The DOPE-PEI conjugate and the DOPE-PEI/siRNA complexes were prepared as previously described21 , 22 (link). Briefly, for the preparation of the phospholipid-PEI/siRNA complexes, fixed amounts of siRNA and varying amounts of phospholipid-PEI conjugates were diluted separately in equal volumes (50 μL) of buffered HEPES glucose (HBG, pH 7.4, nuclease-free water). The siRNA solution was transferred to the polymer solution, mixed by smooth pipetting and incubated for 15-20 min at room temperature. A PEGylated formulation viz. DOPE-PEI/PEG/siRNA was prepared by adding a DOPE-PEI/siRNA complex solution prepared in HBG at N/P 16, to a previously freeze dried lipid film of PEG-PE (DOPE-PEI:PEG-PE was 1:10 w/w) and allowed to stand at room temperature for 30 min with intermittent shaking 26 (link).
The formulations were characterized for particle size distribution and zeta potential by Dynamic light scattering (DLS) using a Malvern Zetasizer ZS90 Nano instrument (Malvern Instruments, Westborough, MA). Briefly, 100μl of freshly prepared formulation was diluted suitably in nuclease free water and the size and zeta measurements were obtained. The morphological properties of the nanopreparations were also evaluated using Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM).
The formulations were characterized for particle size distribution and zeta potential by Dynamic light scattering (DLS) using a Malvern Zetasizer ZS90 Nano instrument (Malvern Instruments, Westborough, MA). Briefly, 100μl of freshly prepared formulation was diluted suitably in nuclease free water and the size and zeta measurements were obtained. The morphological properties of the nanopreparations were also evaluated using Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM).