A MPEG-PAE-Ce6 Nanoparticle Promotes Antitumor Activity Of CAR T Cells Against Hepatocellular Carcinoma

BackgroundMalignancies are the first reason for the death of Chinese residents,the fourth most common cause of cancer related deaths,and the incidence of the disease is sixth.The world health organization estimates that by 2030,more than one million people worldwide will have died of liver cancer.Hepatocellular carcinoma accounts for the 70% of primary liver cancer.As of present,the common treatment of Hepatocellular carcinoma has surgical surgery,tumor ablation,hepatic arterial embolization chemotherapy and system therapy,but the overall survival rate of patients is poor.In recent years,Car T cell immunotherapy has provided a pioneering method for the treatment of liver cancer.GPC3 is highly expressed in Hepatocellular carcinoma,but hardly expressed in normal tissues.It is a relatively safe target for the treatment of Hepatocellular carcinoma by CAR T cells.However,the effect of Car T cells on the physical tumor is still not high due to the obstruction of the dense intercellular mass,the warped tumor blood vessels and the heavy pressure interstitial fluid.Photodynamic therapy using photosensitizer,which is used to kill cells by photochemical reaction,has been widely used in tumor therapy in recent years.Chlorin e6,as a classic photosensitizer,is widely used in the photodynamic therapy of the tumor,but its lack of specificity and the very high concentration of the aggregation-caused fluorescence quenching effect severely limit its clinical application.The size of nanoparticles can vary according to the low p H response in the tumor microenvironment are overcome the above defects of Chlorin e6 by further tumor infiltration,better tumor accumulation and higher treatment efficiency.In this study,we proposed mPEG-PAE-Ce6 nanoparticles,which can be treated with light kinetic energy in the tumor microenvironment,and discussed the effectiveness of the nanoparticles in the immune treatment of Hepatocellular carcinoma patients with GPC3-CAR T cells,and provide a new research thought and direction to provide the efficacy of Car T cell immunotherapy for solid tumors.Methods1.Using mPEG-PAE polymers with low p H effect to encapsulate the photosensitizer Ce6 which is commonly used in photodynamic therapy,to synthesize an mPEG-PAE-Ce6 nanoparticles of tumor microenvironment low p H effect,and using dynamic light scattering,electron transmission microscope scanner,ultraviolet-visible absorption spectrum scanning,and fluorescence emission spectrum scan to characterization of nanoparticles。2.In vitro cell culture experiments,Hep G2 and Huh7 cells were selected as the experimental subjects.First,a green fluorescent lysosomal probe was used to co-locate the mPEG-PAE-Ce6 nanoparticles and investigate whether they could intracellular by tumor cells efficiently.Secondly,ROS probe was used to detect the ROS production efficiency of mPEG-PAE-Ce6 nanoparticles in tumor cells.After then,the anti-tumor treatment effect of mPEG-PAE-Ce6 nanoparticles combined with GPC3-Car T cells was determined by MTT assay,and used ELISA to detect the secretion of INF-γ and IL-2 in the supernatant of cell culture.Finally,the treated tumor cells were photographed by fluorescence microscope using the Calcein-Am /PI double staining live/dead cell method.3.Using Hep G2-GL cells to build the NSG mouse subcutaneous transplantation model,and recording tumor growth by using IVIS Spectrum Imaging System to verify the anti-tumor therapeutic effect of mPEG-PAE-Ce6 nanoparticles combined with GPC3-Car T cells in the mouse liver cancer model.Results1.The synthesized mPEG-PAE-Ce6 nanoparticles are spherical in shape,the particle size is about 100 nm,and with a high encapsulation concentration of Ce6.2.At the p H of 5.5 or 6.5,mPEG-PAE-Ce6 nanoparticles gradually cracked and released Ce6 with the extension of incubation time,and restored its photoactivity and photochemical activity.3.mPEG-PAE-Ce6 nanoparticles can be efficiently autophagy by Hep G2 and Huh7 cells,and it has the same pharmacological effect of free Ce6.4.mPEG-PAE-Ce6 nanoparticle combined with GPC3-Car T cells therapy has a strong lethal effect on Hep G2 and Huh7 cells,and it is better than separately using photodynamic nanoparticle therapy and GPC3-Car T immunotherapy,and can also promote the secretion of INF-γ and IL-2 by tumor cells.5.The mPEG-PAE-Ce6 nanoparticles combined GPC3-Car T cells therapy has better effect on the subcutaneous Hep G2-GL cell transplantation tumor in NSG mice than separately using photodynamic nanoparticle therapy and GPC3-Car T immunotherapy,a nd it has little effect on the health of the mice.ConclusionsWe designed and synthesized an mPEG-PAE-Ce6 nanoparticles with a size of about100 nm and a good low p H response,which can promote the anti-tumor therapeutic effect of GPC3-Car T cells therapy at both the vitro and vivo levels.