"Domino-effect" Chemotherapy Combined With Immune-Therapy Cascade Amplification In The Treatment Of Metastatic Breast Cancer

In tumor immunotherapy,Treg cells are immunosuppressive cells.Generally,the main strategies of chemo immune-therapy for Treg cells are to eliminate them by using chemotherapy drugs.However,the dead Treg cells still exert immunosuppressive effects via the nucleoside adenosine pathway.To improve immunosuppression and chemotherapy sensitivity,we designed a nanosystem to deliver synthetic chemotherapeutics and immunosuppressant to achieve cascade synergy in immunochemotherapy.The homemade curcumin analog(CA-1)was encapsulated by ?-lactalbumin(?-LA),and Treg cell specific antibody(mAb,immunosuppressant),as a therapeutic agent in delivery system,was linked to the drug-loaded protein via matrix metalloproteinase responded peptide(P).After the cleavage peptide responded to matrix metalloproteinase(MMP2),the CA-1@?-LA-P-mAb nanoparticles were separated into CA-1@?-LA and antibody,which can specifically enter cancer cells and Treg cells through membrane fusion and Nrp-1 receptors,respectively.The ?-LA responded to intracellular GSH cleavage to release CA-1,and then lead to mitochondria-mediated apoptosis.In summary,this article delivered chemotherapeutic drugs and immunomodulators to different target cells.Then,its response characteristics,anti-angiogenesis ability and antitumor activity were systematically studied.A series of basic research has been conducted in the following areas:1.Construction and characterization of nanosystemsFirstly,curcumin analog was prepared using 2-pyridinecarboxaldehyde and cyclohexanone.The ?-lactalbumin(?-LA)was hydrolyzed by bacillus licheniformis(BLP),and the disulfide bond in the protein was recrosslinked by DTT to prepare a nanoparticle.The curcumin analog linked to alpha-lactalbumin via self-assembly.Laser nanometer particle size analyzer and Transmission electron microscope(TEM)analysis showed that the CA-1@?-LA-P-mAb nano system was successfully prepared,with an average particle size of 175±8.4 nm and an average Zeta potential of-15.03±3.5 mV.2.The study of MMP2/GSH dual-responsive characteristics in vitroThe enzyme responsiveness of the system to MMP was investigated by HPLC and TEM.Its ability to separate into CA-1@?-LA nanoparticle and mAb was investigated in physiological concentrations of MMP2.In addition,the response of drug-loaded proteins to GSH under GSH conditions was examined.The results showed that CA-1@?-LA-P-mAb was able to achieve programmed release in response to MMP2/GSH.3.The antitumor activity in vitro and the targeting of mAbThe antitumor activity of CA-1@?-LA-P-mAb nanosystem in vitro was studied using mouse 4T1 breast cancer cells.Cell uptake experiments showed that CA-1@?-LA-P-mAb nanosystem had a good targeting ability.Cytotoxicity experiments show that CA-1 and mAb had a synergistic antitumor effect Western blotting investigation of mitochondrial pathway cascade-related protein content has proved that CA-1@?-LA-P-mAb nanosystem can damage mitochondria and cause the release of cytochrome C,thereby inducing apoptosis.In addition,HUVEC(human umbilical vein endothelial cells)was used to study the biocompatibility of the?-LA-P-mAb nanoparticles,the results showed that the blank vector was safe.The targeting of CA-1@?-LA-P-mAb nanosystem in vitro was studied using DU 145(human prostate cancer cells),targeting results in vitro indicated that mAb can specifically target neuromucin-1(Nrp-1).4.Anti-tumor activity in vivo of CA-1@?-LA-P-mAbIn order to study the anti-tumor effect and pathological characteristics of the delivery system,BALB/c female mice bearing 4T1 breast cancer were used as examining the distribution of CA-1@?-LA-P-mAb nanosystem in mice,the relative tumor volume changes,H&E staining pathological sections of mice in each administration group,tumor inhibition rate and Tunel staining analysis.Targeting results in vivo showed that the drug-loading system can rapidly accumulate at the tumor site,CA-1@?-LA-P-mAb nanosystem had a good ability to target the tumors.Through pharmacodynamics and pathological investigations,CA-1@?-LA-P-mAb nanosystem had a good biological safety.The drug delivery system had a significant effect on inhibiting tumor growth in mice.5.Anti-angiogenesis studies of CA-1 in vitroThe anti-angiogenesis study was investigated by using HVEC(human umbilical vein endothelial cells)to study the effect of CA-1@?-LA-P-mAb nanosystem in vitro.The results showed that the synthetic chemotherapeutic curcumin analog CA-1 can rupture blood vessels that have already formed.What's more,immunofluorescene staining was performed on tumor sections to investigate the tumor metastasis of the nanodelivery system in vivo through reducing the expression of VEGF.6.In vivo anti-metastatic researchThe 4T1 tumor-bearing mice were used to investigate the anti-metastatic effect of CA-1@?-LA-P-mAb nano-delivery system in vivo.Through observing lung metastases and pathological sections of lung tissue,it was proved that the number of lung nodules was significantly reduced when the nano-drug delivery system was used for treatment,and the metastasis was effectively suppressed.The combination of immune and chemotherapy successfully activated the immune response,and multiple mechanisms work together to effectively treat the metastatic lesions.Conclusion:This study provided a basis for regulating Treg cell function,thereby improving chemosensitivity.Eventually,C A-1@?-LA-P-mAb nanosystem successfully regulated the immunosuppressive function of Treg cells,which improved the immunosuppression of tumor microenvironment,downregulated the expression of VEGF,and effectively restrained breast cancer metastasis.