In Vivo Fate And Pharmacodynamic Evaluation Of EGFR Mediated Lung Cancer Targeting Delivery Of SiRNA Loaded Novel Cationic Liposomes

Objective:To establish EGFR mediated a novel cationic lipoid modified lung tumor targeting delivery liposomes containing survivin & MRP1 iRNA, and evaluate its pharmaceutical properties, cytotoxicity, intracellular fate, in vivo distribution and antitumor effect after pulmonary inhalation while combined with cisplatin.Method:A novel cationic lipoid, N,N’-dioleoyl tris (2-aminoethyl) amine (NDOA) was synthesized by the substitution and amidation reaction. PEG2ooo-Mal modified DSPE was obtained by the classical synthetic method. The structures and purities of synthesized products were confirmed by TLC, FT-IR, and 1H-NMR. Various kinds of cationic liposomes were prepared via the ethanol injection method and physicochemical properties, such as particle size, Zeta potential and encapsulation efficiency of FAM-siRNA were measured. A kind of non-small-cell adenocarcinoma cell strain and resistant to cisplatin, A549/DDP, was selected due to the overexpressed survivin proteins and compared with A549 cells for the following study. Cell cytotoxicity was investigated by MTT method. Cellular uptake, endosomal escape and cell apoptosis of EGFR-PEG-NCLs-siRNA were performed using flow cytometry (FCM) and confocal laser scanning microscope (CLSM). Gene silencing effect of EGFR-PEG-NCLs-survivin/MRP1 siRNA to A549 cell was detected by RT-PCR. Spray freeze drying method was used to fabricate the inhalation powders containing siRNA loaded cationic liposomes. IVIS Lumina II imaging system was used to characterize the status of cancer and tissue distribution of liposomes on the Luc-A549 tumor bearing mice after pulmonary inhalation and i.v injection. Finally, H&E staining method was employed to investigate the influence of drug administration on the tissues and organs.Results:The results of TLC,1H-NMR and FT-IR confirmed the structure of NDOA and DSPE-PEG2000-Mal with high purity 87.0% and 86.2%, respectively. The mean particle size, zeta potential and FAM-siRNA encapsulation efficiency of EGFR-PEG-NCLs-siRNA were (193.2 ± 3.4) nm, (7.9 ± 0.1) mV and (91.9 ± 0.8)%. The photos of TEM showed smooth surface and homogeneous size distribution with satisfied dispersion. FCM study demonstrated that EGFR modification has significantly increase the cellular uptake of PEG-NCLs (p<0.05). CLSM observation displayed that EGFR-PEG-NCLs could escape from lysosome of A549/DDP. The apoptosis measurement showed that the apoptotic index by EGFR-PEG-NCLs-siRNA group was higher than that of Lipofectamine -2000. The results of RT-PCR indicated EGFR-PEG-NCLs-survivin and MRP1 siRNA showed high gene silencing capacities. Inhalation of EGFR-PEG-NCLs-siRNA showed a higher and prolonged accumulation in tumor area compared with i.v. injection under the IVIS Lumina II imaging system. In addition, i.v. injection of cisplatin combined with pulmonary inhalation of survivin and/or MRP1 siRNA showed remarkable synergetic effect on the tumor growth. H&E staining also indicated that the most potential therapeutic effects on the tumor without distinct normal tissue damages in cisplatin plus EGFR-PEG-NCLs-survivin and/or MRP1 siRNA groups.Conclusion:EGFR mediated a novel cationic lipoid modified lung tumor targeting delivery liposomes containing survivin & MRP1 siRNA were successively fabricated. The results of comprehensive characterization in vitro and in vivo indicated its potential as an efficient siRNA carrier for pulmonary inhalation, especially for tumor therapeutics combined with chemical anticancer agents.