PEN Coated CLIO-NH₂ With SiSnail2 Combined With Cisplatin To Treat Hepatic Carcinoma Through Ferroptosis

Background:As a malignant tumor with the sixth highest incidence of cancer and the second highest mortality rate in the world,hepatic carcinoma has always attracted much attention in clinical and scientific research.Although early hepatocellular carcinoma can be treated by surgical methods such as tumor resection and liver transplantation,and the prognosis is good.However,due to the poor living and eating habits of the Chinese people and the neglect of regular physical examinations,the number of liver cancer patients has gradually increased.Most of the liver cancer patients have developed into the middle or late stages when they are found.The liver itself is rich in blood vessels,and is prone to recurrence and metastasis after surgery;some patients develop various metastases when liver cancer is detected,so the 5-year survival rate of liver cancer patients is only 12.1%due to this series of reasons.Therefore,in clinical practice,radical resection is currently performed for early stage liver cancer.However,for advanced liver cancer or inoperable liver cancer,chemotherapy,radiotherapy,interventional therapy,immunotherapy,and targeted therapy should be considered to convert or downgrade to operable liver cancer to improve the survival rate of patients.Ferroptosis is a type of cell death that was discovered long ago but was not reclassified and named until 2012.Unlike apoptosis in the traditional sense,ferroptosis is considered to be cell death caused by excessive accumulation of intracellular iron resulting in increased lipid peroxides.Cisplatin（DDP）is one of the commonly used drugs in the chemotherapy treatment of liver cancer,but it is not clinically possible to increase the dose of cisplatin to improve the efficacy of chemotherapy in patients with liver cancer,and high-dose cisplatin is usually accompanied by serious adverse reactions.Therefore,it is of great significance to develop new chemotherapy regimens that can be applied to the clinical treatment of liver cancer.In recent years,studies have found that ferroptosis is closely related to the mechanism of DDP in the treatment of cancer.DDP can cause cells to generate a large amount of reactive oxygen species（ROS）,disrupt the balance of oxidative stress in cells,promote intracellular lipid peroxidation,and induce iron production in cells.In addition,the current research on the association between ferroptosis and tumor epithelial-mesenchymal transition（EMT）is becoming more and more close,and Snail2 is a transcription factor that plays an important role in the EMT process of various tumors.Our previous study demonstrated that in liver cancer,Snail2 can recruit a variety of histone-modifying enzymes to the promoter region of E-cadherin,inhibit the expression of E-cadherin through epigenetic modification,thereby inducing EMT phenomenon and promoting tumor invasion and metastasis.In our previous experiments,specifically knocking out or reducing the expression of Snail2by RNA interference technology（RNAi）can effectively inhibit the invasion and metastasis of tumor cells in vivo and in vitro.By rationally applying the close relationship between ferroptosis and DDP,we designed a vector CNP based on the combination of nano-cross linked iron oxide（CLIO-NH₂）and Polyethylenimine-NH₂（PEN）generated after improved transfection reagent PEI.At the same time,carrying si Snail2,which can inhibit the metastasis of liver cancer cells,constitutes CNP@si Snail2 combined with DDP chemotherapy to induce ferroptosis in liver cancer cells,and at the same time kill liver cancer cells,inhibit the EMT phenomenon of tumor cells through si Snail2,weaken metastasis of liver cancer cells achieves the purpose of transforming or downstaging inoperable liver cancer into operable liver cancer by centrally eliminating liver cancer cells.Objective:By designing a nano-iron complex CNP as a carrier,carrying si Snail2,and using it in combination with DDP,it can induce ferroptosis of hepatic carcinoma cells while inhibiting hepatic carcinoma metastasis as much as possible,in order to reduce the stage of advanced hepatic carcinoma and improve opportunities for the patient’s surgery.Method:（1）Cross-linking of 60nm CLIO-NH2 and PEN-25K with different mass ratios,ultrasonication in a water bath at 37°C,and ultra-high-speed centrifugation to obtain carrier CNP.Detect the particle size and point height of the synthesized carrier CNP,and respectively detect the cell entry ability and carrying si Snail2 ability of the carrier CNP prepared with different mass ratios,and select the appropriate mass ratio according to the test results.（2）Different concentrations of carrier CNP were used to carry out killing experiments on hepatic carcinoma cells,and they were detected by CCK8.According to the test results,choose the best concentration.And through flow cytometry,it was confirmed that ferroptosis occurred during the cell death process induced by carrier CNP.（3）Measure the IC10 concentration of DDP through the CCK8 experiment to determine the best concentration of DDP,and then through microscopic observation,CCK8,ROS detection,flow cytometry and other means to confirm that the combination of carrier CNP and DDP can promote the occurrence of cell ferroptosis.（4）Construct an orthotopic tumor model of hepatic carcinoma in BALB/c mice,and inject the mixture of CNP@si Snail2 and DDP into the mice through the tail vein.Check the tumor size after 16 days.（5）Determine the toxicity of the combination drug to the individual by detecting liver function and immunohistochemistry;determine the occurrence of cell death in the tumor by cell death-related immunohistochemistry;detect the migration status of tumor tissue by RT-q PCR and immunohistochemistry.Result:（1）When using 60nm CLIO-NH₂ with a mass ratio of 1:1 to cross link with PEN-25K,the synthesized carrier CNP can enter cells stably,and can carry si Snail2.The ability to enter cells and carry si Snail2 is in an optimal balance state.（2）Different concentrations of carrier CNP have different killing effects on hepatic carcinoma cells.Through CCK8 detection,it was decided to detect the effect of CNP carrying si Snail2 into cells when the CNP concentration is 1μg/ml,in order to eliminate the interference of carrier CNP with cytotoxicity as much as possible.Various cytotoxicity tests can perform when the CNP concentration is 10μg/ml,and it is confirmed that the carrier CNP can induce cell ferroptosis.（3）When the CNP concentration is 10μg/ml,using the IC10 concentration of DDP,the combined use has a stronger killing effect on hepatic carcinoma cells than the two alone,and in the process,the ferroptosis induction effect of the two on cells is also increased.（4）In the hepatic carcinoma orthotopic tumor model of BALB/c mice,it can be clearly seen that the combination of CNP@si Snail2 and DDP has smaller tumors and fewer metastases,which has an excellent therapeutic effect on the in vivo orthotopic model.（5）The liver function and immunohistochemical results of mice showed that CNP@si Snail2 combined with DDP did not increase the toxicity of individuals;the immunohistochemical results related to cell death showed cell death in tumor tissues in the combined group of CNP@si Snail2 and DDP At most,ferroptosis was significant;the tumor tissues were detected by RT-q PCR and immunohistochemistry,and it was found that the expression of Snail2 in the tumors in the CNP@si Snail2combined with DDP group was significantly reduced,and tumor cell migration was less.Conclusion:We designed a combination of nano-crosslinked iron oxide（CLIO-NH₂）and Polyethylenimine-NH₂（PEN）as a carrier carrying si Snail2,and at the same time it can induce ferroptosis in cells.Furthermore,when combined with DDP,Significantly inhibit the growth of hepatic carcinoma cells,induce cell death,and inhibit the metastasis of hepatic carcinoma cells.CNP@si Snail2 can enter tumor cells stably,and under the action of intracellular lysosomes and other organelles,si Snail2 is released,which interferes with the expression of metastasis-related genes in hepatic carcinoma cells;at the same time,the degraded CNP is decomposed into separate iron ions and greatly increase the iron concentration in cells,to generate a large amount of ROS and promote lipid peroxidation,inducing ferroptosis in cells.The DDP used in combination with it can promote the process of ferroptosis and strengthen the killing effect on hepatic carcinoma cells.This experiment fully proves that this kind of combined use can effectively reduce hepatic carcinoma staging,inhibit hepatic carcinoma metastasis,and provide patients with more treatment options.Through this combination,we can also see that the carrier can not only be used as a carrier,but also as a death-inducing agent,playing a greater role,providing further possibilities for the application of nanomaterials,and more will appear in the future.Multiple combined application therapies.