Betulinic Acid-Mediated Redox-Responsive Chemo/Photodynamic Synergistic Therapy For The Research Of Breast Cancer

At present,breast cancer has become a common tumor that threatens women’s health,and traditional chemotherapy is prone to drug resistance,resulting in low efficacy and serious toxic and side effects on the body.Photodynamic therapy（PDT）has become an effective method of cancer treatment due to its advantages of minimally invasive,low toxicity and high selectivity.At the same time,chemo-photodynamic combination treatment with carrier-free nanomedicine can further improve the therapeutic effect through a synergistic effect.The previous research of our research group finds that some small natural molecules not only have excellent anti-tumor activity but also have the characteristics of forming nano-morphology by self-assembly.In addition,the drugs can be slowly released in tumor cells with a high content of glutathione through the disulfide bond.Therefore,in this paper,the natural small molecule betulinic acid（BA）is coupled with the disulfide bond and co-assembled with the photosensitizer chlorin e6（Ce6）to construct the carrier-free nanomedicine with redox stimulus responsiveness（BA-s-s/Ce6 NPs）which is applied to conduct chemo-photodynamic combination therapy for breast cancer in vivo and in vitro.The main research details and results of the paper are shown below:Betulinic acid（BA）and 3,3’-dithiodipropionic acid（DPA）synthesized betulinic acid derivatives（BA-s-s）with the disulfide bond through an esterification reaction.The reaction conditions were the molar ratio of BA:DPA:DCC:DMAP=1:4:1.5:2.5,stirring in the ice bath for 24 h.Normal phase silica gel column chromatography,infrared spectroscopy and nuclear magnetic resonance spectroscopy were used to separate and purify BA-s-s and identify its structure.The method of co-assembly of BA-s-s and Ce6 into nanoparticles（BA-s-s/Ce6 NPs）was the co-precipitation method by SEM and DLS.The ratio of BA-s-s: Ce6 was 4:1 and the concentration was 33.6 mmol/L.The morphology,basic parameters and intermolecular interaction force of BA-s-s/Ce6 NPs were determined by SEM,DLS and UV-vis.The BA-s-s/Ce6 NPs which were the best dispersing spherical particles with a diameter of 211.5 nm could be obtained,and the co-assembled structure was formed by π-π stacking and hydrophobic interaction.The release rate of Ce6 in vitro was calculated by dialysis,and it was found that the drug release of BA-s-s/Ce6 NPs showed redox responsiveness to glutathione（GSH）.The release rate of Ce6 reached 51.83% within 2 hours and 88.83% within 24 hours.BA-s-s/Ce6 NPs could form a colloidal system in distilled water,RPMI-1640 medium and PBS,having good solubility and dispersibility.Using DPBF as the probe,the ability of BA-s-s/Ce6 NPs to produce ¹O₂ in water was measured after irradiation.The results showed that the yield of ¹O₂ was 0.30,which was higher than that of free Ce6.It was observed by the fluorescence inverted microscope that 4T1 cells could fully endocytose BA-s-s/Ce6 NPs within 1 h.Through the MTT experiment,it was found that the BA-s-s/Ce6 NPs combination treatment group made the tumor suppression effect better than the monotherapy through the synergistic effect.When the concentration of Ce6 was 4 μg/m L,the inhibition rate of 4T1 cells could reach 97.62%,and the IC50 value was 0.663 μg/m L.A BALB/c mouse model bearing 4T1 cells was established,and it was found by fluorescence imaging that BA-s-s/Ce6 NPs had better tumor targeting than free Ce6,and the fluorescence was the strongest in the tumor at 0.5 h and 6 h after administration.The examination of the changes in body weight,blood biochemical indexes and organ indexes of mice revealed that the body weight of the mice declined during the administration,but the body weight increased after the administration.There was no significant change in blood biochemical indexes and organ indexes,indicating BA-s-s/Ce6 NPs combination treatment had no significant effect on various organs of mice and had lower toxicity.The tumor volume growth rate of the BA-s-s/Ce6 NPs combination treatment group was the slowest,and the tumor suppression rate reached 66.94%.The treatment effect was better than the monotherapy with 25.99% for the chemotherapy of BA-s-s/Ce6 NPs and 33.96% for the photodynamic therapy.Therefore,a new redox-responsive carrier-free nanomedicine system is established by co-assembling BA-s-s and Ce6 in this study,which highlights the good 4T1 tumor suppressive ability of natural small molecule betulinic acid with redox stimulus responsiveness in chemo-photodynamic combination therapy.