Engineered Neutrophil-derived Exosomes For Targeted Cancer Therapy

Objective:To reveal the anti-tumor effect and mechanism of exosomes(Ex)derived from neutrophils,develop and engineer neutrophil exosome-like nanovesicles(NV)as a drug delivery system,evaluate its efficacy and application safety in targeted tumor therapy,and provide new ideas and methods for neutrophil exosomes and biomimetic nanovesicles in precise tumor treatment.Methods:1.Neutrophil-derived exosomes(N-Ex)were prepared by ultracentrifugation and identified.The uptake of N-Ex by cells was observed by multi-dimensional imaging flow cytometry and laser confocal microscopy.2.The effects of N-Ex on the biological function of tumor cells were investigated by CCK-8,cell colony formation assays,flow cytometry and immunofluorescence staining.The changes of apoptosis related signal pathways were detected by Western blot.The subcutaneous tumor model of BALB/c nude mice was established to investigate the effect of N-Ex on tumor growth and evaluate its biosafety in vivo.Immunohistochemistry and immunofluorescence staining were used to detect the effects of N-Ex on the proliferation and apoptosis of tumor cells.3.Superparamagnetic iron oxide nanoparticles(SPION)were synthesized and modified with transferrin(Tf)to form Tf-SPION.Neutrophil-derived exosomes were magnetically separated via Tf-Tf R interaction(SPION-Ex).CCK-8 and cell colony formation assays,flow cytometry and immunofluorescence staining were used to analyze the effects of SPION-Ex on the biological function of tumor cells under magnetic field.The subcutaneous tumor model of BALB/c nude mice was established to investigate the in vivo anti-tumor effect of SPION-Ex under the magnetic field,and the distribution of SPION-Ex in vivo was detected by small animal imager.4.Doxorubicin(DOX)-loaded neutrophil exosome-like nanovesicles(NNV)were prepared by serial extrusion and magnetically separated after SPION modification(SPION-NNV-DOX).The encapsulation and drug release of DOX were detected by HPLC.The killing effect of SPION-NNV-DOX on tumor cells was detected by CCK-8 assay and immunofluorescence staining.The subcutaneous tumor models of BALB/c nude mice and NCG mice were constructed to detect the anti-tumor effect of SPION-NNV-DOX under the magnetic field and evaluate its safety in vivo.Results:1.Exosomes derived from neutrophils(N-Ex)were successfully extracted by supercentrifugation.Transmission electron microscopy and atomic force microscopy showed that the particle size of N-Ex was 30～200 nm,in the shape of a double concave disc,with a 3D three-dimensional structure.NTA results showed that the particle size of N-Ex was 125±10 nm and Zeta potential was-26±1 m V.DLS results showed that N-Ex polymer dispersion index <0.3,with uniform size and good stability.Western blot results showed that N-Ex stably expressed CD9,CD63,CD81 and Alix but not Calnexin.Cell uptake experiments showed that N-Ex could be efficiently internalized by tumor cells.2.N-Ex inhibited the proliferation and colony formation of tumor cells,promoted the change of mitochondrial membrane potential and apoptosis of tumor cells,but did not affect the biological function of normal cells.Western blot showed that N-Ex expressed Fas L,granzyme and perforin,and N-Ex treatment induced activation of caspase signaling pathway in tumor cells.In vivo experiments revealed that N-Ex treatment inhibited tumor growth and had no significant effect on heart,liver,kidney function and body weight of tumor bearing nude mice.3.Transmission electron microscopy showed that multiple superparamagnetic iron oxide nanoparticles were anchored on the surface of SPION-Ex.NTA results showed that the particle size of SPION-Ex was 140±11 nm and the Zeta potential was-34±1 m V,suggesting that there was no significant change in the morphology and structure of exosomes after magnetic modification.DLS test showed that SPION-Ex polymer had good dispersion and stability in different solutions.Compared with unmodified N-Ex,SPION-Ex inhibited tumor cell growth and promoted apoptosis more strongly in vitro under an external magnetic field.More SPION-Ex accumulated in the tumor site in vivo,significantly inhibiting tumor growth and prolonging survival time.4.After co-incubation of neutrophils with liposome-doxorubicin(DOX-CL),DOX-encapsulated neutrophil exosome-like nanovesicles(NNV-DOX)were successfully prepared by serial extrusion.SPION-NNV-DOX was formed after magnetic modification and separation.Compared with NNV,DOX,and NNV-DOX,SPION-NNV-DOX significantly inhibited tumor cell proliferation and promoted apoptosis in vitro under the external magnetic field.The tumor models of BALB/c nude mice and NCG mice in vivo confirmed that SPION-NNV-DOX almost completely accumulated in the tumor site under magnetic chemotaxis,and had the dual anti-tumor effects of NNV and DOX.DOX treatment alone resulted in changes in heart,liver,kidney function and body weight in mice,while SPION-NNV-DOX treatment did not.Conclusion:This study demonstrates that N-Ex carries apoptosis-related proteins,induces tumor cell apoptosis by activating the caspase signaling pathway,and inhibits tumor growth in vivo.SPION modified N-Ex can be separated rapidly and show better tumor targeting and stronger anti-tumor effect under an external magnetic field.NNV can be used as a new DOX delivery vehicle to achieve efficient and safe targeted combination therapy.This study provides theoretical and experimental basis for the application of neutrophil-derived exosomes and their biomimetic nanovesicles in precision tumor therapy.