Enterotoxin C-loaded Tumor-targeted Liposomes For The Treatment Of Metastatic Lung Cancer

Lung is the target organ for metastasis of many malignant tumors.Breast cancer is often complicated with metastatic lung cancer.At present,radiotherapy and chemotherapy are still the main treatments,but the therapeutic efficacy is not improved and have worse side effects.Cancer immunotherapy is gradually emerging.Superantigens such as bacterial superantigens are an important immunomodulatory for cancer immunotherapy,while some problems such as poor targeting and weak tumor-specific immune responses limit its clinical application.Staphylococcal enterotoxin C(SEC)is a typical bacterial superantigen produced by Staphylococcus aureus,which exhibits multi pharmacological functions including the stimulation of differentiation of naive T cells into CD4~+and CD8~+T cells,and promotion the produce of various anti-tumor cytokines.However,due to weak tumor targeting ability,the SEC can lead to"inflammatory storm"and immune cell depletion,which hinders its clinical translation.In this study,after constructing SEC produced engineered bacteria and following purification of SEC,we designed and prepared a tumor-targeting SEC liposome and proved its anti-tumor effect and safety by investigation in vitro and in vivo.(1)Preparation of recombinant SEC and antitumor activity in vitroThe SEC expression vector p MCSG7-SEC was constructed by Ligation Independent Cloning(LIC) method and the gene sequence of SEC was optimized.The rSEC(Recombinant Staphylococcal Enterotoxin C)was induced in E.coli BL21(DE3),and was purified by one-step affinity chromatography.The main peak of MALDI-TOF/TOF was 30980.704.Cirular dichroism(CD)showed that rSEC maintained complete secondary structure,which was benefitial to exert bioactive properties.The proliferation of mice splenic lymphocytes was greatly stimulated by rSEC at a range of 1μg/m L～10μg/m L.The levels of IFN-γ,IL-2,IL-4 and IL-10 in the cell supernatant were significantly increased when the splenic lymphocytes was stimulated by 10μg/m L rSEC.After co-incubation of 4T1-luc and rSEC-stimulated lymphocytes.(2)Properties of rSEC liposome and targeted liposomeCicular dichroism showed that the secondary structure of rSEC can be maintained by freeze-drying method.The Staphylococcal Enterotoxin C Liposome(SL)was prepared by freeze-drying method.An optimal formulation was screened out with the singal factor design:egg phospholipid/cholesterol(5:1.w/w),egg phospholipid/rSEC(40:1,w/w)and the entrapment efficientcy was as high as 93.20%.SL appeared as spherical particles under TEM with the better stability,the particle size of 159.6±2.491 nm(n=3)and the Zeta potential of-4.31±0.75 m V(n=3).SL and TSL could significantly stimulate splenic lymphocytes to secrete IFN-γ,IL-2,IL-4,and inhibited 4T1-luc cells growth within the concentration of 1μg/m L～10μg/m L.The membrane of 4T1-luc cells was extracted by membrane protein extraction kit and co-extruded with SL to produce tumor-targeted Staphylococcal Enterotoxin C Liposome C liposomes(TSL).TSL appeared as spherical particles under TEM with a 1～10nm layer outside,the particle size of 205.6±2.641nm(n=3)and the Zeta potential of-9.18±0.61 m V(n=3).Western Blot analysis showed that CD44,CD47,Ep CAM markers of 4T1-luc cell membrane were retained on the TSL,which laid the foundations for targeting ability.(3)In vivo pharmacodynamics and tumor-targeting of rSEC and its formulationsIn the mouse model of metastatic lung cancer,TSL significantly reduced the number of lung nodules,stimulated the proliferation of CD4~+T cells and CD8~+T cells in the spleen,and significantly increased the levels of anti-tumor cytokines(such as IFN-γand IL-2)in the lung compared with the PBS and SL groups.Tunel analysis showed that TSL promoted tumor cells apoptosis and exerted a good anti-tumor effect.The anti-tumor effect of TSL was related to the infiltration of CD8~+T lymphocytes in the lung and the polarization of M2 macrophages.Compared with the PBS group,TSL significantly increased the number of CD8~+T lymphocytes in the lung(P<0.05).At the same time,TSL had a good targeting ability to 4T1-luc cells in vitro,but was poor to other cells(e.g.,HT29,RAW264.7,L929).TSL had a good targeting ability for lung tumors in vitro imaging.It had well tumor-targeted capability and reduced accumulation in major organs to reduce adverse effects.Finally,rSEC,SL and TSL had good biosafety in vivo.No hepatotoxicity and nephrotoxicity were observed.And rSEC,SL and TSL had little effect on body weight.In this study,to solve the problems of overmuch side effects and poor targeting ability in super antigens anti-cancer immunotherapy,we selected the model of metastatic lung cancer caused by 4T1 cells,and then prepared the TSL with good tumor targeting characteristic.TSL increased the apoptosis of tumor cells and the infiltration of CD8~+T lymphocytes at the tumor site,meanwhile inhibited the metastasis of cancer cells to other organs and had good biosafety,which indicated prospectful clinical application.This study provides a new preparation to solve the problem of cancer immunotherapy of superantigen.It effectively improves the targeting ability of superantigen and reduces the adverse reactions,which is of great significance in the field of superantigen formulations.