Polyplexes of polyethylenimine and per-N-methylated polyethylenimine-cytotoxicity and transfection efficiency

For non-viral gene delivery, the carriers for DNA transfer into cells must be vastly improved. The branched cationic polymer polyethylenimine has been described as an efficient gene carrier. However, polyethylenimine was demonstrated to mediate substantial cytotoxicity. Therefore, this study is aimed at investigating per-N-methylated polyethylenimine, which is thought to have a much lower cytotoxicity due to its lower charge density. Results from a gel retardation assay and laser light scattering indicated that per-N-methylated polyethylenimine condenses DNA into small and compact nanoparticles with a mean diameter <150 nm. Furthermore, polyplexes of polyethylenimine and per-N-methylated polyethylenimine with DNA had a positive zeta potential and the polymers protected DNA from nuclease-mediated digestion. The transfection efficiency of polyethylenimine and per-N-methylated polyethylenimine was tested in CHO-K1 cells. Using green fluorescent protein as reporter gene and flow cytometry analysis, we demonstrated that per-N-methylated polyethylenimine has a lower cytotoxicity, but also a significantly lower transfection efficiency. Using propidium iodide staining, we could additionally distinguish between viable and dead cells. At NP > or = 12, per-N-methylated polyethylenimine showed a much higher cell viability and the ratio of viable and transfected cells to dead and transfected cells was about 1.5 to 1.7 fold higher than for polyethylenimine. The results of cell viability from flow cytometry analysis were confirmed by the MTS assay. Using luciferase reporter gene for transfection experiments, the gene expression of per-N-methylated polyethylenimine was lower at NP 6, 12 and 18 as compared to polyethylenimine, but at NP 24 it yielded similar levels.     

Novel hyperbranched polyamidoamine nanoparticles for transfecting skeletal myoblasts with vascular endothelial growth factor gene for cardiac repair

We investigated the feasibility and efficacy of hyperbranched polyamidoamine (hPAMAM) mediated human vascular endothelial growth factor-165 (hVEGF₁₆₅) gene transfer into skeletal myoblasts for cardiac repair. The hPAMAM was synthesized using a modified one-pot method. Encapsulated DNA was protected by hPAMAM from degradation for over 120 min. The transfection efficiency of hPAMAM in myoblasts was 82.6 ± 7.0% with cell viability of 94.6 ± 1.4% under optimal conditions. The hPAMAM showed much higher transfection efficiency (P < 0.05) than polyetherimide and Lipofectamine 2000 with low cytotoxicity. The transfected skeletal myoblasts gave stable hVEGF₁₆₅ expression for 18 days. After transplantation of hPAMAM–hVEGF₁₆₅ transfected cells, apoptotic myocardial cells decreased at day 1 and heart function improved at day 28, with increased neovascularization (P < 0.05). These results indicate that hPAMAM-based gene delivery into myoblasts is feasible and effective and may serve as a novel and promising non-viral DNA vehicle for gene therapy in myocardial infarction.     

Transfection efficiency of depolymerized chitosan and epidermal growth factor conjugated to chitosan–DNA polyplexes

An efficient non-viral gene delivery for varieties of cells has been considered essential for gene therapy and tissue engineering. This study evaluated transfection efficiency of chitosan (HW) with molecular weights (Mw) at 470 and degree of deacetylation (DDA) 80% and its depolymerization product (LW) with Mw at 16 kDa and DDA 54%, as well as epidermal growth factor (EGF) conjugated to chitosan–DNA microparticles of both HW and LW by using either disulfide linkage or NHS-PEO₄-Maleimide as a cross linker. The results revealed that the depolymerized LW at chitosan/DNA charge ratio 56:1 and pH 6.9 gave high transfection efficiency in both KB, a cancer cell line, and fibroblast cells at about the same level of Lipofectamine^TM, but the EGF-conjugated chitosan–DNA polyplexes from these methods did not improve transfection efficiency, which may come from the aggregation and fusing of the complexes as shown in scanning electron microscopy. However, this depolymerized LW chitosan showed the potential for further development as a safe and cost-effective non-viral gene delivery vehicle.     

O-Carboxymethyl-chitosan/organosilica hybrid nanoparticles as non-viral vectors for gene delivery

Polymeric non-viral vectors, such as chitosan nanoparticles show good biocompatibility, but low transfection efficiency. The objective of this study was to improve the transfection efficiency of chitosan based non-viral vectors by using o-carboxymethyl-chitosan which is a kind of water-soluble chitosan derivative and also has good biocompatibility. O-Carboxymethyl-chitosan-organosilica hybrid nanoparticles (CMG NPs) were synthesized through a rapid one-step aqueous synthetic approach for gene delivery. The size of nanoparticles was 276 ± 25 nm and zeta potential was 31.6 ± 0.4 mV in deionized water. Zeta potential increased with the decrease of pH, and it had been discovered that pH = 5.5 is the best point for CMG NPs to bond with plasmid DNA. DNA inclusion and integrity was evaluated by gel electrophoresis, and it is indicated that CMG NPs could protect DNA against DNase I and serum degradation. The results of MTT for cell viability and in vitro transfection also support the idea that CMG NPs could be used as efficient and safe vectors for gene delivery.     

Dendrosome-based gene delivery

Gene transfer to humans requires carriers for the plasmid DNA, which can efficiently and safely carry the gene into the nucleus of the desired cells. The purpose of the present study was to design dendrosomes as a novel, non-viral, vesicular, gene delivery vector and to carry out a comparative study of the relative transfection efficiencies of dendrosomes with standard non-viral, gene delivery vectors.

Fourth-generation PAMAM dendrimers were synthesized by double the Michael addition reaction and extensively characterized. The dendrimer–DNA complex was prepared and was confirmed by CD spectroscopy. The dendrosomes were prepared by the reverse phase evaporation method and the entrapment efficiency of the dendrosomal formulation was estimated. In vitro toxicity of the formulation was evaluated by hemolytic toxicity and cytotoxicity studies. Transfection efficiency of the dendrosomal formulations was compared to standard non-viral gene delivery vectors in HEK-293 cell.

The results of hemolytic toxicity cytotoxicity studies demonstrated that the dendrosomes possess negligible toxicity as compared to the other formulations and are suitable for in vivo administration. The results of transfection of HEK-293 cell with PGL2 showed that the dendrosomal formulation DF3 possesses superior transfection efficiency against other delivery systems under study.

Dendrosomes possess tremendous potential as a novel non-viral and non-toxic gene delivery vector.     

Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays

Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection-rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.     

Receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/DNA nanoparticles

Dendritic cells (DCs) are professional antigen presenting cells that induce, sustain, and regulate immune responses. Gene modification of DCs is of particular interest for immunotherapy of diseases where the immunes system has failed or is abnormally regulated, such as in cancer or autoimmune disease. Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA. Among various non-viral vectors, chitosan is considered to be a good candidate for gene delivery system, however, lack of cell specificity and low transfection of chitosan need to be overcome prior to clinical use. In this study, mannosylated chitosan (MC) was prepared to induce the receptor-mediated endocytosis and targeting into antigen presenting cells (APCs), especially DCs having mannose receptors. MC showed great ability to form complexes with DNA and showed suitable physicochemical properties for gene delivery system. It had low cytotoxicity and exhibited much enhanced gene transfer efficiency on the macrophage cell line than chitosan itself. Also, MC/DNA complex was more efficient for transferring IL-12 gene into DCs rather than water-soluble chitosan (WSC)/DNA one, which resulted in better induction of INF-gamma from DCs. Therefore, MC is a promising gene delivery system for repeated administration to maintain sustained gene expression, thereby opening the possibility for immunotherapy.     

Ion induced gas desorption problems in the ISR

Not least among the many stringent conditions which must be satisfied to achieve the proper operation of the Intersecting Storage Rings at CERN are those pertaining to its 2 km of ultrahigh vacuum. Apart from the direct influence of the residual gas molecules through nuclear and Coulomb scattering on the stored beam lifetimes (of the order of days during which the stored protons should travel the astronomical distance of some light days!) there are also indirect processes which additionally involve adsorption/desorption phenomena on the vacuum chamber wall. The beam of high energy protons creates low energy secondary ions in the residual gas. These ions, generated in the space charge potential of 1 kV (10 A)^?1 of proton beam, are accelerated by this potential onto the stainless steel vacuum chamber wall where a variety of adsorption/desorption phenomena may be stimulated. Desorbed molecules are fed back into the residual gas resulting in the possibility of creating an unstable run-away pressure increase which can finally destroy the stored beam. This danger depends on the local parameters of the vacuum system, and most critically, on the state of the vacuum chamber surface.The paper summarises recent observations in this field and describes the results of investigations aimed at producing cleaner surfaces in vacuum chambers. These investigations, which have included high temperature bakeouts, ion bombardment scrubbing by glow discharge, oxidation, etc, will show that under favourable conditions, net negative desorption coefficients can be obtained which turn the Intersecting Storage Rings into a large ion pump. Results, supported and compared with measurements using Auger and Sims surface analysis, are presented mainly from the empirical point of view with the emphasis on practical implications for machines where the surfaces of the vacuum system are subjected to ion bombardment.     

Multifunctional nanorods for gene delivery

The goal of gene therapy is to introduce foreign genes into somatic cells to supplement defective genes or provide additional biological functions, and can be achieved using either viral or synthetic non-viral delivery systems. Compared with viral vectors, synthetic gene-delivery systems, such as liposomes and polymers, offer several advantages including ease of production and reduced risk of cytotoxicity and immunogenicity, but their use has been limited by the relatively low transfection efficiency. This problem mainly stems from the difficulty in controlling their properties at the nanoscale. Synthetic inorganic gene carriers have received limited attention in the gene-therapy community, the only notable example being gold nanoparticles with surface-immobilized DNA applied to intradermal genetic immunization by particle bombardment. Here we present a non-viral gene-delivery system based on multisegment bimetallic nanorods that can simultaneously bind compacted DNA plasmids and targeting ligands in a spatially defined manner. This approach allows precise control of composition, size and multifunctionality of the gene-delivery system. Transfection experiments performed in vitro and in vivo provide promising results that suggest potential in genetic vaccination applications.     

A novel cationic liposome formulation for efficient gene delivery via a pulmonary route

The clinical success of gene therapy for lung cancer is not only dependent on efficient gene carriers but also on a suitable delivery route. A pulmonary delivery route can directly deliver gene vectors to the lung which is more efficient than a systemic delivery route. For gene carriers, cationic liposomes have recently emerged as leading non-viral vectors in worldwide gene therapy clinical trials. However, cytotoxic effects or apoptosis are often observed which is mostly dependent on the cationic lipid used. Therefore, an efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), previously synthesized by our group was first used to prepare cationic liposomes. Physicochemical and biological properties of LHLN-liposomes were investigated. LHLN-liposome/DNA complexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA binding capability. Compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited not only lower cytotoxicity (P < 0.05) but also similar transfection efficiency in A549 and HepG2 lung cancer cells for in vitro tests. When administered by intratracheal instillation into rat lungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higher pulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P < 0.05). These results suggested that LHLN-liposomes may have great potential for efficient pulmonary gene delivery.     

Nanostructure formation of Cu/Si(100) thin film induced by ion beam bombardment

A simple method for fabricating self-organized Cu nano-dots on Si(100) substrate by low energy Ar⁺ ion beam bombardment of a Cu thin film at room temperature over a large area is demonstrated. The morphological evolution has been investigated using scanning electron microscopy and atomic force microscopy. It was found that nano-ripple patterns formed on a Cu grain surface on a 110 nm thick polycrystalline Cu thin film under normal ion incidence. Uniformly distributed Cu nano-dots were obtained by bombardment of 55 nm thick nano-crystalline Cu thin films. The formation mechanism of the Cu nanostructures was discussed with the aid of numerical simulations using a modified damped Kuramoto–Sivashinsky equation.     

Cholesterol tethered bioresponsive polycation as a candidate for gene delivery

The efficient unpacking of viral protein shell gave the inspiration for the synthesized vectors. In this research, novel cholesterol tethered bioresponsive polyethylenimine (PEI) was specially designed via disulfide-containing cross-linker. The cholesterol lipid had proved to increase the permeability of gene vector through cell membrane. The acid–base titration indicated that the synthesized polycation possessed efficient proton sponge effect, which was suggested to increase endosomal release of pDNA complexes into the cytoplasm. The cholesterol tethered polycation could effectively induce DNA condensation and form spherical particles with diameter about 200 nm at N/P ratio of 10. At glutathione concentration of 3 mM, the polyplexes were unpacked due to the bioresponsive cleavage of the disulfide bonds. The in-vitro experiment indicated that the polyplexes showed efficient transfection efficiency to HEK293T cells. All the results indicated that the bioresponsive polycation could be served as an effective trigger to control the release of DNA at the intracellular environment. The novel bioresponsive polycation might have great potential in non-viral gene delivery research and application.     

Magnetic Tweezers‐Based 3D Microchannel Electroporation for High‐Throughput Gene Transfection in Living Cells

A novel high‐throughput magnetic tweezers‐based 3D microchannel electroporation system capable of transfecting 40 000 cells/cm² on a single chip for gene therapy, regenerative medicine, and intracellular detection of target mRNA for screening cellular heterogeneity is reported. A single cell or an ordered array of individual cells are remotely guided by programmable magnetic fields to poration sites with high (>90%) cell alignment efficiency to enable various transfection reagents to be delivered simultaneously into the cells. The present technique, in contrast to the conventional vacuum‐based approach, is significantly gentler on the cellular membrane yielding >90% cell viability and, moreover, allows transfected cells to be transported for further analysis. Illustrating the versatility of the system, the GATA2 molecular beacon is delivered into leukemia cells to detect the regulation level of the GATA2 gene that is associated with the initiation of leukemia. The uniform delivery and a sharp contrast of fluorescence intensity between GATA2 positive and negative cells demonstrate key aspects of the platform for gene transfer, screening and detection of targeted intracellular markers in living cells.     

Nitrogen beam bombardment induce polarity of carbon nanotubes

The multiwalled carbon nanotubes (MWCNTs) were prepared on SiO₂ substrates using chemical vapor deposition (CVD). N ion beam bombardment to MWCNTs was performed at different beam currents of 5–15 mA in an ion-beam-assisted deposition (IBAD) system. Scanning electron microscope (SEM) and Transmission electron microscope (TEM) proved no significant crack and surface morphological change for MWCNTs after N ion beam bombardment. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectrometry (FTIR), and Raman studies indicated that higher N ion beam current (15 mA) or N atomic concentration (8.6%) induced formation of polar N-containing functional groups of N–C and N–H bonds on the surfaces of MWCNTs. The content of N–C and N–H bonds increased with N ion beam current.     

Calculation of the pressure in the unit-cell vacuum system of Taiwan Photon Source

Taiwan Photon Source is a third-generation 3-GeV synchrotron light source under construction. The electron storage ring of circumference 518.4 m contains 24 unit cells and 24 long straight sections. Each cell of length about 14 m contains two bending chambers and two short straight chambers. To estimate the pressure distribution in the cell vacuum system, a program combining an iterative method and a Monte-Carlo method was used to calculate the pressure. In the modeling, the rate of thermal outgassing of the chamber surfaces and the yield of photon-stimulated desorption of the absorbers are obtained from the experimental results. To provide the effective pumping speed of various pumps that depends on the gas, the composition of residual gases from photon-stimulated desorption was assumed to be 80% H₂ and 20% CO. The pressure calculation for the vacuum cell compares the beam cleaning efficiency during the early commissioning stage when the accumulated beam dose attained 1 A h and 100 A h. The effects of the confined pumping design are evaluated through the modeling to assess whether the commissioning can be accelerated. The result of the pressure calculation obtained with the Monte-Carlo method shows that a mean pressure rise per beam current at 1.9 × 10⁻¹⁰ Pa mA⁻¹ after beam cleaning to a beam dose 100 A h is reasonable and within typical specifications of the synchrotron light source.     

Vacuum sealing using surface activation bonding of Si wafer

Bonding technology of Si wafer for vacuum seal is important in MEMS. We have tried the vacuum seal using surface activation bonding without any binder. It is the ultimate bonding technique and gives the precise dimension due to the direct contact. The technique is, however, not easy. We have investigated the surface conditions in order to achieve the bonding. The surfaces cleaned by Ar ion beam bombardment were measured by XPS and AFM. The natural oxide on the Si surface was removed by Ar ion bombardment. The surface roughness depended on the condition and the irradiation time of the Ar ion beam. The surface bonding at room temperature was achieved for the clean surface of the surface roughness less than Ra = 1 nm, but it was not done with the rough surfaces more than Ra > 2 nm. The vacuum sealing was checked using the cavities made in the Si wafer. The cavity part sealed in vacuum was depressed in the atmosphere, which was measured using a needle-contact profiler and a 3D laser profiler. The gas in the cavity was measured with a mass spectrometer by clashing the seal in vacuum. Any other gas except Ar gas closed in the cavity was not detected. We concluded that the vacuum sealing using surface activation bonding of Si wafer was achieved. The sealing condition has not changed even after one year.     

Properties of amorphous carbon films deposited by ion beam methods

Amorphous carbon films were prepared by the ion beam sputtering of graphite, by the ion beam sputtering of graphite with simultaneous ion bombardment of the growing film, and by primary ion beam deposition using a beam from a methane- argon plasma. The films are semiconducting in nature, having band gaps of the order of 1 eV. A nuclear reaction involving an energetic (2 MeV) beam of ¹¹B⁺ was used to obtain hydrogen profiles of the films. It was found that the electrical, optical and mechanical properties of the films could be correlated with the hydrogen content. The observed properties are explained qualitatively in terms of amorphous semiconductor theory.     

Tumorbehandlung mittels Partikeltherapie

Tumor Treatment through Particle Therapy Radiation therapy is used since many decades to treat malicious neoplasms. The ionized beam destroys the genetic construction (DNA) of the cancer cell and prevents further cancer cell division, since the required information is no longer available. The cancer cell dies. The radiation therapy is used only for treatment of local neoplasms (tumor). Metastasizing neoplasms cannot be cured. Especially patients with complicated and deep in the body located neoplasms can be treated, where other conventional radiation therapies do not provide effective results. Infantile cancer types can be treated very gentle to avoid long term side effects. The particles need to be accelerated in a high vacuum environment of < 1*10^‐8 mbar by means of magnets in vacuum tubes and synchrotron apperture up to 75 % of light speed to emit its radioactive dose.     

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.     

Mechanism of low energy S+ ion bombarded p-GaAs (100)

X-ray photoelectron spectroscopy (XPS) and low energy electron diffraction (LEED) have been used to characterize the chemical structure and site location of sulfur atom on p-GaAs (100) treated by bombardment of low energy S⁺ ions over the range from 10 to 100 eV. S⁺ ion bombardment resulted in the formation of Ga–S and As–S species on GaAs surface. It was found that the S⁺ ions with energy above 50 eV were more effective in formation of Ga–S species, which assisted the GaAs (100) surface in reconstruction into an ordered (1 × 1) structure upon annealing. After taking into account physical damage due to the process of ion bombardment, we found that 50 eV was the optimal ion energy to form Ga–S species in the sulfur passivation of GaAs (100). The subsequent annealing process removed both donor and acceptor states that were introduced during the ion bombardment of GaAs, and resulted in a sharp (1 × 1) LEED pattern.