Construction And Antitumor Activity Evaluation Of ROS Responsive Galactose Nanoparticles

Triple negative breast cancer（TNBC）in breast cancer subtypes has a high rate of aggressive metastasis and high mortality,accounting for 10-20%of all breast cancers.So far,chemotherapy is still the main treatment strategy for patients with triple negative breast cancer.In order to achieve more effective treatment effect,stimulus-responsive nanomaterials have been widely used.Objective:In this study,ROS responsive galactolactated nanoparticles（DOX@NPs）were developed as highly effective targeting vectors for doxorubicin（DOX）to inhibit TNBC growth in vitro and in vivo.Methods:The research content of this paper consists of three parts of synthetic characterization,experiments in vitro and in vivo.Synthesis characterization of parts,the synthesis of methyl acrylic galactose and benzene boric acid derivatives by reversible addition-breaking chain transfer（RAFT）polymerization reaction,assembled into a on the hydrophobic and hydrophilic polymer by hydrogen nuclear magnetic spectra confirmed its chemical structure,synthesis of compounds through the dialysis method package preparation doxorubicin has targeted nanoparticles.The morphology of nanoparticles under physiological and oxidative conditions was observed under transmission electron microscopy.The stability of nanoparticles under simulated ROS environment in vivo and physiological conditions were measured.In addition,hemolysis experiments were conducted to investigate the safety of nanoparticles.MTT assay was used to detect the cytotoxicity of DOX@NPs to MDA-MB-231 and4T1 cells in vitro and IC₅₀ values were calculated.Flow cytometry was used to investigate the uptake of the nanoparticles by two breast cancer cells and confocal laser scanning microscope was used to observe the uptake using standard.Annexin V-FITC/PI staining was used to evaluate the apoptosis-inducing ability of DOX@NPs nanoparticles by flow cytometry.Meanwhile,flow cytometry was used to investigate the growth cycle arrest of the nanoparticles on two types of breast cancer cells.In the in vivo study,4T1 tumor-bearing mice were divided into high and low concentration DOX,DOX@NPs group and normal saline group.Tumor size was measured by regular drug administration,and the inhibitory effect of DOX@NPs on the growth of breast cancer in mice was investigated.After the mice were sacrificed,the damage of the nanoparticles on the main organs in the body was observed by staining after the viscera were embedded and sections were collected.After the tumors in each group were collected and weighed,immunohistochemical TUNEL staining was performed to evaluate the induction effect of DOX@NPs on the proliferation and apoptosis of breast cancer cells.Results:The synthesis of the copolymerized compound was confirmed by ¹H NMR,and the nanoparticles were synthesized by dialysis method and observed by transmission electron microscopy.The nanoparticles showed regular spherical shape.The particle size of the nanoparticles changed significantly under different concentrations of H₂O₂,but remained stable under physiological conditions,which confirmed that the nanoparticles had a good response to ROS environment.Hemolysis test showed that the nanoparticles were safe for intravenous administration.In vitro experiments,MTT assay results showed that DOX@NPs had significant cytotoxicity to MDA-MB-231 and 4T1 cells.The IC₅₀values of MDA-MB-231 cells at 24h and 48h were 7 and 4μg/m L respectively.The IC₅₀ values of 4T1 cells at 24h and 48h were32 and 10μg/m L respectively.In addition,flow cytometry results showed that DOX@NPs can significantly improve DOX uptake,effectively induce apoptosis and block cell cycle of breast cancer cells.The results of cell uptake experiments showed that the uptake degree of DOX@NPs was significantly higher than that of free DOX（10μg/m L）at the same concentration.Confocal microscopy observation results were consistent with the above,indicating that the nanoparticles have good targeting properties to the two types of breast cancer cells.The obtain results showed that the percentage of MDA-MB-231 cells at G2/M stage were 27.76%,41.21%（5μg/m L）and 60.19%（10μg/m L）in control and free DOX treatment.DOX@NPs yielded the higher percentage of cells in growth arrest compared to free DOX,which reveal 47.04%（5μg/m L）and 65.78%（10μg/m L）cells in G2/M stages in MDA-MB-231 cells.Moreover,the effect of DOX@NPs accompanied with a reduction of the G0/G1 phase,with no significant impact in the S stage.Similar trends were also found in free DOX and DOX@NPs treated 4T1 cells.A mouse model of breast cancer demonstrated that DOX@NPs can effectively concentrate at the tumor site and inhibit tumor growth in tumor-bearing mice.Compared with the normal saline group,the tumor volume and weight of the high and low concentration free DOX groups were significantly reduced,while the tumor volume and weight of the nano group at the same concentration were more significant.In addition,HE staining and TUNEL staining experiments of tumor tissue sections showed that DOX@NPs significantly increased the necrosis degree of tumor tissue and had a strong inhibitory effect on tumor growth.During the administration period,no abnormality was observed in the body weight of mice,and the observation results of the main organs（heart,liver,spleen,lung and kidney）after staining confirmed the in vivo safety of the nanoparticles.Conclusions:Has obvious anti-breast cancer effect in vivo and in vitro.The nanocarrier can target delivery DOX,release it in the environment of high ROS at the tumor site.DOX@NPs can inhibit the proliferation and promote apoptosis of breast cancer cells after aggregation in tumor site with considerable safety in vivo.In conclusion,the nanoparticles have the potential to be a vector for the treatment of TNBC and provide research ideas for the treatment of triple negative breast cancer.