Surface Modification Of Esophageal Stent Materials By Polyethylenimine And 5-FU For Anti-Tumor Effects

Esophageal cancer is a malignant tumor of digestive tract in esophageal epithelial tissue,which possesses high mortality rate less than 20% survival rate within 5 years.Metal stent implantation is generally accepted as the crucial method for relieving late esophageal obstruction in nonsurgical palliative care.However,the traditional stent materials lack the sustained ability of anti-tumor and anti-inflammation,which will cause esophageal lumen restenosis.Wherein,inhibiting the growth of tumor cells is the key for further inhibiting inflammation caused excessive proliferation of fibroblasts and epithelial cells,and then preventing restenosis.Esophageal drug eluting stent makes its advantage of a certain anti-tumor function through the release of the loaded drug at the lesion.However,the quantity of the loading drugs on the stent surface is limited: with the drug releasing,the loading will gradually decrease,and the effective concentration of the drug will also gradually reduce,resulting in the function loss of the stent on inhibiting the tumor cells.In addition,the exposed stent substrate will not only lose its ability on suppressing cells,but also promote the growth of tumor cells for the materials good biocompatibility,which is an important factor affecting the long-term efficacy of stents.Therefore,it's a preferable strategy to construct modified layer with continuous impedance function on tumor cell growth between the material substrate and the drug layer.One of the main material of esophageal stent used in clinic is 317 L stainless steel(317L SS),thus it's necessary to introduce an organic-polymeric film capable of both binding to a variety of materials and the functional layer.Dopamine is well known for its outstanding ability to bind strongly to virtually all substrates and to provide secondary reactivity for conjugating biomolecules.PEI molecules are rich in imino group,which could be binding to the PDA film via Michael addition and Schiff base reaction.In addition,PEI has been proved to kill cancer cells.5-fluorouracil(5-FU)is an effective anti-cancer drug that is widely applied in clinic.In situ release of 5-FU is more effective to kill tumor cells.However,the current research on the surface modification of esophageal stent materials by PEI and 5-FU functional molecules to prevent restenosis has not been reported.ObjectivesThe PDA film was deposited on a esophageal stent material(317L stainless steel,317 L SS)surface.Then a polyethylenimine(PEI)functional layer on the PDA layer was constructed and the parameters were optimized.Finally,5-FU was immobilized onto the PDA/PEI modified 317 L SS surface to construct a functional layer for continuously impeding cells.In addition,the anti-tumor and anti-restenosis effects of PDA/PEI/5-FU functional layer and its related mechanisms were investigated.Method1.Fabrication,characterization and optimizing of the PDA/PEI/5-FU layersThe PDA film was deposited on 317 L stainless steel substrates,then the PEI was binding to the PDA film via Michael addition and Schiff base reaction to form PEI impedance layer.The PDA/PEI coated 317 L SS were immersed into the 5-FU solution to form PDA/PEI/5-FU layer.The surface morphology and roughness of samples were analyzed by atomic force microscopy.To evaluate the wettability of layers,the water contact angles of samples were detected by a contact angle apparatus.The density of amine groups on layers was determined using Acid Orange II(AO II)colorimetric method.AO/PI double stained Eca109 cells to determine the optimal molecular weight of PEI.The amounts of immobilized 5-FU on the PDA/PEI/5-FU layers and their eluting drugs were determined by typical spectrophotometry.To investigate the modified layers' biomechanical properties,the stress values of each sample were detected by a microcomputer controlled electronic universal testing machine.The weight loss of all the samples was also measured to evaluate the layers' stability.2.Evaluation and mechanism of anti-tumor and anti-inflammatory function of the PDA/PEI/5-FU layers in vitroAO/PI double stained Eca109(esophageal tumor cells),Het-1A(epithelial cells),L929(fibroblast cells)and RAW264.7(macrophage cells)to optimize the concentration of 5-FU.Cell Counting Kit-8(CCK-8)assay was used to measure the effect of PDA/PEI/5-FU on the macrophages activity.AO/DAPI stained macrophages to investigate the effect of inhibiting inflammatory cells of the functional layer.Transwell assay was used to estimate the number of migrated cells.Western Blot was used to detect the expression of E-cadherin and the activity of NF-?B signaling pathway.The effect of functional layer on inhibiting the secretion of inflammatory factors was detected by ELISA assay.ResultsPart ? Surface modification of esophageal stent materials by a polyethylenimine layer aiming at anti-tumor functionThe surface characterization including atomic force microscopy(AFM),amine quantitative and water contact angle measurement indicated successful preparation of the PDA/PEI layer.The Eca109 cells culture results proved that the PDA/PEI layers significantly promoted cells apoptosis and necrosis,suggesting excellent anti-cancer function.In addition,it was also found that the anti-tumor function of the PDA/PEI layers was positively correlated to the immobilized PEI's MW.Part ? Anti-tumor and anti-inflammatory effect of polyethylenimine combined with 5-FU on the surface of esophageal stent materialsThe surface characterization including AFM,drug quantitative characterization and water contact angle measurement indicated successful preparation of the PDA/PEI/5-FU layer.The 5-FU release and biodegradability of each modified layer demonstrated rapid release of the loaded 5-FU and good stability of the left PDA/PEI layer.The layers' biomechanical properties measurement suggested the PDA/PEI/5-FU-1 layer showed similar stress tolerance to 317 stainless steel.The cells culture results suggested that PDA/PEI/5-FU and PDA/PEI layers remarkably inhibited the viability and adhesion of Eca109,Het-1A,L929 and macrophage cells;the released 5-FU made a significant contribution to improving the apoptosis and necrosis of Eca109,Het-1A,L929 cells and RAW264.7.And the PDA/PEI/5-FU layers maintained the consistent impedance function on their surfaces with the PDA/PEI layer after drug release.Part ? Anti-tumor and anti-inflammatory mechanisms of polyethylenimine combined with 5-FUThe results of cells' migration showed that PDA/PEI/5-FU and PDA/PEI groups can be more effective in reducing cell migration and enhancing the expression of E-cadherin in comparison to 317 L SS group.Western blot assay showed that PDA/PEI/5-FU could inhibit the activity of NF-?B signaling pathway.The expression of p65 and Bcl-2 were down-regulated,and the expression of Bax,caspase-3 were up-regulated after PDA/PEI/5-FU treatment.In addition,PDA/PEI/5-FU significantly decreased the secretion of TNF-? and IL-1?,and increased the secretion of IL-4 and IL-10.Conclusion:1.PDA/PEI/5-FU could inhibit adhesion and migration of restenosis related cells,prevent restenosis and induce cell apoptosis through the NF-?B signaling pathway.2.PDA/PEI/5-FU has anti-inflammatory effect that inhibit the proliferation and adhesion of macrophage cells and the secretion of inflammation-related factors.