Reversal Of Ovarian Cancer Multidrug Resistance By LAH4-L1-siMDR1 Nanocomplexes

Objective: As one of the three commonest gynecological malignancies,ovarian cancer has the highest mortality and worst prognosis.For most patients,the cancer cells had metastasized when they were diagnosed due to the lack of specific symptoms and diagnostic methods at an early stage.At least 75% of patients,cancer cells had metastasized(FIGO stages 3/4)when they were diagnosed.And 90% of them died of multidrug resistance to chemotherapeutics.In our study,RNAi technology was introduced and applied to overcome this big problem.LAH4-L1,an amphipathic cationic polypeptide,as the delivery vector to silence MDR1 gene which encodes P-gp in multidrug resistance ovarian cancer cells SKOV-3.We prepared self-assembled LAH4-L1-si MDR1 nanocomplexes(LSMCs)via electrostatic interaction.The effect of gene silencing,following expression of corresponding proteins,reversal of SKOV-3 cells MDR by drugs combination were the focuses of the research.Besides,characterization,p H responsivity,cytotoxicity,idiographic mechanisms of endocytosis and endosomal escape of this nanocomplexes were also evaluated.At last,we tested the effect on cell cycle and migration after transfection.Methods: 1.Characterization of LSMCs(zeta potential,particle size,morphology,si RNA encapsulation and p H responsiveness);2.Downregulation of MDR1 in SKOV-3 by LSMCs(m RNA level and protein level);3.CCK8 assays(cytotoxicity of LSMCs and reversal of multidrug resistance in ovarian cancer after MDR1 gene silence);4.Cellular uptake and its mechanism of LSMCs in SKOV-3 cells;5.Endosomal escape and its mechanism of LSMCs in SKOV-3 cells;6.Cell migration assays;7.Cell apoptosis experiment;8.Cell cycle assay.Results: 1.LSCMs(L/si MDR1=3/1)is a kind of circular/spherical shaped nanoparticle with weekly positive charged,p H responsiveness and size less than 200 nm,which can encapsulate si MDR1 well;2.And efficiencies of MDR1 gene silencing was the highest when the weight ratio of LAH4-L1/si RNA was at 3,with 87.3% gene silencing at m RNA level and 85% down-regulating at P-gp level;3.LSCMs(L/si MDR1=3/1)had lower cytotoxicity.And cells apoptosis can increase from 20.7%(PTX 3?g/ml),27.9%(PTX 7.5?g/ml),39.7%(PTX 15?g/ml),38.4%(DOX 5?g/ml),41%(DOX 10?g/ml)with si MDR1 transfection to 52.5%(PTX 3?g/ml),60.8%(PTX 7.5?g/ml),73.7%(PTX 15?g/ml),50.6%(DOX 5?g/ml),82.9%(DOX 10?g/ml)with LSCMs transfection;4.The process of cellular uptake of LSCMs by SKOV-3 can be completed in nearly 1h,and it was via lipid raft/caveolae and some other pathways with energy-dependent;5.LSMCs can escape from endosomes successfully within 6h transfection,and protonation is one of the important mechanisms of lysosomal escape;6.Cells which were transfected with LSMCs have lower capability of migration comparing with control groups;7.The number of apoptotic cells increased from 9.07% in si MDR1 group to 53.2% in LSMCs group when combined with 7.5?g/ml PTX;8.Compared with control group,cells transfected with LSMCs showed an increase in the proportion of S phase.When combining LSMCs with 7.5?g/ml PTX,cells were blocked both at S and G2 phase.Conclusions: LSMCs can achieve higher silencing efficiency on SKOV-3 cells MDR1 gene downregulation and reduced cells migration ability at the same time.