Antitumor Activity Of Mn²⁺/CpG Polydopamine Nanoparticles

BackgroundMalignant tumor is a disease that seriously threatens human health with extremely high morbidity in the world today,and it is one of the main causes of human death.According to the latest data on global cancer in 2020 released by the World Health Organization’s International Agency for Research on Cancer（IARC）,there were 19.3 million new cancer cases and nearly 10 million deaths worldwide in 2020.At present,the treatment methods of tumor mainly include surgery,chemotherapy and radiotherapy.Among them,surgical resection is often limited by the invasion and spread of cancer cells to adjacent tissues or distant metastasis,while chemotherapy and radiotherapy are also limited by the toxicity to other normal tissues in the body.Among emerging cancer therapies,chemodynamic therapy（CDT）has attracted considerable attention over the past few decades.This therapeutic strategy usually utilizes metal ions,such as Fe3+/2+,Mn2+,Co2+,Cu2+,to initiate the Fenton reaction,which can generate cytotoxic hydroxyl radicals（·OH）at the tumor site,leading to cancer cell apoptosis or necrosis.However,most previous studies have focused more on the redox properties of metal ions while ignoring their functions in the immune system.For example,Ca2+is the second messenger of the immune response;the increase of intracellular Fe2+ level directly triggers the immune response;and Zn2+ is essential for innate and adaptive immune activation.Therefore,metal-involved CDT induced immune activation is one of the directions worthy of further study.Among many metal ions,manganese,as a nutritional inorganic trace element,plays a very important role in various physiological processes such as development,reproduction,neural function and antioxidant defense.Studies have shown that Mn2+can enhance the sensitivity of cyclic GMP-AMP synthase（Cyclic GMP-AMP synthase,cGAS）to doublestranded DNA（dsDNA）and its enzymatic activity,enabling cGAS to generate secondary messenger cGAMP in the presence of low concentrations of dsDNA.And Mn2+can enhance the binding affinity of cGAMP-STING to enhance STING activity and promote the production of interferon（IFN）and the secretion of pro-inflammatory cytokines,thereby triggering the innate immune response and realizing tumor therapy.Recent results show that Mn2+can directly activate cGAS independent of dsDNA and trigger the unique synthesis of 2’3’-cGAMP,expanding its application in tumor immunotherapy.Therefore,a detailed study of the dual functions of manganese ions,that is an initiator of the Fenton-like reaction and an activator of the cGAS-STING pathway,has certain theoretical and practical significance for the effective treatment of tumors.Based on the above research background,this thesis designed and synthesized a kind of Mn2+/CpG polydopamine nanoparticles for tumor therapy.On the one hand,the nanoparticles have Fenton-like reactivity,which can catalyze the generation of ·OH in the hydrogen peroxide inside the tumor to realize chemokinetic therapy;on the other hand,it can activate the cGAS-STING pathway to trigger the innate immune response to realize immunotherapy.In addition,CpG oligonucleotides（CpG ODNs）as immune adjuvants can activate Toll-like receptor 9（TLR-9）expressed on innate immune cells,induce a variety of immune cells（including B lymphocytes,T lymphocytes,NK cells and monocytes/macrophages）maturation,differentiation and proliferation,further triggering a stronger immune response.PurposeIn order to achieve the high effeciency of tumor treatment,a kind of Mn2+/CpGpolydopamine nanoparticles（MnCpGPNPs）was designed and synthesized in this thesis.The nanoparticles can simultaneously realize chemodynamic Therapy,and immunotherapy,providing an experimental and theoretical basis for advancing the application of multifunctional nanomaterials in the biomedical field.MethodChapter 1:Preparation and characterization of MnCpGPNPsIn this chapter,MnCpGPNPs were prepared based on the coordination between manganese ions and CpG ODNs.First,the morphology,size,surface zeta potential and stability of MnCpGPNPs were characterized by transmission electron microscopy（TEM）,scanning electron microscopy（SEM）and Malvern particle size analyzer;subsequently,the structure and composition of MnCpGPNPs were analyzed in detail by X-ray photoelectron spectroscopy（XPS）;at the same time,the content of manganese ions and CpG ODNs in MnCpGPNPs was detected by inductively coupled plasma spectrometer（ICP-AES）and trace ultraviolet spectrophotometer;finally,the Fenton-like reactivity of MnCpGPNPs was evaluated by methylene blue spectrometry and electron paramagnetic resonance spectroscopy（ESR）.Chapter 2:Antitumor activity of MnCpGPNPs in vitroIn this chapter,the reactive oxygen species（ROS）fluorescent probe（DCFH-DA）was used to investigate the Fenton-like reactivity of MnCpGPNPs at the cellular level（mouse colon cancer cells,CT26-WT cells）;subsequently,the in vitro antitumor effects of MnCpGPNPs were evaluated by MTT method,Hoechst/PI staining method,mitochondrial membrane potential probe,Western Blot analysis（Western Blot）,enzyme-linked immunosorbent assay（ELISA）and flow cytometry and its mechanism of action was discussed.Chapter 3:Antitumor activity of MnCpGPNPs in vivoIn this chapter,by establishing the subcutaneous tumor model of mouse colon cancer（CT26-WT）,the mouse CT26-WT double tumor（in situ tumor and distal tumor）model and the mouse preventive research model,the growth curve of the tumor was recorded in detail.The mouse tumor was subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling analysis（TUNEL）staining and hematoxylin-eosin（H＆E）staining to analyze the apoptosis of tumor cells;subsequently,immunocytofluorescence staining and flow cytometry were used to analyze the immune cells within the tumor and in the inguinal lymph nodes;finally,the serum cytokines of tumor-bearing mice were measured by ELISA,and the antitumor effect of MnCpGPNPs in vivo was comprehensively evaluated.ResultChapter 1:The hydrodynamic size of prepared MnCpGPNPs is about 250 nm,with good dispersibility and uniform size.MnCpGPNPs also exhibited pH-responsive release behavior,which could rapidly release Mn2+ and CpG ODNs under acidic conditions.MB spectrometry and ESR spectroscopy showed that MnCpGPNPs had Fenton-like activity and could catalyze H2O2 to generate.OH.Chapter 2:In this chapter,through intracellular localization,MTT assay,Hoechst/PI staining,flow cytometry,ROS fluorescent probe,mitochondrial membrane potential and apoptotic protein expression,it is proved that MnCpGPNPs can enter into tumor cells,and catalyze endogenous H2O2 in tumor cells to generate ROS,and regulate endogenous proteins Bax,Caspase-3 and anti-apoptotic protein Bcl-2 to induce tumor cell apoptosis;through flow cytometry,ELISA and related protein expression,it was verified that CpG ODNs in MnCpGPNPs can promote the Ml-type polarization of RAW264.7 cells and produce cytokines such as TNF-α,IL-2 and IL-6,and induce the maturation of DC cells and enhance the immunostimulatory activity to reverse the immunosuppression of the tumor microenvironment;at the same time,Mn2+ in MnCpGPNPs activates the cGAS-STING pathway,enhances the expression of STING,pIRF3 and IFN-β proteins,further induces the maturation of DC cells,and jointly triggers strong immune response.Chapter 3:In this chapter,three mouse tumor models were established:mouse subcutaneous tumor model,double tumor model and preventive research model.The results showed that MnCpGPNPs not only had good killing effect on primary tumors,but also played a role in distant tumors.In addition,the experimental results also found that MnCpGPNPs have the potential to become nanovaccine to fight tumorigenesis.