The Study Of PH-responsive Nanosystem Carrying Immune Agonist And Anticancer Drug For Combination Therapy On Breast Cancer

Objective Breast cancer is one of the most common malignant tumors in women.It is increasingly threatens women health.Currently,the main treatment methods for breast cancer are including surgery,chemotherapy,radiotherapy,hormonal therapy and immunotherapy.Advanced anti-cancer regimens are being introduced for more effective cancer treatment with improved life expectancy.In this research,immuno-stimulating agent toll-like receptor-7/8(TLR-7/8)agonist-resiquimod and low dose chemotherapeutic agent-Doxorubicin were synergized to demonstrate tumor combination therapy.Methods Section 1 : The preparation and characterization 1)To synthesis of polyhistidine(Polyhistidine,PHIS)-R848 micellar: polyhistidine were coated R848 formed PHIS-R848 micelle through hydrophilicity.2)To synthesis of prodrug HA-DOX: First,EDC used as crosslinking agent was connected succinic dihydrazide via an amide bond,then combined DOX carbonyl reacts with p H-sensitive hydrazone bond(Schiff base reaction).Hyaluronic acid(Hyaluronic acid,HA)was connected DOX via p H sensitive hydrazone bond.3)HA-DOX were packaged PHIS-R848 micelles via plus-to-minus charge interactions,formed a co-carrying nanoparticles which combined DOX and resiquimod.4)We used transmission electron microscopy(TEM),dynamic light scattering and Zeta-potential measurement were applied to characterize their physicochemical property.In order to measure the loading capacity of HA-DOX,we used the ultraviolet spectrophotometry(UV).Section 2 : In vitro experiments 1)Real-time PCR was used to detected expression of IFN-α,IL-6,IL-12p40 and TNF-α m RNA after PHIS-R848 treated CAL-1 cells.2)The CD44 expressed on MCF-7 cells,MDA-MB-231 cells and 4T1 cells weredetected by the flow cytometry.3)The in vitro DOX releases from HA-DOX were studied by dynamic dialysis method..4)The confocal microscopy was used to measure the endocytosis of HA-DOX in MCF-7 cells.5)The cytotoxicity of HA-DOX-PHIS-R848 in MCF-7 cells and 4T1 cells were assessed by CCK-8 assay.6)HA-DOX-PHIS-R848 induce Caspase-9 and Caspase-3 protein expression in MCF-7 cells was analyzed by Western blot analysis.Section 3: In vivo experiments 1)Establish MCF-7 cells tumor-bearing mouse model.2)Synthesis of fluorescently labeled PHIS-R848-Cy5.5 and HA-DOX-PHIS-R848-Cy5.5.3)The distribution of HA-DOX-PHIS-R848 nanoparticles in 4T1 tumor-bearing nude mice was evaluated with in vivo imaging system.4)The In vivo antitumor effect of HA-DOX-PHIS-R848 was evaluated in 4T1 tumor-bearing mice via tail vein injection.Results 1.The PHIS-R848 micelles were successfully synthesized.The PHIS-R848 was almost spherical with size of 153.1±5.6 nm,particles size of polydispersity index PDI was 0.132.The Zeta potential of PHIS-R848 was 40.4±0.7 m V.The prodrug HA-DOX was successfully synthesized and DOX content in HA-DOX was about 5 %.The HA-DOX-PHIS-R848 nanoparticles were successfully synthesized and its had a spherical shape with classic “core-shell” structure.The size of HA-DOX-PHIS-R848 was 213.4±8.4 nm,particles size of polydispersity index PDI was 0.248.The Zeta potential of HA-DOX-PHIS-R848 was-34.5±0.5 m V.2.The cytokines of IFN-α,IL-6,IL-12p40 and TNF-α were increased in the expression of m RNA after PHIS-R848 treated CAL-1 cells.Flow cytometry showed the CD44 was highly expressed on MCF-7 cells,MDA-MB-231 cells and 4T1 cells.The prodrug HA-DOX exhibited obvious p H-sensitive and the release of DOX wasslow at p H 7.4 phosphate buffer saline(PBS)but greatly accelerated at p H 5.5.Confocal experiments showed that HA-DOX possessed a high targetability toward CD44 receptor overexpressing MCF-7 human breast cancer cells..HA-DOX-PHIS-R848 exhibited effectively toxicity in breast cancer cells.HA-DOX-PHIS-R848 could activated Caspase-9 and Caspase-3,which lead MCF-7 cells to apoptotic death.3.At 24 h after administration of Cy5.5-labled PHIS-R848 and HA-DOX-PHIS-R848(PHIS-R848-Cy5.5 and HA-DOX-PHIS-R848-Cy5.5),the fluorescent signals were almost observed in tumor tissues.In vivo anti-tumor efficacy result showed HA-DOX-PHIS-R848 had direct anti-tumor activity.Conclusions In this study,a novel breast tumor targeting co-carrying nanoparticles with delivery of immuno-stimulating agent toll-like receptor-7/8(TLR-7/8)agonist-resiquimod and low dose chemotherapeutic agent-Doxorubicin were synergized to demonstrate breast tumor combination therapy.This p H-responsive nanosystem have appeared as a highly promising platform for combination therapy on breast cancer.