Full-length clone DNA of human CRT cloned into pCMV3 vector was used (HG13539-ACR, Sino Biological Inc., Wayne, PA, USA). For CRT-NP synthesis, a lipid film was hydrated in 10 mM HEPES buffer (pH 7.4) at 55°C, and the lipid suspension was then extruded five times through filters of 200 nm pore size to yield homogeneous liposomes 34 (link). Next, a one-step method for loading the plasmid was developed by adding the pDNA solution in the liposomes vial (1:10, wt/wt), gently mixing them by pipetting, and incubating at room temperature for 30 min. The resultant CRT-NPs were characterized for plasmid encapsulation by the gel retardation assay in 1% agarose pre-cast gels containing ethidium bromide. A control sample of free pCRT as well as blank NPs were loaded onto the gels and the gels were run at 80 V on a Bio-Rad electrophoresis system. DNA dose was 0.2 µg per lane. Approximately 60 minutes after beginning the run, the gels were observed for plasmid migration. The CRT-NPs were also characterized in physiological buffers at room temperature by size (z-average) and zeta-potential using dynamic light scattering (DLS) with a Brookhaven ZetaPALS instrument (Holtsville, NY, USA). Furthermore, transmission electron microscopy (TEM) as previously published was performed to assess the morphology of CRT-NP using the JEOL JEM-2100 TEM (JEOL USA, Peabody, MA, USA) 26 (link).
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