A SiRNA Loaded Epoxy Alkyl Amine Derivative Lipoplex For Pulmonary Administration

Objectives: To reduce the resistance of cancer cells against cisplatin by pulmonary administration of Survivin si RNA and MRP1 si RNA loaded lipoplexes and evaluate si RNA delivery efficiency of lipoplexes.Methods: A series of Epoxy alkyl amine derivative Lipidoids(EAADs) were synthesized and characterized by 1H-NMR and FT-IR. EAADs, PEG-EAADs and EGFR m Ab-PEG-EAADs lipoplexes were prepared by ethanol injection method. Cellular uptake and intracellular distribution were evaluated using flow cytometer and confocal laser scanning microscopy, respectively. Dry powder inhalation was gained by spray freeze drying process. In vitro gene silencing efficiency in A549/DDP cells was subsequently carried out after transfected by Survivin si RNA and MRP1 si RNA loaded EAADs lipoplexes and which were evaluated by MTT test and Western Blot. Furthermore, cell apoptosis and cell cycle were detected using Annexin V-FITC/PI and PI method after transfected by Survivin si RNA. In vivo distribution of Cy5 labeled si RNA was observed through IVIS Lumina Ⅱ after administrated by intravenous injection or pulmonary delivery. Orthotopic model of lung cancer developed for nude rats was used to evaluate in vivo anti-tumor efficiency of Survivin si RNA and MRP1 si RNA loaded EGFR m Ab-PEG-EAADs lipoplexes coadministrated with cisplatin and tumor growth was monitored by IVIS Lumina Ⅱ. Besides, releated blood biochemistry index were examined by blood chemistry to evaluate liver and kidney functions. At last, pathological examinations of tumor tissues were analyzed by HE staining.Results: The fabricated EAADs lipoplex has a uniform particle size about 200 nm with low cell toxicity and improved anti-RNase efficiency. Dry powder inhalation got a physical size of 11.90±0.57 μm, aerodynamic size of 2.45 μm, hygroscopicity of 6.64±1.12%, which has a porous low density characteristics, suitable for pulmonary administration. Celluar uptake efficiency of EAADs-3 lipoplexes in A549/DDP cells was higher than EAADs-1 and EAADs-2 lipoplexes. PEG modification was conducive to celluar uptake. Besides, EGFR m Ab-PEG-EAADs-3 lipoplexes had a relative uniform distribution in the cytoplasm of A549/DDP cells. Moreover, EGFR m Ab-PEG-EAADs-3 lipoplexes had an interesting property to selectively locate in the cytoplasm of A549/DDP cells in our A549/DDP cells & Raw 264.7 cells co-culture system. Survivin si RNA and MRP1 si RNA with high silence efficiency were chosen through Western Blot. After transfected with Survivin si RNA or/and MRP1 si RNA by EAADs-3, PEG-EAADs-3 and EGFR m Ab-PEG-EAADs-3 lipoplexes, the sensitivity of cisplatin to A549/DDP cells was increased and the target protein expression was down-regulated. For A549/DDP cells transfected with Survivin si RNA, cell apoptosis rate was increased and cell cycle was blocked in G2. Pulmonary delievery of EGFR m Ab-PEG-EAADs-3 lipoplexes showed a satisfactory distribution in lung tissue compared with intravenous injection. Pulmonary delivery of Survivin si RNA and MRP1 si RNA loaded EGFR m Ab-PEG-EAADs-3 lipoplexes showed a significant anti-tumor efficiency when combined administrated with DDP. There was no obvious damage of liver and kidney function according to blood biochemistry analysis.Conclusion: EGFR m Ab-PEG-EAADs-3 lipoplexes had a promising potential of si RNA delivery by pulmonary administration to reverse tumor resistance and enhance treatment effect of cisplatin.