Aptamer Modified Cationic Liposomes For Targeting Delivery Of Let-7a To Lung Cancer

Objectives:To prepare and characterize let-7a-loaded cationic liposomes for lung cancer targeting delivery,and investigate its inracellualr fate and in vivo synergistic anti-tumor effect with cisplatin(DDP)after pulmonary administration.Methods:A novel cationic lipoid,N,N'-dioleoyl tris(2-aminoethyl)amin(NDOA)was synthesized by the substitution and amidation reaction method.A zwitterionic compound,carboxyl betaine(CB)was obtained by ring-opening reaction.Their structures were confirmed by 1H-NMR,FTIR and LC-MS.Cationic liposomes composed of NDOA and CB(NLs)were prepared via the ethanol injection method,the physicochemical properties,such as particle size,Zeta potential,encapsulation efficiency of miRNA and the safety in A549 cells and 293T cells were characterized.The uptake and distribution of Apt-NLs in A549 cells were performed using flow cytometry(FCM)and confocal laser scanning microscope(CLSM).The effects of let-7a on the apoptosis and cell cycle were investigated by FCM.Western Blot technique was used to detect the effect of let-7a on the espression of target protein k-Ras.Gene silencing effect of let-7a to A549 cell was detected by RT-PCR.The effect of let-7a on the sensitivity of DDP in A549 cells was observed by MTT method.Spray freeze drying method was employed to fabricate dried powder inhalers containing miRNA-loaded NLs.IVIS Lumina ? imaging system was used to characterize and compare the progress of cancer and tissue distribution of NLs on the Luc-A549 tumor bearing mice after various administration of DDP and/or Apt-NLs-let-7a.Finally,H&E staining method was employed to investigate the influence of drug administration on the tissues and organs.Results:NDOA and CB were synthesized and confirmed by TLC,1H-NMR,FT-IR and LC-MS method.A novel cationic liposome modified with NDOA and CB(NLs)was prepared which have uniform particle size of about 70 nm,Zeta potential of 30 mV and high miRNA encapsulation capacity.In vitro anti-RNase test indicated that NLs played protect for let-7a against RNase degradation from the serum.The dry powder inhalant containing NLs showed regular round appearance and low hygroscopicity with mean physical particle size about 11.5 ?m and aerodynamic particle size about 3.9 ?m,respectively.The study of agarose gel electrophoresis showed that no significant influences on miRNA stability drung the spray freeze drying process.MTT test indicated that NLs had a low cytotoxicity in the concentration lower than 50 ?g/mL.The cellular uptake study displayed that AS 1411 could significantly enhanced the uptake of NLs in A549 cells.The results of CLSM observation showed that our vector could obviously deliver miRNA to cytoplasm of A549 cells and decrease the uptake by RAW264.7 in co-culture system.Annexin V-FITC/P1 double staining showed that the transfection of let-7a could promote the cycle of cancer cells in S phase,which could promote cell apoptosis.Western blot and RT-PCR study were also indicated the silence effect of let-7a on k-Ras protein and targeted mRNA.In addition,let-7a enhanced the efficacy of DDP in MTT test which demonstrate the potentional synergistic effect.The observation of in vivo fluorescence imaging of nude mice and in vitro tissue showed notable increasement of Cy5-miRNA to lung tissue by inhalation of Apt-NLs.DDP combined with Apt-NLs-let-7a had obvious inhibitory effect on tumor growth.Furthermore,H&E staining showed that DDP combined with Apt-NLs-let-7a had the most satisfied inhibition efficacy to the tumor growth with low toxicity on normal tissues and organs.Conclusion:The AS 1411-modified cationic liposomes we prepared showed low cytotoxity and high loading capacity of miRNA with satisfied targeting delivery efficiency,which could significantly down-regulate the expression of related proteins and enhance the anti-tumor effect of DDP by pulmonary inhalation.