| Background: Pancreatic cancer has the characteristics of no obvious clinical symptoms,easy metastasis and strong invasiveness.There is no effective means for advanced treatment,and the mortality rate is high.5-year survival rate is only 8%.Gemcitabine(Gem)is a first-line chemotherapy for pancreatic cancer treatment.Although gemcitabine can improve the quality of life of patients to a certain extent,there are still problems such as the efficacy of single-agent chemotherapy being less than 15%.Therefore,in order to further improve the therapeutic effect of gemcitabine on pancreatic cancer,a new strategy is urgently needed.In recent years,overexpression of myeloid cell leukemia-1(Mcl-1)has been confirmed in a variety of tumors such as pancreatic cancer,and is closely related to the occurrence and development of malignant tumors.Decreasing the expression level of Mcl-1 can not only directly promote the apoptosis of tumor cells,but also significantly increase the chemosensitivity of tumor cells.Therefore,targeted Mcl-1 intervention can greatly improve the patient's postoperative survival rate.Compared with the small molecule inhibitor drugs of Mcl-1,small interfering RNA(si RNA)technology has been widely concerned in gene silencing and drug development due to its high specificity and small side effects.However,si RNA has the disadvantages of weak membrane permeability and easy degradation.Therefore,it is necessary to find a reliable vector for efficient delivery of si RNA to the interior of cells to exert functions of gene silencing.Nowadays,various types of si RNA delivery systems have been developed,which are mainly classified into two types: viral vectors and non-viral vectors.Although viral vectors have high transfection efficiency,their poor specificity,limited encapsulation capacity and biosafety limit their clinical application.The non-viral vector has become an emerging carrier because of its large loading capacity,strong modification,low cytotoxicity and easy storage.Among them,cationic liposome is considered to have potential clinical application due to its simple preparation,retransfection and easy degradation.1,2-dioleoyl-3-trimethylammonium-propane(DOTAP)is the most widely used cationic liposome,which is relatively inexpensive and has good effects in both in vitro and in vivo applications.Objective: In this study,we constructed a cationic liposome-based system(LPs)using DOTAP,cholesterol and protamine for gene delivery.DOTAP can be combined with a negatively charged si RNA by a positively charged cation head,protecting the si RNA from nuclease degradation and allowing it to be released into the cytoplasm.As a common auxiliary lipid,cholesterol can stabilize the lipid bilayer and reduce the toxicity of cationic lipids.Protamine can bind to si RNA through electrostatic interaction and can be used as a carrier itself,and it can enhance the transfection efficiency of lipid gene vector.In addition,the delivery carrier can also encapsulate a chemotherapeutic drug at the same time.Therefore,we used synthetic nanocarriers to co-deliver Gem and Mcl-1 si RNA to form LP-Gem-si Mcl-1,the intervention of targeted Mcl-1 on pancreatic cancer and the synergistic anti-tumor effect of gemcitabine and Mcl-1 si RNA were evaluated by a series of experiments in vitro and in vivo.Methods:(1)We used quantitative real-time PCR(q RT-PCR)and western blot to determine the expression of Mcl-1 in pancreatic cancer,and examined the effect of down-regulating Mcl-1 expression on the sensitivity of gemcitabine chemotherapy.(2)Nanocarriers were prepared using DOTAP,cholesterol and protamine,and Gem and Mcl-1 si RNAs were loaded and their characterization was tested.The particle size,potential and the morphology of nanocarriers were measured by dynamic light scattering(DLS)and transmission electron microscopy(TEM);Ultra Performance Liquid Chromatography(UPLC)and agarose gel electrophoresis were used to detect the encapsulation efficiency of gemcitabine and the adsorption capacity of nanocarriers on si RNA;Confocal microscopy and flow cytometry were used to analyze intracellular uptake and subcellular localization of LPs;Western blot and q RT-PCR to detect the interference efficiency of si RNA on intracellular Mcl-1.(3)In vitro,we measured the effects of LP-Gem-si Mcl-1 on proliferation and apoptosis of pancreatic cancer cells by Cell counting kit-8(CCK-8)and flow cytometry,respectively.(4)In vivo,by establishing a subcutaneous xenograft model of pancreatic cancer in mice,the tumor growth curve and histology were used to detect the anti-tumor effect of LP-Gem-si Mcl-1.The biosafety evaluation of the nanocarriers was carried out by histopathological analysis of the main organs and detection of blood biochemical indicators.Results:(1)We found that Mcl-1 is overexpressed in pancreatic cancer relative to normal tissues and cells,consistent with published articles.By transiently transfecting si RNA to down-regulate the expression of Mcl-1,it can effectively inhibit the proliferation of pancreatic cancer cells PANC-1 and Bx PC-3,and when combined with gemcitabine,enhance the killing effect of gemcitabine on pancreatic cancer cells.(2)Morphological characterization of the nanoparticles,the particle size of the nanoparticles was observed to be about 150-200 nm,spherical and monolayer dispersed;UPLC measured the entrapment rate of gemcitabine was about 19.3%;Agarose gel experiments confirmed LPs has good nucleic acid adsorption capacity;Confocal microscopy and flow cytometry showed that gemcitabine and si RNA encapsulated by LPs can be efficiently taken up by pancreatic cancer cells and localized in lysosomes;It was further determined that LP-si Mcl-1(LPs encapsulated Mcl-1 si RNA)can effectively inhibit the expression of Mcl-1 in cells.(3)In vitro confirmed that LP-Gem-si Mcl-1 can effectively inhibit the proliferation of pancreatic cancer cells and promote the occurrence of apoptosis compared with other treatment groups,the effect was significantly higher than the single-loaded group of LP-Gem(LPs encapsulated Gem)and LP-si Mcl-1.(4)In vivo,it was observed that the drugs carried by LPs can passively target the tumor site and increase the drug concentration at the tumor site.The inhibition of tumor growth by LP-Gem-si Mcl-1 was significantly higher than that of any other treatment groups,and the expression of cell proliferation marker Ki-67 was also significantly inhibited,confirming the synergistic anti-tumor effect between Gem and Mcl-1 si RNA,and LPs nanomaterials have no obvious toxic side effects.Conclusion: In summary,we successfully co-delivered Gem and Mcl-1 si RNA to pancreatic cancer cells using a cationic liposome-based drug and gene co-delivery system,and demonstrated that decreasing Mcl-1 expression can enhance the killing effect of gemcitabine on pancreatic cancer cells,both have synergistic anti-tumor effects.Through this study,we identified the potential value of targeted Mcl-1 intervention in the treatment of pancreatic cancer,and demonstrated the feasibility of combining si RNA and chemotherapeutic drugs for pancreatic cancer with a cationic liposome delivery system,it provided a theoretical basis and laboratory basis for the new treatment strategy of pancreatic cancer. |
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