Figure 2
EGFR-targeted liposomal delivery system for cell transfection with the Picchu biosensor plasmid. (a) Schematic representation of the lipopolyplex used for targeted delivery of sensor DNA. The lipopolyplex comprises: pDNA encoding for the Picchu-X sensor; a bifunctional peptide with a cationic DNA-binding sequence and an EGFR-targeting sequence; a mixture of cationic and neutral lipids (DOPE, DOTMA, cholesterol and the PEG-shielding lipid DODEG-4); and optionally Cy-5 DOTMA. (b) Structures of key lipid components of the lipopolyplex and all four sequences of the bifunctional peptides used to formulate lipopolyplexes. Liposomes used in the experiments were formulated comprising: the helper lipid DOPE (23 mol%), which should fuse with the endosomal membrane and destabilize it;16 the cationic lipid DODEG4 (20; 39 mol%), which has a short ethylene glycol headgroup to provide steric shielding to the nanocomplex without impeding cellular uptake; DOTMA (15 mol%); and cholesterol (23 mol%). (c) For in vitro experiments, Cy5-DOTMA (1 mol%) was added to allow detection of the nanoparticles in cells. Liposome uptake (Cy5/UV ratio represents amount of liposome taken up by all cells (Hoechst staining – UV channel) in the well) in MDA-MB-231 cells treated with lipopolyplexes modified with Y-I or L-T peptides (please see Figure 2b for an explanation of the abbreviations). (d) Quantification of the expression of Picchu-X biosensor (eGFP/UV ratio) in the same cells as in (c) Complexes were prepared at 4:1 peptide/DNA weight ratio. One-way ANOVA to test differences between group means. P values are derived from a post-hoc Tukey’s test subsequently performed to compare means of individual groups while controlling for family-wise error rate. The difference was statistically significant between control and treatment groups, and data were expressed as means±s.e.m. (*P<0.05, **P<0.01, Student’s t-test, N=3).
