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.     

The targeted gene (KDRP-CD/TK) therapy of breast cancer mediated by SonoVue and ultrasound irradiation in vitro

Suicide gene therapy has become an effective therapy for breast cancer, and ultrasound targeted microbubble destruction (UTMD) has become a popular topic in the gene therapy field. In this study, MCF-7 cells with the KDR promoter and LSl74T cells without the KDR promoter were transfected with the recombinant plasmid pEGFP-KDRP-CD/TK using UTMD. The recombinant plasmid pEGFP-KDRP-CD/TK was transfected into MCF-7 and LS174T cells successfully with no significant difference in transfection efficiency (p > 0.05). By RT-PCR, the CD/TK fusion gene was shown to be expressed in MCF-7 cells but not expressed in LS174T cells. In a cytotoxicity experiment, transgenic MCF-7 cells were sensitive to the prodrugs 5-FC and GCV. When both 5-FC and GCV were administered, the rate of cellular inhibition was significantly greater than that achieved when only one of the prodrugs was administered (p < 0.001). Moreover, the inhibition rates achieved administering 5-FC, GCV and both 5-FC and GCV were all significantly greater than the gene transfection rate of 21.92 ± 3.64% (p < 0.001). However, transgenic LS174T cells were not sensitive to any prodrug. These results demonstrated that UTMD is a safe, effective and targeted gene delivery system. Also, the KDR promoter can drive expression of the CD/TK double suicide gene target in MCF-7 cells, and the targeted killing effect of the KDRP-CD/TK gene on MCF-7 cells in vitro has good synergy with expression of the CD/TK fusion gene.     

Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid microbubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin–biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm²) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells.     

Application of high-power ultrasound to enhance fluid/solid particle separation processes

The separation of fine particles from gases or liquids is a topic of permanent industrial attention. The use of ultrasonic energy to assist conventional separation techniques seems to be very promising. The adequate applications of high-intensity ultrasonic fields may contribute to improve the efficiency and capacity of the separation methods presently used. The specific mechanisms to ultrasonically enhance separation processes basically depend on the medium to be treated. In gas suspensions, where very fine particles have to be removed, ultrasonic action involves agglomeration of particles in order to increase their size and, consequently, to improve collection efficiency of conventional filters. In liquid suspensions, agglomeration is, in general, less efficient than in gases. Nevertheless, the ultrasonic energy is useful to dewater fine-particle high-concentration suspensions such as slurries and sludges. This paper deals with the application of acoustic energy to assist fluid/solid separation processes in gas and liquid suspensions and presents some theoretical and experimental results in specific applications.     

Physics of particulate flows: From sand avalanche to active suspensions in plants

Flows of granular media in air or in a liquid have been a research field for physicists for several decades. Sometimes solid, sometimes liquid, these particulate materials exhibit peculiar behaviors, which have motivated many studies at the frontiers between nonlinear physics, soft matter physics and fluid mechanics. This paper presents a summary of the recent advances in the field, with a focus on the development of continuous approaches, which make it possible to treat granular media as a complex fluid and to develop a granular hydrodynamics. We also discuss how the better understanding of granular flows we have today may help to address more complex materials, such as colloidal suspensions or some biological systems.     

A novel approach to quantitative ultrasonic naked gene delivery and its non-invasive assessment

The purpose of this study was to investigate practical, safe, easy-to-use, non-cytotoxic, and reliable parameters to apply to an ultrasound (US) naked gene therapy system. The ultrasound pressure at the point of cell exposure was measured using a calibrated hydrophone and the intensity calculated. An acoustic power meter calibrated using a hydrophone was used to measure the power of the transducer. Four cell types were exposed to US with different exposure times and intensities. Fluorescent microscopy, spectrophotometry, scanning electron microscope, laser scanning confocal microscopy, flow cytometry and histogram analysis were used to evaluate the results of the study. The plasmid of green fluorescent protein (GFP) served as the reporter gene. The energy accumulation E in US gene delivery for 90% cell survival was defined as the optimal parameters (E=3.56+/-0.06), and at 80% cell survival was defined as the damage threshold (E=59.67+/-3.54). US safely delivered GFP into S180 cells (35.1 kHz) at these optimal parameters without obvious damage or cytotoxity in vitro. Exposed cell function was proved normal in vivo. The transfection rate was 35.83+/-2.53% (n=6) in viable cells, corresponding to 90.17+/-1.47% (n=6) cell viability. The intensity of GFP expression showed a higher fluorescent peak in the group of adeno-associated virus GFP vector (AVV-GFP) than in the control group (P<0.001). The effect of US gene delivery and cell viability correlated as a fifth order polynomial with US intensity and exposure time. With optimal parameters, US can safely deliver naked a gene into a cell without damage to cell function. Both optimal uptake and expression of gene depend on the energy E at 90% cell survival. E can be applied as a control factor for bioeffects when combined with other parameters. Stable caviation results in optimal parameters for gene delivery and the transient caviation may cause cell damage, which will bring about a sharp rise of permeabilization. The results may be applied to the development of a novel clinical gene therapeutic system.     

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.     

Sensitive Parameter Predicting Steady-State Pressure Difference of Partially Crosslinked Polyacrylamide Suspension in Core Flow Experiments

Using artificial cores, six different partially crosslinked polyacrylamide (PCPAM) suspensions were used for sandpacked core flow experiments; the results indicated that the migration of the PCPAM particles through porous media was a process of plugging and flooding at the same time. A theoretical model was proposed and the sensitive parameter, G′, was determined by rheological oscillation measurements with a 200 μm plate gap. In addition, the validity of the proposed theoretical model was verified by the results of the core flow experiments and a fitting equation of ΔP ₁ with G′ was obtained which can be used for quantifying the steady-state pressure of PCPAM suspensions flowing through porous media in the core flow experiments.     

动力排气系统细水雾蒸发冷却试验研究

为了掌握设计的细水雾蒸发冷却器对发动机排气的喷雾降温性能,建立了喷雾降温试验台,用设计的屏蔽式气相测温装置和压力损失测量装置,准确测量了排气管内喷雾后的排气温度和压力损失。结果表明,细水雾蒸发冷却器向发动机排气内喷雾80 s,高温排气即可降至稳定温度,排气压力损失比喷雾前减小;雾化压力越大,喷雾流量越大,喷雾降温效果越好,压力损失越小;排气出口对应的液态水饱和温度可视为喷雾降温的极限温度。     

Flow-field measurements of liquid and gaseous phases in the ultra-dense region of diesel sprays

The velocities of both the liquid and the gaseous phases in a diesel spray are determined for the first time simultaneously via laser flow tagging (LFT). The experimental setup and uncertainties introduced by seeding particles in the gas phase are greatly reduced by using phosphorescent tracer molecules, i.e. acetone and biacetyl, for the gaseous phase. In addition, simultaneous droplet velocimetry is achieved by doping the liquid fuel with a lanthanide–chelate complex.The relative velocity of gas and liquid phases can be determined from the data on the spray axis close to the nozzle for the first time. This is an important quantity for modeling droplet break-up and evaporation in ultra-dense sprays.PACS 42.62.-b; 47.61.Jd     

Large Eddy Simulation of a swirl stabilized spray flame

Large Eddy Simulations (LES) of kerosene spray combustion in an axial-swirl combustor have been carried out focusing on the effect of the evaporating droplets on the flame temperature and species concentrations. The LES-PDF methodology is used for both dispersed (liquid) and gas phases. The liquid phase is described using a Lagrangian formulation whilst an Eulerian approach is employed for the gas phase. The predictive capability of LES with sub-grid scale models for spray dispersion and evaporation is assessed placing emphasis on the effect of the unresolved velocity and temperature fields on the droplet evaporation rate. The results of the fully coupled LES formulation exhibit good agreement between the measured and simulated mean velocity fields. The global behaviour of the spray combustion, such as droplet dispersion and evaporation, are captured reasonably well in the simulations. It was found that the large velocity fluctuations observed in the shear layer strongly affect the evaporation rate and thus the temperature distributions. The present work also demonstrated the feasibility of LES to study complex flow features which are typical of gas-turbine combustion chambers.     

分子动力学模拟玻璃微球充气保气性能的机理

为了探讨载能重粒子辐照改善惯性约束聚变中所用玻璃微球的充气性能,以及热弛豫过程中改善其保气性能的微观机制,利用分子动力学的方法对载能重粒子辐照前后的SiO₂玻璃在内部形成气体扩散微通道的机理进行了模拟研究。结果表明,重粒子辐照可以在SiO₂玻璃中形成明显的扩散通道,说明辐照后的玻璃更适合通入Ar等较大尺寸气体;在高温高压的条件下,形成的扩散通道有逐渐关闭的趋势,辐照产生的缺陷能够在一定程度上得到修复。因此,利用重粒子辐照的方法,可在玻璃体中产生充气的微通道,该通道在高温高压下可实现关闭,从而起到开关的作用。模拟结果可为实验上通过重粒子辐照提升微球玻璃充气保气性能的方法提供理论支持,说明该方法具有可行性。     

“Slow” active control of combustion instabilities by modification of liquid fuel spray properties

This paper describes an experimental investigation of the feasibility of using “slow” active control approaches, which “instantaneously” change liquid fuel spray properties, to suppress combustion instabilities. The objective of this control approach was to break up the feedback between the combustion process heat release and combustor pressure oscillations that drive the instability by changing the characteristics of the combustion process (e.g., the characteristic combustion time). To demonstrate the feasibility of such control, this study used a proprietary fuel injector (Nanomiser^TM), which can vary its fuel spray properties, to investigate the dependence of acoustics–combustion process coupling, i.e., the driving of combustion instabilities, upon the fuel spray properties. This study showed that by changing the spray characteristics it is possible to significantly damp combustion instabilities. Furthermore, using combustion zone chemiluminescence distributions, which were obtained by Abel’s deconvolution synchronized with measured acoustic data, it has been shown that the instabilities were mostly driven midway between the combustor centerline and wall, a short distance downstream from the flame holder, where the mean axial flow velocity is approximately zero in the vortex near the flame holder. The results of this study strongly suggest that a “slow” active control system that employs controllable fuel injectors could be effectively used to prevent the onset of detrimental combustion instabilities.     

Spray flow-network flow transition of binary Lennard-Jones particle system

We simulate gas-liquid flows caused by rapid depressurization using a molecular dynamics model. The model consists of two types of Lennard-Jones particles, which we call liquid particles and gas particles. These two types of particles are distinguished by their mass and strength of interaction: a liquid particle has heavier mass and stronger interaction than a gas particle. By simulations with various initial number densities of these particles, we found that there is a transition from a spray flow to a network flow with an increase of the number density of the liquid particles. At the transition point, the size of the liquid droplets follows a power-law distribution, while it follows an exponential distribution when the number density of the liquid particles is lower than the critical value. The comparison between the transition of the model and that of models of percolation is discussed. The change of the average droplet size with the initial number density of the gas particles is also presented.     

Plasma synthesis of semiconductor nanocrystals for nanoelectronics and luminescence applications

Functional nanocrystals are widely considered as novel building blocks for nanostructured materials and devices. Numerous synthesis approaches have been proposed in the solid, liquid and gas phase. Among the gas phase approaches, low pressure nonthermal plasmas offer some unique and beneficial features. Particles acquire a unipolar charge which reduces or eliminates agglomeration; particles can be electrostatically confined in a reactor based on their charge; strongly exothermic reactions at the particle surface heat particles to temperatures that significantly exceed the gas temperature and facilitate the formation of high quality crystals. This paper discusses two examples for the use of low pressure nonthermal plasmas. The first example is that of a constricted capacitive plasma for the formation of highly monodisperse, cubic-shaped silicon nanocrystals with an average size of 35 nm. The growth process of the particles is discussed. The silicon nanocubes have successfully been used as building blocks for nanoparticle-based transistors. The second example focuses on the synthesis of photoluminescent silicon crystals in the 3–6 nm size range. The synthesis approach described has enabled the synthesis of macroscopic quantities of quantum dots, with mass yields of several mg/hour. Quantum yields for photoluminescence as high as 67% have been achieved.     

Detonation waves in bubble-drop media

Wave processes in chemically active multicomponent media: liquid — gas bubbles — liquid drops have been studied experimentally. Existence of detonation waves in multicomponent (bubble-drop) media has been proved. Structure of detonation waves in bubble-drop and bubble media is qualitatively identical: detonation waves are solitary waves with pulsation profile the pressure behind which is close in value to the one in unperturbed medium. Propagation velocity of detonation waves in bubble and bubble-drop media drops with the increase in medium gas phase concentration and with the decrease in carrier liquid viscosity. Presence of liquid drops decreases detonation wave velocity compared with bubble medium that does not contain liquid drops. Detonation wave propagation in multicomponent media causes gas bubbles fragmentation as well as fragmentation of individual liquid drops. The work was financially supported by the Russian Foundation for Basic Research (Grant No. 04-03-33106).     

Prospects of anionic nanolipoplexes in nanotherapy: transmission electron microscopy and light scattering studies

Currently nanosystems composed of polynucleotides and lipid vesicles (nanolipoplexes) are considered to be promising tools for gene therapeutics. Successful in vivo application of these vectors depends on their physicochemical, technological and biological characteristics including morphology, size distribution, molecular interactions and stability. Anionic nanoliposomes (DPPC:DCP:CHOL) were prepared by two different techniques, namely the conventional thin-film hydration method followed by extrusion, and the heating method (HM), in which no volatile solvent or detergent is used. A non-viral and non-cationic gene transfer vector was constructed by incorporating plasmid DNA (pcDNA3.1/His B/lacZ) to the HM-nanoliposomes by the electrostatic mediation of Ca²⁺ ions. Transfection efficiency of the nanolipoplexes was evaluated using a human bronchial epithelial cell line (16HBE14o-) in the presence of serum. Particle characterisation, stability of the formulations and lipid–DNA interaction studies were performed using transmission electron microscopy (TEM) and light scattering. TEM pictures of nanolipoplexes showed presence of two to four closely packed vesicles with signs of fusion. Efficient delivery of plasmid DNA and subsequent β-galactosidase expression was achieved using the anionic nanolipoplexes. Transfection efficiency increased with lipid:DNA ratio up to 7:1 (w/w), where transfection efficiency was 12-fold higher than in untreated cells. Further increase in lipid ratio decreased transfection. These nanolipoplexes appear to be safe, stable and efficient in the protection and delivery of DNA to different cells and tissues.     

Combined “Fluorescence” LDV (FLDV) and PDA Technique for Non‐ambiguous Two Phase Measurements Inside the Spray of a SI‐Engine

Laser velocimetry measurements in the vicinity of reflecting surfaces are still a major problem in many fluid mechanical applications such as measuring close to walls or wall film surfaces, respectively. Moreover, in any kind of two phase flow an unambiguous separation of the gas and the liquid phase is of particular interest. Commonly used techniques like Phase Doppler Analyzers (PDA) with size discrimination are limited to two phase flows where the smallest particle of the dispersed phase is significantly larger than the seeding particles. This condition can rarely be fulfilled in technically relevant spray/air systems for instance in automobile engines or gas turbines. One of the most promising approaches to overcome this problem is a correct phase discrimination using fluorescent tracer particles for the gas phase. In this paper different laser based velocimeters have been compared using the spray of a gasoline injection nozzle as a typical example. The working principle of the “fluorescence” LDV (FLDV) will be explained in detail. Moreover, the quality of the fluorescence signals and of the standard bursts received from Mie‐scattering particles will be compared. Finally, the capabilities of combined FLDV and PDA measurements inside the spray of a SI‐engine at unsteady conditions will be presented. The pros and cons of this technique will be discussed against the background of discriminatory two phase PIV measurements applied to the same spray.     

悬浮液凝固研究进展

悬浮液凝固是将固体颗粒均匀分散于液体中,并对液体进行凝固.它涉及冷冻铸造法多孔材料制备、冻土冻胀、海上浮冰、生物组织冷藏等多个学科和领域.近年来通过悬浮液凝固的方法,人们相继开发出了众多性能优异的新材料,这更为悬浮液凝固的研究注入了新的活力.悬浮液凝固过程中存在众多不同于传统合金凝固的独特现象,如颗粒密堆层、层片晶组织、周期性冰透镜体组织等,但这些凝固微观组织的形成机理尚不清楚.揭示颗粒对悬浮液凝固界面形态演化的影响机理,建立悬浮液体系的凝固理论,不仅帮助人们理解冻土冻胀等自然现象,也为高性能材料的制备与开发提供理论依据.本文首先回顾了悬浮液凝固的研究历史,然后对当前悬浮液凝固微观组织及颗粒动态堆积等方面的研究进展进行述评,并简要介绍本课题组近年来在悬浮液凝固方面的工作进展,最后对悬浮液凝固研究的未来发展进行了展望.     

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.