Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer

The layer-by-layer assembly technique was used to adsorb alternately poly(ethyleneimine) and plasmid DNA onto the surface of a transparent electrode made of indium-tin oxide. The surface with adsorbed poly(ethyleneimine) and DNA was characterized by X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy, and contact angle measurements. These analyses revealed that the alternate adsorption process generated a multilayered assembly of cationic poly(ethyleneimine) and anionic DNA. For the spatially and temporally specific gene transfer, cells were cultured on the plasmid-loaded electrode and then a short electric pulse was applied to the cell-electrode system. It was shown that, upon electric pulsing, the plasmid was released from the electrode and transferred into the cells, resulting in efficient gene expression even in primary cultured cells. Transfection could be effected for hippocampal neurons after 3-day culture on the plasmid-loaded electrode, which indicated the feasibility of selecting the time of transfection. Our results also showed that electroporation could be performed in a spatially specific manner by using a plasmid-arrayed electrode, demonstrating the feasibility of the method for the fabrication of transfected cell microarrays.     

The electroactive multilayer films of polyelectrolytes and Prussian blue nanoparticles and their application for H2O2 sensors

Prussian blue (PB) nanoparticles were immobilized in polyelectrolyte (PE) multilayers of various compositions and thickness. Films containing nanoparticles and poly(allylamine hydrochloride) (PAH) were formed using the layer-by-layer adsorption method. A layer of branched poly(ethyleneimine) (PEI) was used to anchor the multilayer structure at the surface of a gold electrode. The films exhibited electroactive properties, increasing with the number of deposited PB layers. The properties of PEI/(PB/PAH) _n multilayers were then compared with the ones containing additionally the conductive polymer poly(3,4-ethylenedioxythiophene)–poly(styrenesulfonate) (PEDOT:PSS). We found that the addition of the conductive, water-soluble polymer enhances the electroactive properties of the multilayer films. It also increased sensitivity of the multilayer-covered electrodes for electrochemical detection of hydrogen peroxide.     

Gene transfection into adherent cells using electroporation on a dendrimer-modified gold electrode

Gene transfection into adherent cells from plasmid DNA (pDNA)-arrayed substrates known as gene transfection arrays appears to be a promising tool for the high-throughput analysis of gene functions and protein-protein interaction networks. We tested the ability of electric pulse-stimulated gene transfection from a substrate to overcome low expression efficiency and cross contamination between spots on arrays. We prepared the electrodes used for electric pulse-stimulated gene transfection by sequentially loading a gold thin layer, a self-assembled monolayer of a carboxylic acid-terminated alkanethiol (COOH-SAM), and poly(amidoamine) (PAMAM) dendrimers, either through electrostatic interactions or by covalent linkage to COOH-SAM and then to pDNA. When dendrimers were loaded onto the electrode using electrostatic interactions, the gene-expression efficiency of adherent cells increased as the generation numbers of the dendrimers that we used increased. Gene expression was rarely observed in adherent cells when dendrimers were covalently immobilized onto the electrode. Additionally, we successfully demonstrated site-specific gene transfer using a dendrimer-array electrode with no cross contamination between spots on the electrode.     

碳纳米管修饰电极同时测定邻苯二酚和对苯二酚

用十二烷基磺酸钠(SDS)分散碳纳米管(CNTs),通过层层组装(LBL)聚二甲基二烯丙基氯化铵(PDDA)和CNTs构筑PDDA/CNTs多层膜电极.利用紫外-可见光谱法对PDDA/CNTs多层膜的组装过程进行监测,用循环伏安法(CV)和差分脉冲伏安法(DPV)研究了邻苯二酚和对苯二酚同时存在时PDDA/CNTs多层膜电极上的电化学行为.结果表明,碳纳米管修饰电极对邻苯二酚和对苯二酚有较好的电催化活性和电分离作用,邻苯二酚和对苯二酚无需经过分离即可同时被检出.在修饰电极上的线性范围如下:邻苯二酚为2.0×10-6~1.4×10-4mol/L,线性相关系数R=0.9991;对苯二酚为2.0×10-6~1.4×10-4mol/L,线性相关系数R=0.9987.     

非病毒型DNA纳米微球层层组装构建多层膜用于原位基因传递

高效安全的基因传递体系是基因技术发展的关键问题. 基于聚阳离子的基因纳米微球是一种典型的非病毒型基因载体, 能够在体内外有效转染细胞. 本文通过层层组装方法构建装载基因纳米微球的可降解多层膜, 这种固相基因传递体系能实现材料表面的贴壁细胞的原位转染. 与装载裸DNA的多层膜相比, 基因纳米微球多层膜能更有效地原位转染贴壁细胞, 这主要是因为DNA在此多层膜中仍处于与聚阳离子缔合的状态. 构建于聚乳酸三维支架表面的基因纳米微球多层膜亦能实现支架表面贴壁细胞的原位转染. 这种结构可控、易制备的基因纳米微球多层膜为精确控制基因纳米微球传递提供了一种新方法, 也为基因治疗进一步应用于组织工程、介入治疗和医用植入体提供了一种可能的技术手段.     

Combining hydrogen-bonding complexation in solution and hydrogen-bonding-directed layer-by-layer assembly for the controlled loading of a small organic molecule into multilayer films

We have combined hydrogen-bonding complexation in solution and layer-by-layer assembly for the controlled loading of a water-insoluble small organic molecule, bis-triazine (DTA), an azobenzene derivative containing multiple hydrogen bond donors and acceptors, into layer-by-layer multilayer films of poly(acrylic acid) and diazo-resin. UV-visible spectroscopy indicates that DTA has been loaded into multilayer films, with the loading amount increasing linearly with the number of layers. The loading amount can be well tuned either by changing the concentration of DTA or the solvent composition at the complexation step. Fourier transform infrared spectroscopy has revealed that both the complexation and layer-by-layer assembly are driven by hydrogen bonding. After photo-cross-linking and immersion in dimethyl sulfoxide to release DTA, the film can serve as an absorbent for DTA. This study provides a new unconventional layer-by-layer assembly that combines hydrogen-bonding complexation in solution and hydrogen-bond-driven layer-by-layer assembly at the interface. This method provides a new route to load a variety of water-insoluble functional organic molecules into layer-by-layer films.     

Efficient gene delivery in differentiated human embryonic stem cells

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.     

Effect of architecture on the formation of surface multilayer structures at the air-solution interface from mixtures of surfactant with small poly(ethyleneimine)s

The impact of ethyleneimine architecture on the adsorption behavior of mixtures of small poly(ethyleneimines) and oligoethyleneimines (OEIs) with the anionic surfactant sodium dodecylsulfate (SDS) at the air-solution interface has been studied by surface tension (ST) and neutron reflectivity (NR). The strong surface interaction between OEI and SDS gives rise to complex surface tension behavior that has a pronounced pH dependence. The NR data provide more direct access to the surface structure and show that the patterns of ST behavior are correlated with substantial OEI/SDS adsorption and the spontaneous formation of surface multilayer structures. The regions of surface multilayer formation depend upon SDS and OEI concentrations, on the solution pH, and on the OEI architecture, linear or branched. For the linear OEIs (octaethyleneimine, linear poly(ethyleneimine) or LPEI(8), and decaethyleneimine, LPEI(10)) with SDS, surface multilayer formation occurs over a range of OEI and SDS concentrations at pH 7 and to a much lesser extent at pH 10, whereas at pH 3 only monolayer adsorption occurs. In contrast, for branched OEIs BPEI(8) and BPEI(10) surface multilayer formation occurs over a wide range of OEI and SDS concentrations at pH 3 and 7, and at pH 10, the adsorption is mainly in the form of a monolayer. The results provide important insight into how the OEI architecture and pH can be used to control and manipulate the nature of the OEI/surfactant adsorption.     

Self-assembly of S-layer-enveloped cytochrome c polyelectrolyte multilayers

We report a study of the electrostatic layer-by-layer self-assembly of electroactive polyelectrolyte multilayers incorporating the redox protein cytochrome c (cyt c) combined with recrystallization of the bacterial cell wall surface layer from Bacillus sphaericus CCM 2177 SbpA (S-layer). The polyelectrolyte multilayer assembly was prepared on flat gold electrodes with a nanometer-scale roughness that allowed monitoring of the film formation throughout all the assembly stages by atomic force microscopy measurements in liquid with respect to topography and forces. The deposition of alternating layers of sulfonated polyaniline and cyt c was carried out by adsorption from the corresponding solutions on a cyt c monolayer electrode. The electroactivity of cyt c within the assembly was confirmed by cyclic voltammetry. We showed that the surface properties of the electrode terminating layer change after each adsorption step accordingly. We also found that S-layer recrystallization on the top of the multilayer film was feasible while electroactivity of cyt c within a polyelectrolyte matrix was partially maintained. This approach offers a new strategy to design a biocompatible and permselective outer envelope of a polyelectrolyte multilayer, promising sensor applications.     

SiW_(12)O_(40)~(4-)在玻碳电极上的多层组装及电催化性能研究

本文采用层层自组装法制备包含杂多酸SiW_(12)O_(40)~(4-)(SiW_(12))和聚合物阳离子聚二烯丙基二甲基氯化铵(PDDA)的多层膜修饰电极,并用循环伏安法研究其对BrO_3~-和NO_2~-体系还原的电催化性能.结果表明:SiW_(12)修饰电极对BrO_3~-和N_2O~-的还原具有明显的电催化作用.催化电流随修饰层数的增加而明显增加.     

辣根过氧化物酶活性膜结构及生物电催化性能

通过分子沉积法研究了在聚对苯二甲酸乙二醇酯（PET）表面及金电极表面组装辣根过氧化物酶（HRP）/聚对苯乙烯磺酸钠（PSS）多层生物活性膜，用原子力显微镜(AFM)研究了组装膜的表面形貌，并研究了组装膜的形貌、粗糙度和活性关系.应用循环伏安法（CV）研究了组装HRP膜后电极对H2O2的电化学催化还原作用.实验发现，采用亚甲基蓝（MB）溶液为介质，在H2O2浓度为0.2～5.0 mmol•L－1时，其响应电流对H2O2浓度变化基本呈线性.     

Amphiphilic Block Copolymer Micelles for Gene Delivery

Gene therapy is a promising method to treat acquired and inherited diseases by introducing exogenous genes into specific recipient cells. Polymeric micelles with different nanoscopic morphologies and properties hold great promise for gene delivery system. Conventional cationic polymers, poly(ethyleneimine)(PEI), poly(L-lysine)(PLL), poly(2-dimethyla-minoethyl methacrylate)(PDMAEMA) and novel cationic polymers poly(2-oxazoline)s(POxs), have been incorporated into block copolymers and decorated with targeting moieties to enhance transfection efficiency. In order to minimize cytotoxicity, nonionic block copolymer micelles are utilized to load gene through hydrophilic and hydrophobic interactions or covalent conjugations, recently. From our perspective, properties(shape, size, and mechanical stiffness, etc.) of block copolymer micelles may significantly affect cytotoxicity, transfection efficiency, circulation time, and load capacity of gene vectors in vivo and in vitro. This review briefly sums up recent efforts in cationic and nonionic amphiphilic polymeric micelles for gene delivery.     

Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells

This paper describes the fabrication of microarrays that enable the parallel electroporation of small interfering RNAs (siRNAs) into mammalian cells. To optimize the conditions of microarray preparation and electric pulsing, a self-assembled monolayer was formed on a gold electrode, and a cationic polymer was adsorbed by the entire surface of the monolayer. siRNA was then adsorbed by the cationically modified electrode through electrostatic interactions. Human embryonic kidney cells stably transformed with the expression construct of green fluorescent protein (GFP) were used to examine the electric pulse-triggered transfer of GFP-specific siRNA. A single electric pulse was applied to the cells cultured on the electrode at a field strength of 240 V cm(-1). The expression of GFP was significantly suppressed in a sequence-specific manner two days after pulsing. Microscopic observation and flow-cytometric analysis revealed that the expression of GFP was attenuated in the majority of cells in a loading-dependent manner. Moreover, the effect of siRNA could be temporally controlled by changing the culture periods before pulsing. When a micropatterned self-assembled monolayer was used as a platform for loading siRNA in an array format, gene silencing was spatially restricted to the regions where specific siRNA was loaded. From these results, we conclude that array-based electroporation provides an excellent means of individual transfer of siRNAs into mammalian cells for high-throughput gene function studies.     

Bio-chip for spatially controlled transfection of nucleic acid payloads into cells in a culture

Transfection of siRNA and plasmid nucleic molecules to animal, microbial and plant cell cultures is a critical process in various research areas, including drug discovery, functional genomics and basic life science research. Till recent times, transfection of these exogenous molecules have been global in nature i.e. targeting all the cells in a culture and lacking capability to spatially confine the transfection to small populations of cells within a single culture. However, in emerging areas like high-throughput screening of large molecule libraries, there is a critical need to transfect multiple different molecules to locally specified regions of a single cell culture and monitor phenotypical changes in these different cell populations. In this study, we present a cell-based biochip that utilizes a microelectrode array to generate localized current density fields that induce electroporation to a targeted group of cells for site-specific transfection of exogenous molecules. More specifically, we optimize the transfection efficiency and viabilities for spatially controlled transfection of Alexa-Fluor-488 conjugated siRNA molecules into NIH3T3 fibroblast cell cultures. Optimal electroporation parameters are established at current density values ranging between 0.05-0.07 microA microm(-2) for high transfection efficiencies (>60%) while maintaining viability (>80%) on individual microelectrodes. Additionally, exogenous plasmid molecules are electroporated for site-specific GFP expression and monitored over 48 h in-situ. The microelectrode array technology reported here can therefore be potentially used for targeting specific cells in a culture with spatial precision and transfecting siRNA and plasmids. The microfabrication approach lends itself to significant high-throughput applications in drug-discovery research.     

Assembly and Electrochemical Study of Transition Metal Substituted Polyoxometalates SiW11 NiO39^6- and SiW11 MnO39^6- on 4-Aminothiophenol Modified Glassy Carbon Electrode

Via layer-by-layer assembly, the polyoxometalates of Keggin type, SiW11 O39 Ni(H2O)^6- (SiNiW11) and SiW11 O39Mn( H2O)^6-( SiMnW11 ) were first immobilized on a 4-aminothiophenol (4-ATP) modified glassy carbon electrode surface. The electrochemical behavior of these polyoxometalates was investigated. They exhibited some special properties in the films, which are different from those in a homogeneous aqueous solution. Their reaction mechanism in a multilayer film is proposed. The electrocatalytic behavior of these muhilayer film electrodes for the reduction of BrO3^- and NO2^- were comparatively studied.     

Preparation of poly(glutamic acid) shielding micelles self-assembled from polylysine-b-polyphenylalanine for gene and drug codelivery

A novel amphiphilic cationic block copolymer polylysine-b-polyphenylalanine(PLL-b-PPhe) was synthesized and self-assembled into micelles in aqueous solution,then shielded with poly(glutamic acid)(marked as PG/PLL-b-PPhe) to codeliver gene and drug for combination cancer therapy.Here,doxorubicin(DOX) was selected to be loaded into PLL-b-PPhe micelles and the drug loading efficiency was 8.0%.The drug release studies revealed that the PLL-b-PPhe micelles were pH sensitive and the released DOX could reach to 53.0%,65.0%,72.0% at pH 7.4,6.8 and 5.0,respectively.In order to reduce positive charge and cytotoxicity of PLL-b-PPhe micelles,PG was used as shelding,simultaneously condensed with Bcl2 siRNA to form gene carrier system.Compared with PEI,PG/PLL-b-PPhe had excellent gene transfection efficiency,especially when the molar ratio of PLL to PPhe was 30:60 and the mixed mass ratio of PLL-b-PPhe to gene was 5:1.More importantly,DOX and Bcl2 siRNA gene codelivery system displayed remarkable cytotoxicity against B16 F10 cells.Confocal laser scanning microscopy(CLSM) and flow cytometry were used to characterize endocytosis of the codelivery system,and confirmed that both DOX and Bcl2 siRNA had been endocytosed into B16 F10 cells.The above results indicated that gene and drug codelivery was a promising strategy in future cancer therapy.     

Construction of glucose biosensor based on sorption of glucose oxidase onto multilayers of polyelectrolyte/nanoparticles

A new approach to constructing an enzyme-containing film on the surface of a gold electrode for use as a biosensor is described. A basic multilayer film (BMF) of (PDDA/GNPs) _n /PDDA was first constructed on the gold electrode by electrostatic layer-by-layer self-assembly of poly(diallyldimethylammonium chloride) (PDDA) and gold nanoparticles (GNPs). Glucose oxidase (GOx) was then sorbed into this BMF by dipping the BMF-modified electrode into a GOx solution. The assembly of the BMF was monitored and tested via UV-vis spectroscopy and cyclic voltammetry (CV). The ferrocenemethanol-mediated cyclic voltammograms obtained from the gold electrode modified with the (PDDA/GNPs) _n /PDDA/GOx indicated that the assembled GOx remained electrocatalytically active for the oxidation of glucose. Analysis of the voltammetric signals showed that the surface coverage of active enzyme was a linear function of the number of PDDA/GNPs bilayers. This result confirmed the penetration of GOx into the BMF and suggests that the BMF-based enzyme film forms in a uniform manner. Electrochemical impedance measurements revealed that the biosensor had a lower electron transfer resistance (R _et) than that of a sensor prepared by layer-by-layer assembly of PDDA and GOx, due to the presence of gold nanoparticles. The sensitivity of the biosensor for the determination of glucose, which could be controlled by adjusting the number of PDDA/GNPs bilayers, was investigated.     

Metal-enhanced fluorescence using silver nanoparticles-embedded polyelectrolyte multilayer films for microarray-based immunoassays

Metal-enhanced fluorescence (MEF) of quantum dots (QDs) and its potential application in microarray-based immunoassays was investigated using silver nanoparticles (AgNPs) prepared by the in situ photoreduction of Ag⁺ inside a multilayer film consisting of poly(ethyleneimine) (PEI) and hyaluronic acid (HA). UV–Vis spectroscopy, X-ray diffraction, atomic force microscopy, scanning electron microscopy, and transmission electron microscopy confirmed the formation of well-dispersed AgNPs within the multilayer films, the thickness and the amount of which depended on the number of HA layers. Using AgNPs-containing hybrid multilayered films, it was possible to observe the MEF effect of adsorbed QDs, which could be tuned by the thickness of interlayer spacer film prepared of the layer-by-layer assembly of PEI and poly(styrene sulfonate). When the MEF-inducing hybrid film was used as a platform for immunoassay, a significant improvement in the fluorescence signal and sensitivity of the biosensing were observed in the presence of AgNPs in comparison with films that did not contain the nanoparticles.     

Hydrogen bonds in polycation improve the gene delivery efficiency in the serum-containing environment

Cationic polymers with high charge density could effectively condense the DNA and achieve gene transfection; however, it often brings non-negligible cytotoxicity. Notably, the high charge density gene vector fails in the serum environment, limiting further application in vivo. In this paper, an efficient and reliable non-viral gene vector of poly (amidoamine) (PAA) was designed by introducing diacryolyl-2,6-diaminopyridine (DADAP) onto the PAA backbone through Michael-addition polymerization, which provides high transfection efficiency in a serum-containing environment. Diacryolyl-2,6-diaminopyridine and cationic parts provided multiple interactions between gene vectors and DNA, including hydrogen bond and electrostatic interactions. The introduction of hydrogen bonding can effectively reduce the charge density of polyplexes without reducing the DNA condensing ability, incorporating the diaminopyridine group and cationic part in PAA chains successfully consolidated cellular uptake, endosome destabilization, and transfection efficiency for the PAA/DNA complexes with low cytotoxicity. The constructed vector with multiple interactions presented 6 times higher transfection efficiency in serum-free and 9 times in serum-containing environment than that of branched polyethyleneimine (PEI 25K) in 293T cells in vitro. Therefore, introducing the hydrogen band to form low charge density polyplexes with high transfection efficiency and low cytotoxicity has a great potential in gene delivery.     

Reversible disulfide cross-linking in layer-by-layer films: preassembly enhanced loading and pH/reductant dually controllable release

This paper describes the fabrication of polyelectrolyte multilayer film which combines preassembly of poly(allylamine hydrochloride) (PAH) and 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p',p' ',p' '-tetrasulfonic acid tetrasodium hydrate (TPPS) in aqueous solution with the layer-by-layer (LbL) assembly of the PAH-TPPS complex and cross-linkable polyelectrolyte, PAASH60, which is a poly(acrylic acid) with 60% of its carboxylic acid grafted of thiol groups. During preassembly, TPPS was incorporated into PAH chains. After oxidative cross-linking to form disulfide bonds in between the layers, the multilayer with preassembly of the PAH-TPPS complex allowed for release and loading of TPPS in a reproducible way. The release of TPPS from the loaded film was a pH-controlled process. To compare with the conventional multilayer, the reloading capacity was greatly enhanced, which was related to the charge binding sites that formed by release of TPPS from the multilayer. Moreover, the release of TPPS could also be achieved by breaking off the cross-linking through reduction of disulfide bonds, and the release rates could be controlled by the reductive efficiency of the reductants in the media. In this way, the release of TPPS is pH/reductant dually controllable, thereby facilitating a new route to multistimuli controllable materials.