A Study On Cyclodextrin-based Nanoparticle For Co-delivery Of Ginsenoside Rg3 And Quercetin To Achieve Immunotherapy Against Colorectal Cancer

Colorectal cancer(CRC)is a common gastrointestinal malignant tumor occurring in the colorectal region.Conventional therapy has the disadvantages of low curative effect,high toxicity and easy recurrence.It is urgent to develop new treatment methods.In recent years,immunotherapy,which activates the immune system to prevent,control and kill cancer cells,has become a new anti-cancer strategy.Studies have shown that some chemotherapeutic drugs can activate the anti-tumor adaptive immune response by inducing cancer cell apoptosis,which is called immunogenic cell death(ICD).Therefore,ICD by promoting the occurrence of CRC cells is an effective means to induce adaptive immune response against CRC.At present,there is a clear lack of ICD inducing drugs,so there is an urgent need to develop new inducing drugs to enrich ICD based immunotherapy.In this study,the potential of Rg3 to induce ICD was proved by studying the occurrence process of Ginsenoside Rg3(Rg3)induced ICD and the expression of biomarkers.It was proved by laser confocal and flow cytometry that Rg3 can effectively stimulate endoplasmic reticulum stress in CRC cells(CT26and HCT116).When endoplasmic reticulum stress occurs,Rg3 induces the transport of calreticulin(CRT)from endoplasmic reticulum to cell membrane surface;When autophagy occurs,Rg3 induces the secretion of adenosine triphosphate(ATP);In the late stage of apoptosis,Rg3 promotes the release of high mobility group protein B1(HMGB1)to the outside of cells.Therefore,the above results prove that Rg3 can induce ICD.It is reported that reactive oxygen species(ROS)produced by tumor cells can induce endoplasmic reticulum stress,which is considered to be a major reason to promote ICD.Therefore,this paper next explores the production of ROS induced by quercetin(QTN).Flow cytometry showed that QTN could increase the apoptosis rate of CRC cells and increase the content of intracellular ROS,thus inhibiting the growth of CT26 and HCT116 cells;This result provides a theoretical basis for the joint application of Rg3 and QTN.This paper further explores the formulation strategy of Rg3 and QTN,and determines the optimal proportion of the combination of Rg3 and QTN through MTT experiment.From the markers of apoptosis,ICD and the activation of induced dendritic cells(DCs),it can be seen that when the molar ratio of Rg3 and QTN is 1:1,it has the best anticancer effect in vitro and can promote the ICD effect of "1 + 1 > 2".However,the different physicochemical properties of Rg3 and QTN limit their synergistic antitumor effect in vivo.At present,the latest development of nano drug delivery system has greatly promoted the in vivo administration of chemotherapeutic drugs in cancer treatment.According to the literature,there is no study on the co delivery of Rg3 and QTN in CRC nano preparations.In this paper,based on cyclodextrin(CD),a PEGylated amphiphilic cyclodextrin nanoparticles(NPs)targeting folate(FA)was developed,which co encapsulated Rg3 and QTN with the best molar ratio(1:1).Double drug loaded nano preparation(CD-PEGFA.Rg3.QTN)achieved synergistic effect in anticancer effect in vitro.Finally,CD-PEG-FA was studied Rg3.In vivo antitumor effect of QTN.The experimental results showed that the nano preparation improved the pharmacokinetics and in vivo distribution of the two drugs in the mouse in situ CRC model.In addition,CD-PEGFA.Rg3.QTN can promote the occurrence of ICD in tumor cells,break the immune tolerance tumor microenvironment,enhance the antitumor effect of PD-L1 antibody,and significantly prolong the survival time of tumor bearing mice.The combined strategy of "dual drug loaded nanoparticles + PD-L1 antibody" in this paper is expected to provide a new treatment for CRC patients.