Drug Carrier Nanosystem Based On Polymeric Copper C Helator For Combining Antiangiogenesis And Immunotherapy For Targeted Treatment Of Breast C Ancer

ObjectiveBreast cancer is one of the most common cancers in women and its incidence increased continuously in recent years,causing a great threat to public health.Some previous investigations have shown that angiogenesis and immunosuppression play important roles in the growth and metastasis of breast cancer.Therefore,antiangiogenesis combined with immunotherapy has been considered as a promising strategy for breast cancer treatment.In this work,a copper chelating coil-comb block copolymer named as RPTDH was constructed with PEG-b-PGA as the backbone and then used to prepare nanoparticles for efficiently loading TLR7/8 specific agonist resiquimod（R848）through hydrophobic interactions.Thus obtained RPTDH/R848nanoparticles with pH-sensitivity and tumor-targeting ability can combine antiangiogenesis and immune activation to treat breast cancer synergistically.ContentsThis research was mainly divided into three parts as follows.In the first part,RPTDH/R848 nanoparticles were prepared and characterized.RPTDH was firstly synthesized and chemically characterized,and its copper-chelating ability was evaluated meanwhile.Next,RPTDH nanoparticles were prepared for loading R848,and their pH-sensitivity and drug release behavior were also evaluated in vitro.In the second part,synergistic effects of RPTDH/R848 nanoparticles against breast cancer were assessed in vitro,and furthermore their antiangiogenesis mechanisms were also preliminarily discussed.In the third part,a mouse model of breast cancer was constructed and used to evaluate tumor-targeting ability and antitumor activity of RPTDH/R848 nanoparticles in vivo.Methods1.Preparation and characterization of RPTDH/R848 nanoparticles.RPTDH was synthesized via a multistep process and chemically characterized by the FT-IR and ¹H NMR spectra.The sulfur content was analyzed by the inductively coupled plasma mass spectrometry（ICP-MS）.The copper-chelating ability of RPTDH was evaluated by the UV spectrometry and atomic absorption spectro metry.The nanoprecipitation method was used to prepare RPTDH and RPTDH/R848 nanoparticles.The loading content and encapsulation efficiency of R848 were detected using the ultra performance liquid chromatography（UPLC）method.The size,size distribution and surface charge properties of RPTDH/R848 nanoparticles were detected separately using the dynamic laser scattering method and Zeta potential analyzer,and their morphology of nanoparticles was observed by a transmission electron microscope（TEM）.The pH-sensitivity of RPTDH nanoparticles was investigated using the ¹H NMR,TEM and fluorescent pyrene probe methods.Moreover,the in vitro releases of R848 from RPTDH/R848 nanoparticle were investigated in different pH aquoues media by the dynamic dialysis method.2.In vitro studies of RPTDH/R848 nanoparticles.The cytotoxicity of RPTDH and RPTDH/R848 nanoparticles in breast cancer cells were detected by the MTT assay.The antiangiogenic effects of RPTDH/R848 nanoparticles were detected in human umbilical vein endothelial cells（HUVECs）by the wound-healing,transwell and tube-formation assays.To investigate antiangiogenesis mechanism of RPTDH/R848 nanoparticles,the expressions of transcription factor nuclear factor kappa B（NF-κB）in the nuclear and cytoplasm in HUVECs and breast cancer MDA-MB-231 cells were detected by western blotting analysis,and the subcellular distribution of NF-κB was also observed by a laser confocal scanning microscopy.The immune activation effects of RPTDH/R848 nanoparticles were assessed in human plasmacytoid dendritic CAL-1 cells.The mRNA expressions of TNF-α,IFN-α,IL-6,and IL-12p40 were detected by the quantitative Real-time PCR（qRT-PCR）,the protein expressions of IL-1α,VEGF,IL-8 and MMP-2 were analyzed by the ELISA kits,and the expressions of costimulatory molecules CD80,CD86 and CD83 were detected using the flow cytometry.3.In vivo studies of RPTDH/R848 nanoparticles.4T1 cells or 4T1-Luc cells were injected into the mouse mammary fat pad to construct a mouse model of metastatic breast cancer.IR780,a near-infrared dye,was loaded into RPTDH nanoparticles to prepare RPTDH/IR780 nanoparticles.To evaluate tumor-targeting ability,the accumulations of RPTDH/IR780 nanoparticles in the liver,kidney,spleen,lung,and tumor tissues in 4T1 tumor-bearing mice were detected after intravenous injection using an in vivo imaging system.The in vivo antitumor effects of RPTDH/R848 nanoparticles were evaluated in 4T1-Luc tumor-bearing mice.After treatments,the mice were intraperitoneally injected with D-Luciferin,and then their main organs（liver,kidney,spleen and lung）and tumors were removed and imaged under an in vivo imaging system,thus to investigate the in vivo antimetastasis effect of RPTDH/R848 nanoparticles.The histopathological changes of main organs and tumor were examined using the hematoxylin and eosin（H&E）staining.In addition,the tumor apoptosis,immune activation and antiangiogenesis effects were also assessed by the immunohistochemical method to further evaluate the synergistic antitumor effects of RPTDH/R848 nanoparticles in vivo.Results1.A novel polymer copper chelator named as RPTDH was successfully synthesized and its chemical structure was confirmed.The UV and atomic absorption spectra showed that RPTDH had a strong chelating and removal ability for Cu²⁺.RPTDH and RPTDH/R848 nanoparticles were successfully prepared and the TEM images showed that these nanoparticles had a regular spherical shape with small sizes.The results of ¹H NMR,TEM and fluorescent pyrene probe method verified that RPTDH nanoparticles had a significant pH-sensitivity.R848,a specific agonist for TLR7/8,was efficiently encapsulated into RPTDH nanoparticles via hydrophobic interactions and showed significant pH-responsive release from RPTDH/R848nanoparticles e.g.,the release rate of R848 notably increased with the pH value of release medium decreasing.2.RPTDH and RPTDH/R848 nanoparticles displayed a selective cytotoxicity in breast cancer cells through copper chelation.RPTDH/R848 nanoparticles significantly inhibited the mobility,invasion and vascular tube formatio n of HUVECs.RPTDH/R848 nanoparticles notably suppressed the NF-κB nuclear translocation in both HUVECs and MDA-MB-231 cells,and subsequently down-regulated the expressions of downstream proangiogenic factors,thus suppressing tumor angiogenesis.Furthermore,RPTDH/R848 nanoparticles remarkably enhanced the mRNA expressions of TNF-α,IFN-α,IL-6,and IL-12p40,and the protein expressions of CD80,CD86 and CD83 in CAL-1 cells,demonstrating their induction effects on the maturation and activation of dendritic cel s.3.A mouse model of metastatic breast cancer was constructed successfully.At24 h after intravenous injection,RPTDH/IR780 nanoparticles were mainly accumulated in the primary breast tumor and lung metastases of 4T1 tumor-bearing mice,hereby confirmed their excellent tumor-targeting ability.The in vivo studies showed that RPTDH/R848 could synergistically inhibit the tumor growth and metastasis in 4T1-Luc tumor-bearing mice through angiogenesis and immune activation.In addition,no visible histopathological injuries were found in the main organs of tumor-bearing mice after treatments.ConclusionsIn this study,a polymeric copper chelator RPTDH was synthesized and used to prepare nanoparticles possessing tumor-targeting ability and pH-responsive disintegration property for efficient loading and controlled release of R848.RPTDH/R848 nanoparticles showed a selective cytotoxicity in breast cancer cells via copper chelation and significant inhibitory activities on the mobility,invasion and vascular tube formation of HUVECs.The regulation of NF-κB signaling pathway was possibly involved in the antiangiogensesis of RPTDH/R848 nanoparticles.In breast tumor-bearing mice,RPTDH/IR780 nanoparticles displayed excellent tumor-targeting ability,and RPTDH/R848 nanoparticles notably suppressed the tumor growth and metastasis.In summary,RPTDH/R848 nanoparticles will be likely to become a therapeutic agent for treatment of metastatic breas t cancer through combining antiangiogenesis and immune activaton,and our research work provides a new strategy for clinical breast cancer treatment.