Dual‐Polymer‐Functionalized Nanoscale Graphene Oxide as a Highly Effective Gene Transfection Agent for Insect Cells with Cell‐Type‐Dependent Cellular Uptake Mechanisms

Efficient and safe gene transfection carriers, especially for hard‐to‐transfect cells, are urgently demanded in basic biological research and gene therapy applications. Many insect cell lines widely used in molecular cell biology exhibit relatively low transfection efficiencies when treated by conventional non‐viral agents. Herein, we develop a novel gene delivery vector by coating graphene oxide (GO) with both polyethylene glycol (PEG) and polyethylenimine (PEI), obtaining a dual‐polymer‐functionalized nanoscale GO (nGO‐PEG‐PEI) to transfect insect cells. While exhibiting remarkably reduced cytotoxicity compared with PEI, nGO‐PEG‐PEI, when used as the plasmid DNA transfection agent to treat Drosophila S2 cells, offers ≈7‐fold and ≈2.5‐fold higher efficiency compared with those achieved by using bare PEI and Lipofectamine 2000, a widely used commercial transfection agent, respectively. Interestingly, the advantages of nGO‐PEG‐PEI are even more dramatic when transfecting cells with lower‐quality linearized DNA. It is revealed that nGO‐PEG‐PEI/pDNA complexes enter insect cells via a unique pathway working even at a low temperature, rather different from their entry into mammalian adherent cells. Our results encourage the development of nano‐GO‐based gene carriers to treat special types of hard‐to‐transfect cells (e.g., insect cells), and indicate that nanomaterials would enter cells by cell‐type‐dependent mechanisms, which merit significantly more future attentions.     

From oleic acid-capped iron oxide nanoparticles to polyethyleneimine-coated single-particle magnetofectins

Various inorganic nanoparticle designs have been developed and used as non-viral gene carriers. Magnetic gene carriers containing polyethyleneimine (PEI), a well-known transfection agent, have been shown to improve DNA transfection speed and efficiency in the presence of applied magnetic field gradients that promote particle–cell interactions. Here we report a method to prepare iron oxide nanoparticles conjugated with PEI that: preserves the narrow size distribution of the nanoparticles, conserves magnetic properties throughout the process, and results in efficient transfection. We demonstrate the ability of the particles to electrostatically bind with DNA and transfect human cervical cancer (HeLa) cells by the use of an oscillating magnet array. Their transfection efficiency is similar to that of Lipofectamine 2000?, a commercial transfection reagent. PEI-coated particles were subjected to acidification, and acidification in the presence of salts, before DNA binding. Results show that although these pre-treatments did not affect the ability of particles to bind DNA they did significantly enhanced transfection efficiency. Finally, we show that these magnetofectins (PEI-MNP/DNA) complexes have no effect on the viability of cells at the concentrations used in the study. The systematic preparation of magnetic vectors with uniform physical and magnetic properties is critical to progressing this non-viral transfection technology.     

Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe₃O₄) were prepared by chemical coprecipitation method using Fe²⁺, Fe³⁺ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe₃O₄ nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A₂₆₀/A₂₈₀ ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.     

Electron paramagnetic resonance as an effective method for a characterization of functionalized iron oxide

Iron(II, III) oxide magnetic nanoparticles (NPs) have been coated with (3-Chloropropyl) trimethoxysilane and subsequently functionalized with 4-Amino-2,2,6,6-tetramethylpiperidine-N-oxyl and Amoxicillin. Finally, the functionalized iron oxide NPs have been coated with natural polymer, chitosan, in order to prevent NPs agglomeration in aqueous environment. The product was characterized by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). It was studied by the electron paramagnetic resonance method and the parameters describing the magnetic properties of the investigated nanoparticles, such as g-factor and line width, were calculated.     

Characterization of PEI-coated superparamagnetic iron oxide nanoparticles for transfection: Size distribution,colloidal properties and DNA interaction

Superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyethylenimine. Here, we briefly describe the synthesis as well as DNA:PEI:SPION complexes and the characterization of the compounds according to their particle size, ζ-potential, morphology, DNA complexing ability, magnetic sedimentation, and colloidal stability. PEI coating of SPIONs led to colloidally stable beads even in high salt concentrations over a wide pH range. DNA plasmids and PCR products encoding for green fluorescent protein were associated with the described beads. The complexes were added to cells and exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency.     

Transfection using hydroxyapatite nanoparticles in the inner ear via an intact round window membrane in chinchilla

Hydroxyapatite nanoparticles (nHAT) are known to have excellent biocompatibility, and have attracted increasing attention as new candidates of non-viral vectors for gene therapy. In our previous studies, nHAT carrying a therapeutic gene and a reporter gene were successfully transfected into the spiral ganglion neurons in the inner ear of guinea pigs in vivo as well as in the cultured cell lines, although the transfection efficiencies were never higher than 30%. In this study, the surface modification of nHAT with polyethylenimine (PEI) was made (PEI–nHAT, diameter = 73.09 ± 27.32 nm) and a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) gene and neurotrophin-3 (NT-3) gene was constructed as pEGFPC2–NT3. The PEI modified nHAT and the recombinant plasmid was then connected to form the nHAT-based vector–gene complex (PEI–nHAT–pEGFPC2–NT3). This complex was then placed onto the intact round window membranes of the chinchillas for inner ear transfection. Auditory brainstem response (ABR) was tested to evaluate auditory function. Green fluorescence of EGFP was observed using confocal microscopy 48 h after administering vector–gene complexes. There was no significant threshold shift in tone burst-evoked ABR at any tested frequency. Abundant, condensed green fluorescence was found in dark cells on both sides of the crista and around the macula of the utricle. Scattered EGFP signals were also detected in vestibular hair cells, some Schwann cells in the cochlear spiral ganglion region, some outer pillar cells in the organ of Corti, and a few cells in the stria vascularis. The density of green fluorescence-marked cells was obviously higher in the vestibular dark cell area than in other areas of the inner ear, suggesting that vestibular dark cells may have the ability to actively engulf the nHAT-based vector–gene complexes. Considering the high transfection efficiency in the vestibular system, PEI–nHAT may be a potential vector for gene therapy of inner ear diseases, especially vestibular disorders, and deserves further study.     

Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina

Background  

Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI) can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli β-galactosidase or enhanced green fluorescence protein (EGFP) using PEI.     

Hydrophobically modified chitosan/gold nanoparticles for DNA delivery

Present study dealt an application of modified chitosan gold nanoparticles (Nac-6-Au) for the immobilization of necked plasmid DNA. Gold nanoparticles stabilized with N-acylated chitosan were prepared by graft-onto approach. The stabilized gold nanoparticles were characterized by different physico-chemical techniques such as UV-vis, TEM, ELS and DLS. MTT assay was used for in vitro cytotoxicity of the nanoparticles into three different cell lines (NIH 3T3, CT-26 and MCF-7). The formulation of plasmid DNA with the nanoparticles corresponds to the complex forming capacity and in-vitro/in-vivo transfection efficiency was studied via gel electrophoresis and transfection methods, respectively. Results showed the modified chitosan gold nanoparticles were well-dispersed and spherical in shape with average size around 10～12 nm in triple distilled water at pH 7.4, and showed relatively no cytotoxicity at low concentration. Addition of plasmid DNA on the aqueous solution of the nanoparticles markedly reduced surface potential (50.0～66.6%) as well as resulted in a 13.33% increase in hydrodynamic diameters of the formulated nanoparticles. Transfection efficiency of Nac-6-Au/DNA was dependent on cell type, and higher β-galactosidase activity was observed on MCF-7 breast cancer cell. Typically, this activity was 5 times higher in 4.5 mg/ml nanoparticles concentration than that achieved by the nanoparticles of other concentrations (and/or control). However, this activity was lower in in-vitro and dramatically higher in in-vivo than that of commercially available transfection kit (Lipofectin®) and DNA. From these results, it can be expected to develop alternative new vectors for gene delivery.     

Silica encapsulation of luminescent silicon nanoparticles: stable and biocompatible nanohybrids

This article presents a process for surface coating and functionalization of luminescent silicon nanoparticles. The particles were coated with silica using a microemulsion process that was adapted to the fragile silicon nanoparticles. The as-produced core–shell particles have a mean diameter of 35 nm and exhibit the intrinsic photoluminescence of the silicon core. The silica layer protects the core from aqueous oxidation for several days, thus allowing the use of the nanoparticles for biological applications. The nanoparticles were further coated with amines and functionalized with polyethylene glycol chains and the toxicity of the particles has been evaluated at the different stages of the process. The core–shell nanoparticles exhibit no acute toxicity towards lung cells, which is promising for further development.     

Effects of the agglomeration state on the Raman properties of Co3O4 nanoparticles

The features of the Raman spectra of Co₃O₄ 30‐nm nanoparticles depend strongly on their agglomeration state. When measured at low incident laser power, the spectrum of isolated nanoparticles corresponds to that found in bulk materials, whereas the agglomerated nanoparticles present a clear red‐shift and broadening of the Raman bands. On the other hand, when measured at even lower power, both agglomerated and isolated nanoparticles show the same spectrum of microscopic particles. These effects have been studied by variations of the 532‐nm laser power and the environmental temperature. The thermal dependence of Raman spectra of agglomerated nanoparticles is different to that of isolated nanoparticles but is comparable to the one of bulk material. The different behaviour of the nanoparticles at different agglomeration state is associated to the transmission of phonons among the particles. On the other hand, an increase of the laser power causes a larger number of acoustic phonons, producing a variation of the vibration anharmonicity of the nanoparticles. This increase is more pronounced in the agglomerated nanoparticles, due to the transmission of phonons, causing a much intense modification of the Raman spectrum produced by the laser power. These results clearly indicate that the agglomeration state of the nanoparticles affects their Raman properties. Copyright © 2012 John Wiley & Sons, Ltd.     

Plasma-activated core-shell gold nanoparticle films with enhanced catalytic properties

Catalytically active gold nanoparticle films have been prepared from core-shell nanoparticles by plasma-activation and characterized by high-resolution transmission electron microscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. Methane can be selectively oxidized into formic acid with an O₂–H₂ mixture in a catalytic wall reactor functionalized with plasma-activated gold nanoparticle films containing well-defined Au particles of about 3.5 nm in diameter. No catalytic activity was recorded over gold nanoparticle films prepared by thermal decomposition of core-shell nanoparticles due to particle agglomeration.     

Molecular dynamics simulation of the formation of twin boundaries during agglomeration of nanoparticles

The processes controlling early stages of agglomeration of nanoparticles have been investigated by the molecular dynamics method. It has been established that the formation of boundaries with twin misorientation is the main mechanism of structural relaxation during primary agglomeration of nanoparticles. It has been shown that an increase in the temperature leads to an increase in the number of twin boundaries and that their mutual arrangement depends on the misorientation of the nanoparticles. In the case where twin boundaries are noncoplanar, structure relaxation results in the formation of pentagonal twin boundaries. The role of twinning in the formation of interfaces upon compaction of nanoparticles has been discussed.     

Effect of Cationic Polyelectrolyte on the Flow Behavior of Hydroxypropyl Methyl Cellulose/Polyacrylic Acid Interpolymer Complexes

Association of polyacrylic acids and nonionic polymers in solutions via hydrogen bonding results in formation of novel polymeric materials. These complexes are novel individual compounds and their properties are entirely different from the properties of their component polymers. In this study, the effects of a cationic polyelectrolyte (polyethylenimine, PEI) on the interpolymer complexation of hydroxypropyl methyl cellulose (HPMC) with polyarcylic acid (PAA) in aqueous media were studied. Results showed that at low pH, interpolymer complex (IPC) formation was observed between HPMC and PAA at a 3:0.5 polymer ratio. Under basic conditions, the viscosity of the IPC increased accompanied with the transition from coiled structure to an extended conformation of associates. Addition of PEI to the same system caused some structural changes in the polymer solution mixtures depending on the pH of the system. PEI worked as a destructive agent for the HPMC/PAA interpolymer complexes and associates. This behavior was attributed to the complex formation between PEI and HPMC via hydrogen bonding.     

Color-tunable magnetic and luminescent hybrid nanoparticles: Synthesis, optical and magnetic properties

A facile method for synthesizing color-tunable magnetic and luminescent hybrid bifunctional nanoparticles is presented. A series of CdSe/ZnS core-shell quantum dots (QDs) with different sizes were successfully fabricated and self-assembled to Fe₃O₄ magnetic nanoparticles (MNP), which were subsequently coated with a polyethyleneimine (PEI) layer to prevent large aggregates. The hydrophobic QDs capped with trioctylphosphine oxide (TOPO) formed a coating surrounding MNP, and were transferred into hydrophilic phase by PEI with high efficiency. The samples were characterized by TEM, FT-IR, XRD, EDS, UV-vis spectrophotometer, fluorescent spectrophotometer and PPMS. Results show that the original properties of the nanoparticles were well-preserved in the hybrid structure. All MNP-QDs hybrid nanoparticles showed paramagnetic behavior and the nanocomposites were still highly luminescent with no shift in the PL peak position.     

Surface modification of gold nanorods using layer-by-layer technique for cellular uptake

Gold nanorods (NRs), rod-shaped gold nanoparticles, were modified with bovine serum albumin (BSA) and polyethylenimine (PEI) using layer-by-layer technique. From absorption spectroscopy and zeta potential measurements, it was obvious that NRs were wrapped with these polymers without aggregation of NRs. Following BSA modification, the surface-modified NRs (BSA-NRs) were well-dispersed without aggregation in biochemical conditions, verified from absorption spectroscopy. Further modification with PEI provided positively charged NRs (PEI-NRs). A transmission electron microscopy image of PEI-NRs revealed that the surface modification did not affect changing the shape of the initial NRs. In addition, the PEI-NRs retained the colloidal stability of BSA-NRs in biochemical conditions. We have evaluated that transfection activity of PEI-NRs with HeLa cells. From results of gene expression experiments, it was obvious that the stabilization of NRs by BSA and further modification with PEI realized transfection using NRs into cultured cells. Moreover, the cellular uptake of NRs enabled cellular imaging using light scattering from the NRs.     

功能性ZnS纳米荧光探针的合成及其光谱性质研究

报道了功能性ZnS纳米荧光探针的合成与表征 ,同时研究了纳米粒子的荧光衰变寿命 ,考察了该纳米荧光探针的吸收光谱和荧光发射光谱特性。与此同时 ,研究了不同蛋白质和核酸对该纳米荧光探针的吸收光谱和荧光发射光谱的影响 ,实验表明 ,蛋白质的加入使得该纳米荧光探针的吸收和发射光谱强度均增强 ,而核酸的加入则使其吸收和发射光谱强度均减弱。据此 ,可望将该纳米荧光探针应用于生物大分子的分析测定     

Dynamic and Electro-Optical Nonequivalence of the NH Bonds of the Amino Group in Complexes of Dichloro-Substituted Anilines

The IR spectra of 1:1 and 1:2 complexes of dichloro-substituted anilines with various proton acceptors have been investigated and the parameters of the absorption bands of the stretching and deformation vibrations of their amino group have been determined. Vibrational and electro-optical problems have been solved for free and H-bonded molecules of the studied amines within the framework of the R-NH₂ model of a valence-free field. The geometric, dynamic, and electro-optical parameters of the amino group of dichloro-substituted anilines in complexes of different compositions have been determined. It is shown that the hydrogen bond in 1:1 complexes appears to be stronger than in 1:2 complexes.     

Influence of size and surface capping on photoluminescence and cytotoxicity of gold nanoparticles

Hydrophilic and homogeneous sub-10 nm blue light-emitting gold nanoparticles (NPs) functionalized with different capping agents have been prepared by simple chemical routes. Structure, average, size, and surface characteristics of these NPs have been widely studied, and the stability of colloidal NP solutions at different pH values has been evaluated. Au NPs show blue PL emission, particularly in the GSH capped NPs, in which the thiol-metal core transference transitions considerably enhance the fluorescent emission. The influence of capping agent and NP size on cytotoxicity and on the fluorescent emission are analyzed and discussed in order to obtain Au NPs with suitable features for biomedical applications. Cytotoxicity of different types of gold NPs has been determined using NPs at high concentrations in both tumor cell lines and primary cells. All NPs used show high biocompatibility with low cytotoxicity even at high concentration, while Au-GSH NPs decrease viability and proliferation of both a tumor cell line and primary lymphocytes.     

Amine-capped gold nanoparticles: reaction steps during the synthesis and the influence of the ligand on the particle size

Alkylamine-capped gold nanoparticles were synthesised using the Leff method. In order to investigate the influence on the complexes formed during the synthesis and the final size of the protected nanoparticles the chain length of the stabilizing ligands varied. Phase transfer and amine complex formation were studied in solution with X-ray absorption spectroscopy. The phase transfer complexes retain the quadratic planar conformation whilst all alkyl amines form a linear coordinated gold(I) complex. After reduction, the particles were characterised in solution with EXFAS, UV–Vis spectroscopy, and compared to TEM measurements. The size of the particles was determined from the coordination number of the first gold shell. The position of the UV–Vis plasmon band and the distance of the first gold shell were used to qualitatively compare the effects of the ligands on the particle size. The size of the particles decreases with increasing chain length.     

Dispersions of silica nanoparticles in ionic liquids investigated with advanced rheology

The colloidal stabilities of dispersions of unmodified and surface-functionalized SiO₂ nanoparticles in hydrophobic and hydrophilic imidazolium-based ionic liquids were studied with advanced rheology at three temperatures (25, 100, and 200 °C). The rheological behavior of the dispersions was strongly affected by the ionic liquids hydrophilicity, by the nanoparticles surface, by the concentration of the nanoparticles in the dispersion as well as by the temperature. The unmodified hydrophilic nanoparticles showed a better compatibility with the hydrophilic ionic liquid. The SiO₂ surface functionalization with hydrophobic groups clearly improved the colloidal stability of the dispersions in the hydrophobic ionic liquid. The temperature increase was found to lead to a destabilization in all studied systems, especially at higher concentrations. The results of this study imply that ionic liquids with tailored properties could be used in absorbers directly after reactors for gas-phase synthesis of nanoparticles or/and as solvents for their further surface functionalization without agglomeration or aggregation.