The nebulisation was performed for 10 min. The NGI was operated at a flow rate of 15 L/min, after cooling at 4��C for at least 1 h. The AeroEclipse was set on continuous operational mode, thus providing a near constant delivery rate independent from respiratory activity. After nebulisation, the residual amount of nanocomplex selleck chemical Rapamycin suspension was measured and collected. All the NGI stages and the throat were carefully rinsed with 1 ml of clinical grade H2O (Baxter). Aliquots of the recovered material were used for cell transfections, particle sizing and for DNA quantification experiments. Flow cytometry RTN formulations containing the pEGFP-N1 plasmid (Clontech-Takara Bio Europe) were nebulised through the NGI.
Pre-nebulisation samples (PN), the leftover (LO) in the nebuliser reservoir upon completion of the nebulisation and the aerosolised samples, collected from the throat and the different stages of the NGI and diluted with 1 ml of clinical grade H2O, were used for transfections. 30 ��l of each sample was added to 500 ��l OptiMEM (Invitrogen) and then to cells seeded in 24-well plates, grown at 80% confluence. After 48 h incubation, the cells were trypsinised and filtered with a cell strainer (? 70 ��m). Cells of duplicate wells were pooled together in PBS containing 3% FBS, before analysing EGFP expression by flow cytometry (LSRII, DB Bioscience Europe, Oxford, UK). Cell viability was determined by propidium iodide staining (Sigma) and at least 10,000 live cells were acquired. Quantification of DNA released on agarose gel RTN formulations were nebulised as described above.
All the samples pre- and post-nebulisation, and from the different NGI stages were collected. The DNA was released from the nanocomplexes as described previously [47]. Briefly, 100 ��l of samples were mixed with the same volume of 2�� DNA release buffer (20 mM Tris-Cl, pH 8.0, 200 mM NaCl, 50 mM EDTA and 1% SDS) and incubated at 50��C for 35 min. The DNA was then purified with the Wizard? DNA Clean-Up System (Promega) according to the instructions of the manufacturer. 25 ��l of each sample was loaded and run onto 1% agarose gel. The gels were visualised under UV light and the images were used for DNA quantification. The band densities, following the rolling ball background subtraction set at 20, were analysed on ImageJ programme (http://www.NIH.gov) and the yield of the recovered DNA was calculated. The yield was expressed as percentage AV-951 of the sum of the density of DNA (��) in each lane corrected for the volume of the sample collected (V), divided by the density of DNA of the starting nanocomplex suspension (��PreNeb) also corrected by the volume (VPreNeb).