A new polymer-based approach for in vivo transfection in postnatal brain.

BACKGROUND: Gene delivery within the central nervous system at postnatal age is one of the most challenging tasks in neuroscience and currently only a few effective methods are available. COMPARISON WITH EXISTING METHODS: For postnatal central nervous system cells, viral approaches are commonly used for genetic engineering but they face several biosafety requirements for production and use making them less accessible to the community. Conversely, lipid-based methods are widely used in cell culture but face limitation in vivo mainly due to the inflammatory responses they induce. To this aspect, the use of a transgenic mouse line can represent a credible answer to the community working on rat models still requires an effective and successful solution to circumvent these difficulties. NEW METHOD: We describe a new polymer-based gene delivery system allowing persistent and robust in vivo transfection with low DNA amount, reduced inflammation and high diffusion. The expression profile along the brain, the stability, the diffusion of the DNA together with the quantity of cells transfected were evaluated through in vivo approaches. RESULTS: With a single low-volume injection, we targeted different cell types within the rat brain. We measured the diffusion rate ranging from 1 to 5 mm based on the injected volume, in the three-dimensions axis. Finally, we modified brain susceptibility to epileptic seizures using a specific knock-down of the neuronal specific potassium-chloride transporter 2. CONCLUSIONS: This safe and easy system opens perspectives for non viral gene delivery in the rat brain with perspectives to study brain function in vivo.

High binding yet accelerated guest rotation within a cucurbit[7]uril complex. Toward paramagnetic gyroscopes and rolling nanomachines.

The (15-oxo-3,7,11-triazadispiro[5.1.5.3]hexadec-7-yl)oxidanyl, a bis-spiropiperidinium nitroxide derived from TEMPONE, can be included in cucurbit[7]uril to form a strong (K(a)∼ 2 × 10(5) M(-1)) CB[7]@bPTO complex. EPR and MS spectra, DFT calculations, and unparalleled increased resistance (a factor of ∼10(3)) toward ascorbic acid reduction show evidence of deep inclusion of bPTO inside CB[7]. The unusual shape of the CB[7]@bPTO EPR spectrum can be explained by an anisotropic Brownian rotational diffusion, the global tumbling of the complex being slower than rotation of bPTO around its "long molecular axis" inside CB[7]. The CB[7] (stator) with the encapsulated bPTO (rotator) behaves as a supramolecular paramagnetic rotor with increased rotational speed of the rotator that has great potential for advanced nanoscale machines requiring wheels such as cucurbiturils with virtually no friction between the wheel and the axle for optimum wheel rotation (i.e. nanopulleys and nanocars).

Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps.

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-ß-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated ß-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.