Study On The Mechanism Of Folate-mediated Targeted Delivery Of SiPLK1 By Leucine-bearing Polyethylenimine To Treat Gastric Cancer

Gastric carcinoma is one of the most common malignant tumors,which is highly invasive and harmful.It is the second most common disease that causes cancer-related death worldwide.In recent years,with the continuous development of scientific and technological means,a deeper understanding of the pathogenesis and causes of gastric cancer has been achieved.The incidence of gastric cancer has shown a downward trend year by year.In advanced stage,it is a progressive gastric cancer,which leads to poor treatment and poor prognosis of gastric cancer.At present,the treatment of gastric cancer is still based on traditional methods such as surgery,adjuvant chemotherapy and radiotherapy.Although the emerging immunotherapy and monoclonal antibody targeted therapy have also achieved relatively good clinical results,they are still hindered by insufficient accuracy and chemo-resistance caused by long-term use,so people have been looking for more effective and accurate treatment strategies.Polo-like kinase 1(PLK1)is a serine/threonine kinase that is an early trigger for the transition from G2 to M.It supports the functional maturation of centrosomes in the early and late stages of G2 and the establishment of bipolar spindles.PLK1 is over-expressed in many cancers,including gastric cancer.Downregulating the expression level of PLK1 can not only induce G2/M phase arrest and apoptosis and thus inhibit cancer cell proliferation,but also enhance sensitivity to chemotherapy and radiotherapy,but also reduce its roles in carcinogenesis and transformation,tumor initiation and survival,induction of epithelial-mesenchymal transition(EMT),and tumor migration and invasion.Therefore,downregulating the expression level of PLK1 in cells is expected to be an effective therapeutic target for gastric cancer.Compared with ATP competitive or peptide small molecule inhibitors,RNA interference therapy based on small interfering RNA(siRNA)has irreplaceable advantages such as high specificity and low toxic and side effects.The field of drug development has received widespread attention.,siRNA is a 21-25 nt double-stranded RNA that forms a RISC complex with intracellular proteins.The guide strand is paired with the complementary mRNA sequence through the RISC complex After binding,siRNA can induce mRNA silencing through RNase-mediated degradation to Inhibit protein expression.However,siRNA itself is negatively charged and unstable,and has the problems of poor cell penetration and susceptibility to degradation by nucleases.Therefore,a stable and efficient gene delivery system is required to achieve successful internalization and stable existence of nucleic acids and exert gene silencing effectsAt present,delivery systems for siRNA are mainly divided into viral systems and non-viral systems.Compared with viral vectors,non-viral vectors have low immunogenicity and good biocompatibility,high gene load,strong modifiability and low carrier toxicity.Such advantages can effectively overcome many limitations of viral vectors.Cationic polymers are widely used as drug delivery systems due to their positive surface properties,higher loading efficiency,lower biological toxicity and easy modification.Polyethyleneimine(PEI)is one of the most widely used cationic polymers.It has the following excellent characteristics:PEI has a high cationic charge density on the surface,and can be assembled by electrostatic interaction with negatively charged nucleic acid molecules.Protonated amino nitrogen atoms will exist at every two carbon atoms,making the polymer have a "proton sponge effect" to improve the ability of the complex's endosomes to escape.Second,branched PEI(BPEI)has a high degree of branching and has tertiary amino groups.To make the vector more stable and the transfection efficiency higher,many gene-designed gene vectors supply PEI as the core;in addition,the surface of PEI is easy to be modified with a variety of ligand molecules to make it a more stable multifunctional delivery vector.It can be seen that because of its good physical and chemical properties,PEI has the potential to play an important role in gene delivery.Although PEI has high transfection efficiency,unmodified PEI still has the disadvantages of high cytotoxicity,low blood compatibility,and lack of biodegradability,which makes it extremely limited in clinical application Therefore,construction of an efficient,low-toxic,and targeted delivery system by appropriate modification of on PEI surface is of great significance to achieve the precise delivery of gene drugsIn this paper,folate(FA)and N-acetyl-L-leucine(N-Ac-L-Leu)were used to modify the polyethyleneimine(PEI)vector,and a targeted nanocomplex FA-N-Ac-L-Leu-PEI(NPF)for gastric cancer SGC-7901 cell line was successfully constructed.FA can selectively bind to FA receptors that are overexpressed in most human cancers Based on specific recognition,FA modifications have been shown to enhance siRNA silencing of FA receptor overexpressing cell lines in vitro and in vivo,N-Ac-L-Leu can decrease the positive charge density of PEI and stabilize the complex,which has better transfection effect and lower cytotoxicity.The NPF carrier can bind to the negatively-charged PLK1 siRNA through the positive charge on the surface,so PLK1 siRNA was delivered by a NPF/siPLKl nanocomplex,the gene loading capacity and targeting of the vector and the effect of PLK1 silencing was evaluated on gastric cancer through a series of experiments.First,the successful synthesis of the NPF vector was characterized by nuclear magnetic resonance(1HNMR),infrared spectroscopy(FTIR),and GPC.Each PEI molecule could successfully graft 81 N-Ac-L-Leu and 16 folic acid,and the NPF and siPLKl can effectively assemble into stable nanoparticles at a mass ratio of 3.0,and has excellent ability to condense and protect nucleic acids from nuclease degradation.It was found by TEM electron microscopy and size potential measurement that NPF/siRNA can form a spherical structure.Next,the transfection ability of siRNA and pEGFP and the targeted ability mediated by FA was assessed by flow cytometry and microscopy.The efficiency of NPF/siPLKl internalization into cells was significantly higher than that of unmodified FA vectors.Real-time quantitative PCR and western blotting showed NPF-mediated siPLKl transfection can effectively silence gene or protein expression levels.MTT and colony formation experiments revealed that NPF-mediated siPLKl transfection exhibited significant anti-proliferative activity in vitro,mainly by inducing cell cycle arrest and apoptosis.The arrest of tumor cells in the G2 phase was 45%,And the apoptosis rate was 28.3%.SiPLK1 can reduce the expression of endogenous apoptosis pathway-related proteins pro-caspase3 and 9,and mitochondrial membrane potential,indicating that apoptosis induced by siPLKloccurs through the endogenous mitochondrial pathway.Transwell analysis and wound healing experiments proved that PLK1 silencing reduced cell migration and invasion ability,and the expression of Notch-1 protein related to migration and invasion capacity was also significantly inhibitedIn summary,SGC-7901 cell line with high expression of folate receptor was used as a model and targeted delivery of siRNA with FA and N-Ac-L-Leu modified PEI can effectively inhibit the invasion and proliferation of SGC-7901 cell,and induce cell apoptosis and cycle arrest.This study confirms that the PLK1 gene can be used as a potential target for gastric cancer treatment,and gene delivery by targeted nanoparticle can be a promising clinical treatment strategy in gastric cancer.