Serum Albumin-modified Bacterial Outer Membrane Vesicles For Tumor Therapy

Microorganisms are an important member of fermentation engineering,which has been applicated in medicine field.Among them,bacterial outer membrane vesicles(OMV)produced by Gram-negative bacteria carry maternal immune components,such as pathogen associated molecular pattern(PAMP),to activate the body’s immune system and exert tumor-killing effects.However,intravenous lipopolysaccharide(LPS)can cause discomfort,such as systemic inflammatory response and sepsis,limiting its application in tumor therapy.Therefore,there is an urgent need to develop appropriate and effective virulence reduction strategies to improve the safety of OMV.In recent years,methods to improve the safety of OMV have been genetic engineering methods,by knocking out toxic genes but reducing the original immunogenicity of OMV,and solvent methods,which may have solvent residues.In order to avoid the shortcomings of the above two methods,there is an urgent need to develop a toxicity reduction strategy that responds to the tumor microenvironment(TME),in which OMV is only exposed to the tumor site at a fixed point to exert the immune effect.Based on the high expression of Matrix metalloproteinases(MMP)in the tumor microenvironment,in this study,MMP cleavable linker(c L)was selected to modify serum albumin(SA)on the surface of OMV,which dynamically masks the danger signals on the surface of OMV and improves the safety of OMV.In addition,in order to enhance the efficacy,the chemotherapeutic drug doxorubin was combined with OMV-c L-SA to achieve the combined therapeutic effect of chemotherapy and immunotherapy.The specific research results of our study were as follows:(1)Construction of a safe and effective therapeutic platform OMV-c L-SA by surface modification of SA.Based on the tumor microenvironment overexpression of MMP,which was selected to remove the peptide chain 6-Mal-PLGVR-NHS,modification of SA on the surface of OMV dynamically shielded the danger signals of OMV,and improved OMV storage stability.In vivo experiments showed that OMV-c L-SA reduced the systemic toxicity induced by OMV.In addition,based on the inherent prolonged half-life property of SA,the half-life of OMV-c L-SA was 7.2 times longer than that of OMV,significantly prolonging the circulation time of OMV in vivo.In addition,the results of in vivo tumor suppression experiments showed that the selection of MMP cleavable peptide c L could achieve the effective release of OMV at the tumor site and exert the immunotherapeutic effect.(2)To enhance the tumor suppression effect,DOX was encapsulated into OMV-c L-SA,a safe immune platform obtained as described above,to achieve the effect of chemotherapy and immune combination therapy.Firstly,SA was treated by TCEP to open its disulfide bond and expose the hydrophobic region,and the hydrophobic drug DOX was loaded inside SA to produce SA@DOX.Secondly,the former SA@DOX was combined with OMV to get the therapeutic platform OMV-c L-SA@DOX.In vitro 3D cell sphere assay demonstrated that OMV-c L-SA@DOX could effectively inhibit 3D cell sphere growth.In addition,its safety was examined by in vivo experiments,which showed that OMV-c L-SA@DOX could avoid the cardiotoxicity caused by free DOX.Through in vivo distribution experiments,the results of in vivo imaging showed a significant increase in OMV enrichment at tumor sites at 72 h,the tumor/liver ratio in group of OMV-c L-SA@DOX was 12.6 times that of OMV group,providing a theoretical basis for the later in vivo tumor suppression effect.(3)OMV-c L-SA@DOX was a broad-spectrum anticancer platform applied for multiple tumor models.The results of RNA transcriptome sequencing showed that OMV-c L-SA@DOX upregulate immuno-active related genes,downregulate immuno-suppressive related genes,and reverse the regulation of immunosuppressive microenvironment signaling pathways,such as T cell receptor signaling pathway,antigen processing and presentation,and m-TOR signaling pathway of reversing M2 to M1,to inhibit tumor growth.Subsequently,to investigate the tumor suppression efficacy of OMV-c L-SA@DOX in hot tumor model(CT26),the results showed that OMV-c L-SA@DOX could regulate TME via increasing CD4~+T and CD8~+T,decreasing Tregs,and reversing M2to M1 to retard the tumor growth in hot tumor model(CT26).Moreover,to inhibite the tumor growth,metastasis,and recurrence in cold tumor model(4T1),and post-operative luc-4T1tumor recurrence,OMV-c L-SA@DOX needed with anti-PD1,which significantly reversed the tumor immunosuppressive microenvironment,such as the increase of CD8~+T from1.97±0.13%to 2.77±0.25%and M1/M2 from 1.06±0.05%to 1.27±0.04%.