葡聚糖磁性纳米颗粒外加磁场对人树突状细胞基因转染效率的研究

目的研究葡聚糖磁性纳米颗粒（the dextran coated magnetic iron oxide nanoparticles,DMN）在外加钕一铁一硼稀土固定磁场的作用下对人树突状细胞转染效率以及安全性的影响。方法先通过磁力计对DMN进行分析;再将修饰有多聚赖氨酸（Poly-L—Lysine,PLL）的DMN携带绿色荧光蛋白pEGFP—Cl质粒报告基因,在钕-铁-硼稀土周定强磁场的作用下,体外转染人树突状细胞,用荧光显微镜直接观察和流式细胞仪检测来评价外加磁场对DMN作为人树突状细胞转染载体效率的影响;在转染后采用MTT比色法测定在磁场干预下的DMN对人树突状细胞增殖和功能的影响以了解其细胞毒性。结果DMN的核心直径〈30nm,具有明硅的超顺磁性,比饱和磁化强度也明显高于相同Fe3O4含量的普通磁块;DMN作为基因载体在外加磁场作用下,转染12h即可将报告基因转染至人树突状细胞内并成功表达,在荧光显微镜下可观察到绿色荧光细胞,24h转染率可达到最高（约为27％）,转染效率较未加磁场组提高了2～4倍。而且转染后的人树突状细胞增殖活性及功能未因DMN外加磁场及其作用时间的长短而受到影响。结论超顺磁性的DMN在外加磁场作用下可以明显、安全、有效地提高对人树突状细胞的转染效率。     

Spatial gradient effects of 120 mT static magnetic field on endothelial tubular formation in vitro

This study investigated the spatial magnetic gradient effects of static magnetic fields (SMF) on endothelial tubular formation by applying the maximum spatial gradient to a target site of culture wells for cell growth. The respective maximum values of magnetic flux density (B(max)), magnetic flux gradient (G(max)) and the magnetic force product of the magnetic flux density and its gradient (a parameter of magnetic force) were 120 mT, 28 mT/mm and 1428 mT(2)/mm. The effects of gradient SMF on tubular formation were compared with those of uniform SMF that has no spatial gradients on the entire bottom area of culture wells. Five experimental groups of 25 samples each were examined: (1) sham exposure (control); (2) peak gradient exposure in the peripheral part; (3) peak gradient exposure in the central part; (4) uniform exposure to 20 mT; (5) uniform exposure to 120 mT. The SMF or sham exposure was carried out for 10 days. Photomicrographs of tubular cells, immunostained with an anti-platelet-endothelial cell adhesion molecule-1 (PECAM-1 [CD31]) antibody as a pan-endothelial marker, were analyzed after the 10-day culture. Gradient SMF in the peripheral or central part was found to significantly promote tubular formation in terms of the area density and length of tubules in each peak gradient/force part of the wells, compared with the sham exposure. In contrast, uniform SMF did not induce any significant change in the tubular formation. These findings suggest that tubule formation can be promoted by applying the peak gradient/force to a target site of culture wells.     

Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer

BACKGROUND: Gene delivery by the use of magnetic forces, so-called magnetofection, has been shown to enhance transfection efficiency of viral and non-viral systems up to several-hundred-fold. For this purpose gene carriers, such as polyethylenimine (PEI), are associated with superparamagnetic nanoparticles and complexed with plasmid DNA. Gene delivery is targeted by the application of a magnetic field. METHODS: To investigate the underlying mechanism, we studied the impact of the applied magnetic field on the transfection process of PEI-coated superparamagnetic iron oxide gene vectors (magnetofectins) using various cell lines. In particular, we addressed the question whether accelerated sedimentation of magnetofectins is the driving force or if the magnetic field itself directly influences the endocytic processing of the magnetofectins. The cellular uptake mechanism of magnetofectins was studied by electron microscopy and transfection experiments in the presence of various inhibitors that operate at different steps of endocytosis. RESULTS: In this study we could show that cellular uptake of magnetofectins proceeds obviously by endocytosis. Cellular uptake of magnetofectins behaves almost analogously as compared with PEI polyplexes. Besides unspecific endocytosis, apparently clathrin-dependent as well as caveolae-mediated endocytic uptake is involved. CONCLUSIONS: The magnetic field itself does not alter the uptake mechanism of magnetofectins. Obviously, the magnetic forces lead to an accelerated sedimentation of magnetofectins on the cell surface and do not directly affect the endocytic uptake mechanism. So further improvement of magnetic field application could lead to efficient targeting of gene expression into the desired organ and tissue in vivo.     

Magnetizable needles and wires--modeling an efficient way to target magnetic microspheres in vivo

The in vivo targeting of tumors with magnetic microspheres is currently realized through the application of external non-uniform magnetic fields generated by rare-earth permanent magnets or electromagnets. Our theoretical work suggests a feasible procedure for local delivery of magnetic nano- and microparticles to a target area. In particular, thin magnetizable wires placed throughout or close to the target area and magnetized by a perpendicular external uniform background magnetic field are used to concentrate magnetic microspheres injected into the target organ's natural blood supply. The capture of the magnetic particles and the building of deposits thereof in the blood vessels of the target area were modeled under circumstances similar to the in vivo situation. This technique could be applied to magnetically targeted cancer therapy or magnetic embolization therapy with magnetic particles that contain anticancer agents, such as chemotherapeutic drugs or therapeutic radioisotopes.     

脉冲电磁场对C2C12 成肌细胞增殖影响的实验研究

目的:研究不同强度脉冲电磁场干预对成肌细胞增殖的影响。方法:10Hz脉冲低频电磁场刺激经复苏后培养贴壁良好的C2C12成肌细胞,根据不同磁场强度和作用时间将其分为A、B、C组,无磁场干预的为对照组。采用RT-q PCR检测不同磁场强度下成肌细胞标记基因Myf5、Myo D及Pax7的m RNA的表达。结果:经RT-q PCR检测三种基因的表达情况,Myf5 m RNA在1.5 m T磁场强度下照射第五天表达最高;0.5 m T磁场强度下Myf5 m RNA的表达与对照组相比无统计学意义(P>0.05);1.0 m T磁场强度下Myf5 m RNA表达与对照组比较差异具有统计学意义(P<0.05);1.5 m T磁场强度下Myf5 m RNA表达与对照组比较差异具有统计学意义(P<0.05)。对照组Myo D m RNA的表达要比磁场作用下表达要高。三个磁场强度下Myo D m RNA表达与对照组相比均无统计学意义(P>0.05)。0.5 m T、1.0 m T磁场强度下Pax7 m RNA的表达要比对照组要高,与对照组相比具有统计学意义(P<0.05);1.5 m T磁场强度下Pax7 m RNA表达与对照组相比无统计学意义(P>0.05)。结论 :1.5 m T脉冲电磁场强度下作用5天对体外培养的成肌细胞Myf5 m RNA标记基因增殖促进作用最强。     

Enhancement of transfection efficiency by protamine in DDAB lipid vesicle-mediated gene transfer.

We have previously developed a simple gene transfection procedure mediated by cationic lipid vesicles for animal cells, in which a commercially available cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. In the present study, we examined enhancement of transfection efficiency for this method by adding protamine to plasmid DNA solution before the formation of DNA/lipid vesicle complexes. Both free-base protamine and protamine sulfate provided enhanced transfection efficiency and expression level, but the optimal amount of the two protamines was different. The enhancement in transfection efficiency and expression level by protamines was observed in all the cell lines (COS-7, Hela, NIH3T3, MDCK, and BHK-21C13) and all the plasmids (pCMVbeta, pmiwZ, and pCH110) tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only DDAB lipid vesicles. Protamines seemed to protect DNA from degradation by DNase and promote DNA delivery into a nucleus.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

DNA transfer into vascular smooth muscle using fusigenic Sendai virus (HJV)-liposomes

Manipulation of the genetic machinery of cells both in vitro and in vivo is becoming an ever more important means of elucidating pathways of molecular and cellular biochemistry. In addition, gene therapy has been proposed as a novel and potentially powerful treatment for both inherited and acquired diseases. Successful gene transfer and gene blockade generally depend on high efficiency delivery of exogenous DNA or RNA into living cells, and much effort has therefore been focused on the development of methods for achieving this delivery in a safe and effective manner. We describe here our application of fusigenic Sendai virus (HVJ)-liposome technology toward the effective delivery of DNA into vascular smooth muscle cells (VSMC) in cell culture. Cellular uptake and intracellular distribution of oligodeoxynucleotide (ODN) after transfection with HVJ-liposome complexes was characterized using fluorescent (FITC)-labeled ODN, and the biologic effect of HVJ-liposome mediated transfection was demonstrated via inhibition of DNA synthesis in cultured VSMC using antisense ODN against basic fibroblast growth factor.     

阳离子脂质体的转染机制及转染效率影响因素

阳离子脂质体是一种非常具有发展前景的基因载体。简要介绍了阳离子脂质体的结构特点;着重讨论了阳离子脂质体作为基因载体时介导基因转移的机制以及在转染过程中对基因转染效率产生影响的主要因素。     

Influence of magnetic field on activity of given anaerobic sludge

Two modes of magnetic fields were applied in the Cr⁶⁺ removal sludge reactors containing two predominated strains—Bacillus sp. and Brevibacillus sp., respectively. The magnetic field mode I* of 0–4.5 or 0–14 mT between pieces was obtained by setting the magnetic pieces with the surface magnetic density of 0–6 or 0–20 mT into the reactor, and the magnetic field mode II* of 6, 20, or 40 mT on the return line was obtained by controlling the working distance of the permanent magnet outside the sludge return line. The effects of different magnetic fields on the activity of the given anaerobic sludge were studied by comparing with the control (absent of magnetic field). The results showed that the magnetic field of 0–4 mT improved the activity of given sludge most effectively, U_{max·\textCH4} U_{{\max \cdot {\text{CH}}_{4} }} (the peak methane-producing rate) and the methane producing volume per gCOD_Cr reached 64.3 mlCH₄/gVSS.d and 124 mlCH₄/gCOD_Cr, which increased by 20.6 and 70.7%, respectively, compared with the control. And the magnetic field of 20 mT took second place. It could be concluded that the input of some magnetic field could improve the activity of anaerobic sludge by increasing the transformation efficiency of COD_Cr matters to methane, and the total organic wastage did not increase.     

Efficiency of cytoplasmic delivery by non-cationic liposomes to cells in vitro: A confocal laser scanning microscopy study

It is necessary to understand liposomal uptake mechanisms and intracellular distribution in order to design more efficient gene (drug) carrier systems. Until now, a few studies have been carried out using confocal laser scanning microscopy (CLSM) to investigate the cellular uptake and transfection mediated with liposomes. So, by CLSM, we demonstrated that artificial virus-like envelope (AVE) vesicles labeled with rhodamine-PE (Rh-PE), carbocyanine (DiI) and carboxyfluorescein (CF) were investigated into the cytoplasm of two human cell lines, Mewo (human melanoma cell line) and HepG2 (human hepatoma cell line) cells grown in DMEM medium supplemented with different percentages (0%, 30%, and 100%) fetal calf serum (FCS). The liposome uptake was dependent on the cell line, in view that the whole process of liposomes associated with cells (uptake) is a two-step process involving binding and endocytosis. Based upon the various assays used to measure cellular uptake of liposomes, we conclude the efficacy of cytoplasmic delivery by AVE-liposomes to cells in culture.     

Induction of stress proteins by electromagnetic fields in cultured HL-60 cells.

HL-60 cells in culture were exposed for 2 h to a sinusoidal 0.1 or 1 mT (1 or 10 Gauss) magnetic field at 60 Hz and pulse labeled after exposure with radioactive isotopes by incubation by using either [(35)S]methionine, [(3)H]leucine, or [(33)P]phosphate. The radioactive labels were incorporated into cellular proteins through synthesis or phosphorylation. Proteins were extracted from electrostatically sorted nuclei, and the heat shock/stress proteins (sp) were analyzed for synthesis and phosphorylation by two-dimensional polyacrylamide gel electrophoresis. In the control cultures (no exposure to the magnetic field), sp 72c (cognate form) was faintly observed. A 0.1 mT exposure did not show sp metabolism to be different from that of the controls; however, after a 1 mT exposure of the HL-60 cells, sp 70i (inducible form) was synthesized ([(35)S]methionine incorporation). Sp 90 was not synthesized at either field level, but was phosphorylated ([(33)P]phosphate incorporation) in the 1 mT exposure. Sp 27 (isoforms a and b) was induced after a 1 mT exposure as reflected by labeling with [(3)H]leucine. These sps were not detected after a 0.1 mT exposure. After a 1 mT exposure and labeling with [(33)P], sp 27 isoforms b and c were phosphorylated whereas isoform 'a' was not observed. Sps 70i, 72c, and 90 were identified by commercial sp antibodies. Likewise, polypeptides a, b, and c were verified as sp 27 isoforms by Western blotting. Statistical evaluation of sp areas and densities, determined from fluorographs by Western-blot analysis, revealed a significant increase in sps 90 and 27a after a 1 mT magnetic field exposure. The 1 mT magnetic field interacts at the cellular level to induce a variety of sp species. Bioelectromagnetics 20:347-357, 1999. Published 1999 Wiley-Liss, Inc.     

Measurement and analysis of static magnetic fields that block action potentials in cultured neurons

To characterize the properties of static magnetic fields on firing of action potentials (AP) by sensory neurons in cell culture, we developed a mathematical formalism based on the expression for the magnetic field of a single circular current loop. The calculated fields fit closely the field measurements taken with a Hall effect gaussmeter. The biological effect induced by different arrays of permanent magnets depended principally on the spatial variation of the fields, quantified by the value of the gradient of the field magnitude. Magnetic arrays of different sizes (macroarray: four center-charged neodymium magnets of ?14 mm diameter; microarray: four micromagnets of the same material but of ?0.4 mm diameter) allowed comparison of fields with similar gradients but different intensities at the cell position. These two arrays had a common gradient value of ?1 mT/mm and blocked >70% of AP. Alternatively, cells placed in a field strength of ?0.2 mT and a gradient of ?0.02 mT/mm produced by the macroarray resulted in no significant reduction of firing; a microarray field of the same strength but with a higher gradient of ?1.5 mT/mm caused ?80% AP blockade. The experimental threshold gradient and the calculated threshold field intensity for blockade of action potentials by these arrays were estimated to be ?0.02 mT/mm and ?0.02 mT, respectively. In conclusion, these findings suggest that spatial variation of the magnetic field is the principal cause of AP blockade in dorsal root ganglia in vitro. © 1995 Wiley-Liss, Inc.     

Impact of pre-sowing magnetic field exposure of seeds to stationary magnetic field on growth, reactive oxygen species and photosynthesis of maize under field conditions

Impact of pre-sowing exposure of seeds to static magnetic field were studied on 1 month old maize [Zea mays _. var: HQPM.1] plants under field conditions. Pre-standardized magnetic field strength of 100 mT (2 h) and 200 mT (1 h), which were proven best for improving different seedling parameters under laboratory condition, were used for this study. Magnetic field treatment altered growth, superoxide radical level, antioxidant enzymes and photosynthesis. Among the different growth parameters, leaf area and root length were the most enhanced parameters (78–40%, respectively), over untreated plants. Electron paramagnetic resonance spectroscopy study showed that superoxide radical was reduced and hydroxyl radical was unaffected after magnetic field treatment. With decrease in free radical content, antioxidant enzymes like superoxide dismutase and peroxidase were also reduced by 43 and 23%, respectively, in plants that emerged from magnetically treated seeds. Measurement of Chlorophyll a fluorescence by plant efficiency analyzer showed that the potential of processing light energy through photosynthetic machinery was enhanced by magnetic field treatment. Performance index of the plant enhanced up to two-fold and phenomenological leaf model showed more active reaction centers after magnetic field treatment. Among the two field strengths used, 200 mT (1 h) was more effective in altering all these parameters. It is concluded that pre-sowing magnetic field treatment can be effectively used for improving plant growth and development under field conditions.     

Novel non-glycerol-based cytofectins with lactic acid-derived head groups.

We report herein the design, synthesis, and transfection biology of a novel series of non-glycerol-based cationic lipids with lactic acid-derived head groups The synthetic procedure adopted herein for preparing 1-hydroxy-prop-2-yl head-group-based monocationic transfection lipids 1-7 is fairly straightforward and potentially applicable in designing other cationic lipids with lactic acid-derived head groups. A striking anchor-length dependency was observed in NIH3T3 cells in the sense that except lipid 4, all the other lipids were essentially transfection-inefficient. Ethidium bromide assay for the lipid:DNA interactions is consistent with the general observation that significant lipid:DNA interactions do not guarantee on improved transfection efficiency cationic lipid mediated gene delivery. Given its remarkable transfection properties and low cellular toxicity, lipid 4 is likely to find future use in the area of liposomal gene delivery.     

Magnetic nanoparticles with surface modification enhanced gene delivery of HVJ-E vector

To enter the realm of human gene therapy, a novel drug delivery system is required for efficient delivery of small molecules with high safety for clinical usage. We have developed a unique vector "HVJ-E (hemagglutinating virus of Japan-envelope)" that can rapidly transfer plasmid DNA, oligonucleotide, and protein into cells by cell-fusion. In this study, we associated HVJ-E with magnetic nanoparticles, which can potentially enhance its transfection efficiency in the presence of a magnetic force. Magnetic nanoparticles, such as maghemite, with an average size of 29 nm, can be regulated by a magnetic force and basically consist of oxidized Fe which is commonly used as a supplement for the treatment of anemia. A mixture of magnetite particles with protamine sulfate, which gives a cationic surface charge on the maghemite particles, significantly enhanced the transfection efficiency in an in vitro cell culture system based on HVJ-E technology, resulting in a reduction in the required titer of HVJ. Addition of magnetic nanoparticles would enhance the association of HVJ-E with the cell membrane with a magnetic force. However, maghemite particles surface-coated with heparin, but not protamine sulfate, enhanced the transfection efficiency in the analysis of direct injection into the mouse liver in an in vivo model. The size and surface chemistry of magnetic particles could be tailored accordingly to meet specific demands of physical and biological characteristics. Overall, magnetic nanoparticles with different surface modifications can enhance HVJ-E-based gene transfer by modification of the size or charge, which could potentially help to overcome fundamental limitations to gene therapy in vivo.     

Generation of magnetic nonviral gene transfer agents and magnetofection in vitro

This protocol details how to design and conduct experiments to deliver nucleic acids to adherent and suspension cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of nucleic acids and cationic lipids or polymers (nonviral gene vectors), which are associated with magnetic (nano) particles. These magnetic complexes are sedimented onto the surface of the cells to be transfected within minutes by the application of a magnetic gradient field. As the diffusion barrier to nucleic acid delivery is overcome, the full vector dose is targeted to the cell surface and transfection is synchronized. In this manner, the transfection process is accelerated and transfection efficiencies can be improved up to several 1,000-fold compared with transfections carried out with nonmagnetic gene vectors. This protocol describes how to accomplish the following stages: synthesis of magnetic nanoparticles for magnetofection; testing the association of DNA with the magnetic components of the transfection complex; preparation of magnetic lipoplexes and polyplexes; magnetofection; and data processing. The synthesis and characterization of magnetic nanoparticles can be accomplished within 3-5 d. Cell culture and transfection is then estimated to take 3 d. Transfected gene expression analysis, cell viability assays and calibration will probably take a few hours. This protocol can be used for cells that are difficult to transfect, such as primary cells, and may also be applied to viral nucleic acid delivery. With only minor alterations, this protocol can also be useful for magnetic cell labeling for cell tracking studies and, as it is, will be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized transfection efficiency in any cell type.     

Quenching of an electric arc in a vacuum gap with a uniform transverse magnetic field

The breaking ability of a vacuum arc interrupter with a uniform transverse magnetic field formed by a system of permanent magnets was investigated experimentally. The vacuum interrupter with a 0.5-μF shunting capacitor switched off a dc current of up to 150 A at magnetic fields of 100–180 mT. At magnetic fields of 120–160 mT, the breaking ability of the vacuum interrupter was increased to 300 A by introducing a nonuniformity in the magnetic field distribution near the contact surface.     

A comprehensive review on histone-mediated transfection for gene therapy

Histone has been considered to be an effective carrier in non-viral gene delivery due to its unique properties such as efficient DNA binding ability, direct translocation to cytoplasm and favorable nuclear localization ability. Meanwhile, the rapid development of genetic engineering techniques could facilitate the construction of multifunctional fusion proteins based on histone molecules to further improve the transfection efficiency. Remarkably, histone has been demonstrated to achieve gene transfection in a synergistic manner with cationic polymers, affording to a significant improvement of transfection efficiency. In the review, we highlighted the recent developments and future trends in gene delivery mediated by histones or histone-based fusion proteins/peptides. This review also discussed the mechanism of histone-mediated gene transfection and provided an outlook for future therapeutic opportunities in the viewpoint of transfection efficacy and biosafety.     

Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.