Effects Of Sevelamer-Induced Low Phosphate Stress Technique On Hepatocellular Carcinoma Progression And Its Molecular Mechanism

Hepatocellular carcinoma(HCC)is a common digestive tract tumor,with its incidence and mortality rates ranking among the highest in tumors.For unresectable HCC,transarterial chemoembolization(TACE)is the safest and most effective treatment option.However,arterial embolization can induce hypoxia in the tumor microenvironment,which in turn promotes neovascularization and leads to recurrence.The selection of appropriate embolization materials may help improve TACE efficacy and inhibit tumor recurrence.In recent years,scientists have discovered that the abnormally elevated concentration of inorganic phosphate in the tumor microenvironment is closely related to angiogenesis and the occurrence and development of tumors.However,no well-designed studies have reduced the phosphate burden to suppress tumor progression.This study developed an interventional therapy for liver cancer based on phosphate binder embolization materials.It can effectively reduce the phosphate burden of tumors and improve the chemical environment after tumor embolization.Mechanistic studies have revealed that low-phosphate stress inhibits tumor progression through multiple pathways.In addition,an attempt was made to explore how phosphate starvation may enhance the drug sensitivity of liver cancer to systemic treatments(sorafenib),providing new candidate options for the comprehensive treatment of liver cancer.The main research contents and results are as follows:(1)Sevelamer microspheres with phosphate-binding ability were prepared by simple ball milling method.The morphology,particle size,structure,and ability of binding phosphate of the microspheres were characterized by a series of characterization methods.The results showed that the sevelamer microspheres were polyhedral with a diameter of about 1 μm.The morphology of the sevelamer microsphere post-absorption of phosphate greatly changed from the individual ultrafine particle to hard aggregates as phosphate ions raised adjacent crystal face swelling and connection,and eventually,the individual particle disappeared,leaving a continuous film.The guinea pig maximization test(GPMT),in vivo acute systemic toxicity,and serum biochemical parameters assays suggested that sevelamer microspheres showed good biocompatibility.In vivo experiments have found that sevelamer specifically embolized the tumor-feeding artery after administration of sevelamer via the hepatic artery,thereby causing vascular necrosis and reducing metastasis and recurrence of the tumor.Meanwhile,it can specifically reduce the inorganic phosphate in the tumor.We name this technology “Transarterial Sevelamer Embolization”.Experiments on cells in vitro have shown that phosphate starvation induced by sevelamer can significantly down-regulate the expression of oncoproteins to inhibit angiogenesis,tumor cell migration and invasion.Metabolomics analysis revealed that phosphate starvation also inhibits tumor anaerobic glycolysis and glutamine metabolism.The above results show that sevelamer microspheres can be used as a new type of embolic agent to improve the anti-tumor effect after TACE embolization.(2)Phosphate-responsive citrated sevelamer nanoparticle microspheres for complete vascular embolization were prepared by anion exchange reaction to overcome the problem that sevelamer microspheres are difficult to penetrate deep into the tumor tissue.The citrated sevelamer had a particle size range of around 881.21±371.98 nm.In vitro drug release experiments showed that citrated sevelamer nanoparticles had slower rates of phosphate absorption.The morphology of the intermediate and final phosphate-saturated sevelamer nanoparticles was recorded by scanning electron microscope(SEM),note that citration-induced sevelamer nanoparticle aggregates into microparticles,much bigger and denser than sevelamer did,which indicates modulation of phosphate absorption might be more favorable for complete vascular embolization.In vivo experiments showed the organic citrate anion-affiliated sevelamer nanoparticle postpones phosphate adsorption,as well as it’s swelling and aggregation,in favor of flowing into the end of the capillary vessel.However,pristine sevelamer nanoparticles embolize mainly the larger arterial blood vessels and have difficulty diffusing deep into tumor tissues.Therefore,the complete embolization of blood vessels can be achieved by controlling the polymerization of sevelamer induced by phosphate,which is of great value in preventing postoperative recurrence.(3)Meanwhile,according to the above-mentioned anion exchange reaction,the sevelamer was used for drug delivery to load arsenite(AS2O3).It was confirmed by inductively coupled plasma mass spectrometry(ICP-MS)that sevelamer could effectively load AS2O3 to form sevelamer arsenite nanoparticles.In vitro drug release experiments showed that phosphate could trigger the release of chemotherapeutic drug AS2O3 from sevelamer arsenite nanoparticles,and after ion substitution sevelamer tends to form a densely packed aggregation.AS2O3 is released slowly at the desired rate from the system in vivo experiments.In addition,the sevelamer arsenite nanoparticle treated group induced extremely high necrosis of cells.Therefore,we successfully prepared sevelamer arsenite nanospheres with both phosphate starvation and sustained release effect and developed a new D-TACE technology.(4)Exploring the synergistic anti-tumor effect of sevelamer-induced low phosphate stress and sorafenib on human liver cancer Hep G2 cells could further expand the application scope of phosphate starvation technology in liver cancer treatment.Firstly,wound healing and invasion assays showed that the combined treatment of phosphate starvation and sorafenib inhibited the migration and invasion of Hep G2 cells.Furthermore,the combined treatment of phosphate starvation and sorafenib showed strong anti-tumor activities and angiogenesis inhibition in an in vivo liver cancer xenograft mouse model.Western blot analysis showed that the expression levels of MMP-9 and p-ERK were significantly lower in the combination group compared to the other groups.Then,a sorafenib-resistant Hep G2-SR cell line and an acquired sorafenibresistant xenograft model were established,and the results from cell and animal experiments indicate that low phosphate stress can re-sensitize drug-resistant cells to sorafenib by regulating the expression of p-AKT and HIF-1α proteins.In conclusion,this study developed an interventional therapy using sevelamer as an embolic agent,which can regulate the expression and metabolism of proteins related to tumor progression,thereby inhibiting the progression of liver cancer.On this basis,modification of the sevelamer surface enables complete vascular occlusion and drug loading,thereby achieving therapeutic effects.Finally,the combination of sevelamer and sorafenib enhanced the drug sensitivity,providing relatively comprehensive basic data for its future application in the treatment of advanced HCC.