Liposomal magnetofection.

In a magnetofection procedure, self-assembling complexes of enhancers like cationic lipids with plasmid DNA or small interfering RNA (siRNA) are associated with magnetic nanoparticles and are then concentrated at the surface of cultured cells by applying a permanent inhomogeneous magnetic field. This process results in a considerable improvement in transfection efficiency compared to transfection carried out with nonmagnetic gene vectors. This article describes how to synthesize magnetic nanoparticles suitable for nucleic acid delivery by liposomal magnetofection and how to test the plasmid DNA and siRNA association with the magnetic components of the transfection complex. Protocols are provided for preparing magnetic lipoplexes, performing magnetofection in adherent and suspension cells, estimating the association/internalization of vectors with cells, performing reporter gene analysis, and assessing cell viability. The methods described here can be used to screen magnetic nanoparticles and formulations for the delivery of nucleic acids by liposomal magnetofection in any cell type.

Non-viral VEGF(165) gene therapy--magnetofection of acoustically active magnetic lipospheres ('magnetobubbles') increases tissue survival in an oversized skin flap model.

Adenoviral transduction of the VEGF gene in an oversized skin flap increases flap survival and perfusion. In this study, we investigated the potential of magnetofection of magnetic lipospheres containing VEGF(165)-cDNA on survival and perfusion of ischemic skin flaps and evaluated the method with respect to the significance of applied magnetic field and ultrasound. We prepared perfluoropropane-filled magnetic lipospheres ('magnetobubbles') from Tween60-coated magnetic nanoparticles, Metafectene, soybean-oil and cDNA and studied the effect in an oversized random-pattern-flap model in the rats (n= 46). VEGF-cDNA-magnetobubbles were administered under a magnetic field with simultaneously applied ultrasound, under magnetic field alone and with applied ultrasound alone. Therapy was conducted 7 days pre-operative. Flap survival and necrosis were measured 7 days post-operatively. Flap perfusion, VEGF-protein concentration in target and surrounding tissue, formation and appearance of new vessels were analysed additionally. Magnetofection with VEGF-cDNA-magnetobubbles presented an increased flap survival of 50% and increased flap perfusion (P < 0.05). Without ultrasound and without magnetic field, the effect is weakened. VEGF concentration in target tissue was elevated (P < 0.05), while underlying muscle was not affected. Our results demonstrate the successful VEGF gene therapy by means of magnetobubble magnetofection. Here, the method of magnetofection of magnetic lipospheres is equally efficient as adenoviral transduction, but has a presumable superior safety profile.

Searching for the right timing of surgical delay: angiogenesis, vascular endothelial growth factor and perfusion changes in a skin-flap model.

BACKGROUND: The angiogenic potential of vascular endothelial growth factor (VEGF) and its oxygen pressure-dependent regulation suggest a strong connection between this growth factor and the 'delay phenomenon'. In this study we focused on the chronological changes in VEGF concentration and flap perfusion in order to optimise the duration of surgical delay. METHODS: The VEGF concentration in skin and underlying muscle was measured in oversized, random-pattern flaps on 38 male Sprague-Dawley rats after 3, 5 or 7 days of surgical delay. Additionally, flaps were raised 5 or 7 days past preconditioning. The effect on flap perfusion was measured using indocyanine green fluoroscopy and the size of surviving and necrotic areas of the flaps were analysed. Microvessel density was assessed using a monoclonal CD31 antibody, and vessel diameter and morphometry were appraised by means of corrosion casting. RESULTS: VEGF expression in the distal half of the flaps was significantly increased 3 days after preconditioning and perfusion was significantly enhanced after day 5. An interval of 5 days between preconditioning and flap transposition resulted in a significantly reduced average necrosis rate. Microvessel density was significantly increased and vessel diameters were enlarged (P<0.05). CONCLUSIONS: We illustrated the chronology of events from the ischaemic procedure to the rise in VEGF concentration and changes in flap perfusion, and demonstrated vasodilatation and the formation of new vessels. Most significantly, we were able to further specify the optimal length of surgical delay based on alterations on a molecular level as well as changes in vascularisation and perfusion.

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.