cubic of (100), (110) and (111) crystal planes contacting liquid methane was examined using non-equilibrium molecular dynamics simulations. An investigation on the thermal rectification effect was performed by measuring the thermal boundary conductance (TBC) at the S-L interface. Thermal rectification can be defined as the differences in the TBC at the interface between the two opposite heat flow directions; one is from the liquid to solid and vice versa. The thermal rectifications are up to 13% for (110) crystal plane, followed by 6% and 0.3% for (111) and (100) crystal planes, respectively. It was found that the TBC at the S-L interface was influenced by the magnitude of the adsorption of liquid molecules at the vicinity of the interface. The results show that due to the different temperature distribution, different magnitude of the adsorption of liquid molecules is generated for the two opposite heat flow directions. On the surface of the solid walls for (110) crystal plane, where lattice-scale corrugation exists, it was found that there exists difference in distance between the surface layers of the solid and liquid across the interface between the cases of the two opposite heat flow directions, which affects the TBC at the interface. The present results suggest that the factors that influence the thermal rectification at the S-L interface are the magnitude of the adsorption of liquid molecules and the surface structure of the solid walls that differ significantly among the three types of crystal planes.     

Low molecular weight polyethylenimine for efficient transfection of human hematopoietic and umbilical cord blood-derived CD34+ cells

With the emerging role of hematopoietic stem cells as potential gene and cell therapy vehicles, there is an increasing need for safe and effective nonviral gene delivery systems. Here, we report that gene transfer and transfection efficiency in human hematopoietic and cord blood CD34+ cells can be enhanced by the use of low molecular weight polyethylenimine (PEI). PEIs of various molecular weights (800-750,000) were tested, and our results showed that the uptake of plasmid DNA by hematopoietic TF-1 cells depended on the molecular weights and the N/P ratios. Treatment with PEI 2K (m.w. 2000) at an N/P ratio of 80/1 was most effective, increasing the uptake of plasmid DNA in TF-1 cells by 23-fold relative to Lipofectamine 2000. PEI 2K-enhanced transfection was similarly observed in hematopoietic K562, murine Sca-1+, and human cord blood CD34+ cells. Notably, in human CD34+ cells, a model gene transferred with PEI 2K showed 21,043- and 513-fold higher mRNA expression levels relative to the same construct transfected without PEI or with PEI 25 K, respectively. Moreover, PEI 2K-treated TF-1 and human CD34+ cells retained good viability. Collectively, these results indicate that PEI 2K at the optimal N/P ratio might be used to safely enhance gene delivery and transfection of hematopoietic and human CD34+ stem cells.     

A composite gene delivery system consisting of polyethylenimine and an amphipathic peptide KALA

BACKGROUND: Animal viruses such as enveloped virus carry multi-functional proteins in the virion that can mediate more than two distinct steps of a gene delivery process during the transfer of viral genome into host cells. We tested if the aspects of the viral gene delivery mechanism could be mimicked by forming composite formulae from multi-functional synthetic gene carriers having complementary action modes. METHODS: Polyethylenimine (PEI) was chosen as the component responsible for endosome escape and DNA condensation and KALA for cellular entry and DNA condensation. Compact DNA-carrier particles consisting of the core part where DNA chains were tightly condensed by PEI and the outer layer lined with KALA were formulated, characterized and compared with monolithic cationic formulae in terms of gene delivery efficiency and mechanism. RESULTS: High-level gene expression was observed when C2C12 cells were transfected with DNA that was first partially condensed with PEI and, then, fully with KALA. In these formulae KALA mediated enhanced cellular entry of DNA by facilitating endocytic vesicle formation, while PEI provided an effective endosomolytic capacity. An optimal PEI/KALA formula showed transfection efficiencies better than or comparable to the commercial cationic liposome in various cell types in culture and in vivo. CONCLUSIONS: Gene delivery by combining the membrane-active property of KALA with the endosomolytic activity of PEI can be more efficient than that by either of the properties alone. It appears that, in these formulae, the predominant role of KALA is to facilitate cellular entry of DNA by providing a fusogenic capability, rather than an endosomolytic activity.     

Comparative filter binding study of H5 to nucleosome core particles,H1, H5 depleted chromatosomes and DNA fragments

The filter-binding technique with PEI treated glass fiber is used to study the interaction of histone H5 to core particles, chromatosomes and DNA derived from it. By working at very low concentrations of interacting particles we are able to study the effective binding process independent of interfering insoluble complexes. The interactions are characterized by a very high affinity. An intrinsically higher affinity of H5 for cores and chromatosomes versus chromatosome derived DNA is demonstrated. Both chromatosomes and DNA derived from these bind about twice the amount as compared to core particles, which saturate at about one H5 per core particle.Abbreviations GH5 globular domain of histone H5 - PEI polyethyleneimine     

Interaction of the photosystem 1 reaction center with antenna chlorophyll in a pigment-protein complex isolated from pea chloroplasts

Using a specially developed phosporoscopic attachment to spectropolarimeter, light induced spectra of circular dichroism (CD) in region 600-750 nm were measured for a pigment protein complex of photosystem 1 (PC-1) isolated from pea chloroplast (chlorophyll : P700 = 40). Minor components at 672 and 678 nm are observed in light induced spectra besides the components of dimer splitting of P700 Qy transition at 691 and 698 nm. Haussian deconvolution of light induced CD spectra of P700 and low temperature CD spectrum of PC-1 indicates that minor components are due to forms of antenna chlorophylls Chl672 and Chl678, rotational strength of that is changed by 2-4% as a result of P700 oxidation. Long term incubation of PC-1 with Triton X-100 inhibits P700 and destroys longwave optically active chlorophyll forms. A strong relation between dichroic density of 693 nm band in CD spectrum of PC-1 and the value of light induced absorption change at 698 nm could be used to determine P700 concentration on the basis of CD spectrum of PC-1. Such a relation shows that Chl693 is an important component of photo-system 1 reaction center. It is suggested that P700 is not an isolated dimer but it is included in the local complex from 8-10 chlorophyll molecules (Chl672, Chl678, Chl686, Chl693).     

Responses of Marine Bacteria Under Starvation Conditions at a Solid-Water Interface

Size changes during starvation of 17 marine bacterial isolates at a solid-water interface and in the liquid phase were examined. Twelve rod-shaped, hydrophilic bacteria decreased in size more rapidly at the solid surface than in the liquid phase, a result parallel to that observed previously for one of the strains at an air-water interface. On the other hand, three rod-shaped, hydrophobic bacteria diminished in size more rapidly in the liquid phase than at the solid-water interface. The rapid size decrease (defined here as the dwarfing phase) in either situation appeared to be an active process which occurred more rapidly when the cells were in an early stage of logarithmic growth at the onset of starvation. Dwarfing was reversibly inhibited by low temperature and low pH but was not inhibited by chloramphenicol. Three coccoidal bacteria showed little tendency to become smaller upon starvation in the liquid phase or at a surface.     

Specific and nonspecific interactions of integration host factor with oligo DNAs as revealed by circular dichroism spectroscopy and filter binding assay.

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.     

Modeling of direct recovery of lactic acid from whole broths by ion exchange adsorption

Lactic acid fermentation process with L. casei CRL 686 was performed. The static adsorption isotherm over a strong anionic exchange resin, Amberlite^TM IRA-400 was measured, and the static binding capacity parameters were quantified. Early recovery of lactic acid from this lactate producer from unclarified culture broth was performed in a liquid solid fluidized bed, with the resin as the solid adsorbent, and the dynamic adsorption capacity was calculated. Good agreement was found between static and dynamic binding capacity values. The fluidized bed height was twice the settled bed height and the overall process was controlled by the liquid solid mass transfer. This operation was also simulated by continuously well stirred tanks arranged in series and superficial solid deactivation as in a gas solid catalytic reactor. The deactivation process takes into account liquid channeling and agglomerations of solid induced by the viscosity of the broth and also by the cells during the adsorption. These patterns were also verified by experimental observations, and are in agreement with the results found in the literature. The breakthrough data together with others from previous works were satisfactorily fitted until the 90% dimensionless concentration was reached for both culture broths. The model could be used in future studies on predictions about the liquid solid fluidized bed behavior and other different operating conditions.     

Interfacial properties and structural analysis of the antimicrobial peptide NK-2.

The structure of the antimicrobial peptide NK-2 has been studied at the air-water interface and in different solutions using spectroscopic methods such as circular dichroism (CD) and infrared reflection absorption spectroscopy (IRRAS) as well as specular X-ray reflectivity (XR). NK-2 adopts an unordered structure in water, buffer, and in the presence of monomeric cationic and noncharged amphiphiles. However, it forms a stable alpha-helix in 2,2,2-trifluoroethanol (TFE) and in micellar solutions of anionic, cationic as well as nonionic amphiphiles, whereas only in sodium dodecyl sulfonate solutions the alpha-helical structure can also be found below the critical micellar concentration (cmc). The amphiphilic molecule NK-2 is surface active and forms a Gibbs monolayer at the air-buffer interface. In contrast, no adsorption was observed if NK-2 is dissolved in water. During the adsorption process in buffer solutions, NK-2 undergoes a conformational transition from random coil in bulk to alpha-helix at the interface. This change of the peptide's secondary structure is known to be associated with its antimicrobial activity. A comparison of the experimental IRRA spectra with the simulated spectra indicates that the adsorbed NK-2 alpha-helix lies flat at the interface. This is confirmed by XR measurements which show that the thickness of the NK-2 layer is approximately 17 A, which is the average diameter of a alpha-helix, indicating that only a monomolecular adsorption layer is formed.     

Effect of cosolvents on the adsorption of peptides at the solid-liquid interface

The adsorption of a peptide at solid surfaces is the result of a complex interplay of interactions between the peptide, solvent, and surface. In this work, Monte Carlo simulations were performed to evaluate the effect of the solvent hydrogen bonding ability on the adsorption of the peptide ASP(1)-ASP(2)-ILE(3)-ILE(4)-ASP(5)-ASP(6)-ILE(7)-ILE(8) at a charged surface consisting of CH(2) atoms with a fixed lattice arrangement. Various water-alcohol mixtures were used as solvent because alcohols are known to alter the dielectric constant, hydrophobicity, and hydrogen bonding capacity of water. Solvent-solvent, solvent-surface, solvent-peptide, and peptide-surface interactions were studied independently and correlated with the observed peptide behavior at the solvent-surface interface. We concluded that the behavior (and orientation) of the peptide at the surface is directly related to changes in water-water hydrogen bonding properties in water-alcohol mixtures. In the presence of increasing concentrations of methanol, the strength of solvent-peptide and solvent-surface interactions was reduced, and as a result, a stronger interaction between the peptide and the surface was observed. Stronger solvent-peptide and solvent-surface interactions were responsible for a weaker interaction of the peptide with the surface in the presence of increasing concentrations of glycerol. These results suggest that by changing solvent conditions it is possible to finely tune the orientation of a macromolecule at solid/liquid interfaces.     

Molecular dynamics simulations of PEI mediated DNA aggregation

Understanding the molecular mechanism of polycation induced DNA aggregation and condensation is important for optimal design of gene delivery carriers. In this work, we performed a series of all-atom molecular dynamics (MD) simulations to investigate polyethylenimine (PEI) mediated DNA aggregation. We found that PEIs condense DNA through two mechanisms: polyion bridging and electrostatic screening of the DNA charges. At PEI/DNA charge ratio >1, PEIs can completely neutralize DNAs at a short distance (～12 ? from the C1' atoms), and this distance is found to be insensitive to the exact value of the charge ratio. When excess PEIs are added to a formed DNA-PEI aggregate, they are found to bind to the aggregate and increase its cationic charge. The added PEIs can also replace the PEIs previously bound to the aggregate. The excess PEIs, however, do not change the spacing of the DNAs in the aggregates. Our simulation results shed light on the mechanisms of PEI, and more generally polycation, mediated DNA aggregation and condensation.     

Cholesterol biosensors prepared by layer-by-layer technique

The analysis of formation, deposition and characterization of cholesterol oxidase (COX) layer-by-layer films were performed. Initially, a layer of polyanion, poly(styrene sulfonate) (PSS) was adsorbed followed by a layer of polycation, poly(ethylene imine) (PEI) on each solid substrate from aqueous solutions. The alternating layers were formed by consecutive adsorption of polycations (PEI) and negatively charged proteins (COX) and cholesterol esterase (CE). A strong interaction between protein and polyelectrolyte improves the stability of the alternating multilayer; however, it can change a native protein conformation and impair the protein activity. The PSS/PEI/COX, PSS/PEI/COX/PEI/CE, PSS/PEI/COX-CE/PEI etc. layered structures were prepared on the surface of a platinum electrode, ITO coated glass plate, quartz crystal microbalance, quartz plates, mica and silicon substrates. Optical and gravimetric measurements based on an ultraviolet–visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of molecular layered of the proteins. The surface morphology of such bilayer films was investigated by using atomic force microscopy. The electrochemical redox processes of the enzyme-layered films deposited either on platinum or ITO coated glass plate were investigated. The response current of cholesterol oxidase electrode with concentration of cholesterol was investigated at length.     

Further characterization of cooperative interactions of monoclonal antibodies

We reported previously that mixtures of some monoclonal antibodies directed against the glycoprotein hormone human chorionic gonadotropin (hCG) had a higher affinity for the antigen than either monoclonal antibody separately. The synergistic interaction could no longer be detected when one of the antibodies was replaced with its F(ab) fragment. This cooperative interaction has now been further characterized. One-half of 10 possible pairs prepared from five IgG1 monoclonal antibodies against hCG result in a synergistic interaction. The addition of an IgG2b monoclonal antibody to one of the IgG1 monoclonal antibodies also induces a cooperative interaction, which shows that the effect is not subclass restricted. Cooperative interactions between antibodies are also not restricted to solution conditions; adsorption of one antibody to a solid support appears to increase the cooperative effect. Indeed, one pair of antibodies that failed to bind hCG synergistically in solution did so when one antibody was bound to a solid surface. The liquid phase antibody also has an effect on the specificity of the solid phase antibody. The sensitivity of the solid phase assay system has enabled us to develop a rapid method of determining if two monoclonal antibodies can bind to an antigen simultaneously. A quantitative theoretical model has been devised that successfully predicts the cooperative behavior observed between antibodies and should be useful in devising conditions that result in sensitive solid phase radioimmunoassays.     

Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV–vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV–vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.     

Heparan sulfate targets the HIV-1 envelope glycoprotein gp120 coreceptor binding site

Human immunodeficiency virus (HIV) attachment to host cells is a multi-step process that involves interaction of the viral envelope gp120 with the primary receptor CD4 and coreceptors. HIV gp120 also binds to other cell surface components, including heparan sulfate (HS), a sulfated polysaccharide whose wide interactive properties are exploited by many pathogens for attachment and concentration at the cell surface. To analyze the structural features of gp120 binding to HS, we used soluble CD4 to constrain gp120 in a specific conformation. We first found that CD4 induced conformational change of gp120, dramatically increasing binding to HS. We then showed that HS binding interface on gp120 comprised, in addition to the well characterized V3 loop, a CD4-induced epitope. This epitope is efficiently targeted by nanomolar concentrations of size-defined heparin/HS-derived oligosaccharides. Because this domain of the protein also constitutes the binding site for the viral coreceptors, these results support an implication of HS at late stages of the virus-cell attachment process and suggest potential therapeutic applications.     

Wettability of polymers by mucin aqueous solutions

The wettability of poly(methyl methacrylate) and polyethylene by water and aqueous mucin solutions have been studied by sessile drop and under-water captive air bubble contact angles, respectively. From the sessile drop and octane under-water contact angles the polymer-water interfaces have been characterized in terms of works of adhesion and acid-base (polar) interactions. A large water-air contact angle hysteresis observed with poly(methyl methacrylate) surfaces has been attributed to side-chain beta relaxations of polymer ester methyl groups. The wettabilities of the polymers by mucin aqueous solutions have been studied as a function of protein concentration and related to the surface tensions. A positive slope of adhesion tension vs surface tension line, characteristic of polar surfaces, was found with poly(methyl methacrylate). By contrast, a change in the slope, explained as a change in mucin relative adsorption densities at solid/liquid and solid/vapour interfaces, was observed with polyethylene. This adhesion tension behavior appeared to be in agreement with previous data we have published concerning the quantity and state of mucin which are adsorbed to polymers characterized by different surface properties.