Preparation and characterization of magnetic microbubbles and coating with lentiviral particles

The phospholipid solution containing DPPE and DPPC (Lipoid) for production of magnetic microbubbles (MMB) was prepared as described elsewhere¹⁰. To generate a 150 and 250 µg Fe/ml MMB solutions, MNP (SO-Mag or PEI-Mag) corresponding to a total iron weight of 150 or 250 µg were added to 1 ml lipid solution in 1.5 ml glass vials with screw caps and silicon/PTFE membranes (Schubert & Weiss Omnilab GmbH, München, Germany). The mixtures were then covered with perfluorocarbon gas and shaken for 20s in a CapMix (3M ESPE, Seefeld, Germany). Lentiviral particles were added to the MMB solutions in an optimal VP:MNP²⁵. In detail, 3.3*10⁶ VP/µg Fe, corresponding to 5*10⁸ VP/ml MMB solution, were added and left to associate for 10 min before use. Unless otherwise stated, amounts of lentiviral MMB applied in different cell culture experiments were individually calculated to achieve a cell:VP ratio of 1:5. Size and density of MB in solution with or without associated MNP (SO-Mag or PEI-Mag) and lentivirus (pCHIV.eGFP) were measured in 1:1000 dilutions in Hank's balanced salt solution (HBSS; Biochrom, Berlin, Germany) using the Casy Counter (Schärfe Systems, Roche Diagnostics, Mannheim, Germany). From the resulting MMB concentrations (MB/ml) mean iron content (µg Fe/MMB) and MNP content (MNP/MMB) were calculated. ζ-potential of MMB in HBSS was measured by photon correlation spectroscopy using a Malvern 3000 HS Zetasizer (Malvern, Herrenberg, Germany). Magnetic moments were assessed by magnetic responsiveness measurements as described by Heidsieck⁴². Lentivirus binding was verified by flow cytometry analysis of pCHIV.eGFP-MMB using a FACS Canto II (BD Biosciences, Heidelberg, Germany). Magnetizability of MMB as well as lentivirus binding capacity was determined by exposing lentiviral MMB solutions to a magnetic field for 15 min and applying MMB-free supernatant as well as MMB concentrates to endothelial cell culture. Culture plates were subsequently placed over a neodymium iron boron magnet (IBA Bio TAGnology, Goettingen, Germany) for 30 min and exposed to ultrasound (30 s, 1 MHz, 2 W/cm2, 50% duty cycle) using an ultrasonic device from Rich-mar (G. Heinemann, Schwaebisch Gmuend, Germany). Cells were washed with PBS supplemented with calcium and provided with growth medium. GFP expression was detected 72 h later by fluorescence microscopy and flow cytometry.