Research On New Indocyanine Green/atovaquone Carrier-free Nanodrug In Enhancement Of Photothermal Therapy For Breast Cancer

Photothermal therapy is a new tumor treatment method,which kills tumor cells by using light sources such as near-infrared(NIR)light to irradiating photothermal agents in tumor areas to convert light energy into thermal energy.It is developing rapidly in the field of cancer treatment because of short treatment time,high efficiency,low toxicity,minimally invasive and other advantages.The heat tolerance of tumor is a key issue affecting the photothermal therapy effect,mainly due to the rapid increase in the expression of heat shock proteins(HSPs)during photothermal therapy,which improves the heat tolerance of cells and reduces the thermal damage of photothermal therapy to tumor cells,thereby reducing the effect of photothermal therapy.The expression of HSPs relies on the energy supply of adenosine triphosphate(ATP),and inhibition of ATP synthesis can lead to downregulation of HSPs expression.Mitochondria are an important source of energy supply within cells,and interference with mitochondrial oxidative phosphorylation can inhibit ATP synthesis,thereby reducing intracellular ATP levels.Therefore,inhibition of mitochondrial oxidative phosphorylation in tumor cells can limit the supply of ATP in cells,thereby inhibiting the expression of HSPs,which is an effective way to reverse tumor heat tolerance in the process of photothermal therapy,enhancing the sensitivity of tumor cells to photothermal therapy and enhance the effect of tumor photothermal therapy.Based on the above relevant research,this project designed a carrier-free nanodrug(IR820/ATO NPs)self-assembled by photothermal molecule new indocyanine green(IR820)and oxidative phosphorylation inhibitor atovaquone(ATO),which was used to limit the synthesis of intracellular ATP by inhibiting mitochondrial oxidative phosphorylation and downregulate the expression of HSPs to reduce the thermal tolerance of tumor cells and enhance photothermal therapy.The main research contents,methods and conclusions are as follows:First,the in vitro basal properties of IR820/ATO NPs were characterized.The composition of IR820/ATO NPs was analyzed by ultraviolet-visible spectrophotometry(UV-Vis),and the results showed that the nanodrug successfully loaded two drug components.The microstructure and stability of IR820/ATO NPs were characterized by transmission electron microscopy(TEM)and dynamic light scattering(DLS),and the results showed that IR820/ATO NPs were uniform,well-dispersed,spherical nanoparticles with an average size of 112.6 nm,zeta potential of-26.0 mV and good stability.Photothermal heating experiment proved that IR820/ATO NPs showed good heating effect under the irradiation of in vitro laser.The results of the hemolysis test showed that the hemolysis rate of IR820/ATO NPs is less than 5%,which indicated that it had good blood compatibility.Secondly,the 4T1 mouse breast cancer cells were used to characterize the cell-level antitumor effect of IR820/ATO NPs.Uptake experiments showed that the cellular uptake efficiency of 4T1 cells in the IR820/ATO NPs group was higher than that of single drug IR820,which proved that this nanomedicine could enter cells more effectively.Intracellular ATP content detection and western blot experiments of HSPs demonstrated that the nanodrug could inhibit the synthesis of ATP to reduce the level of intracellular ATP,thereby reducing the expression levels of HSP70 and HSP90.MTT experiments and apoptosis experiments proved that the nanodrug under the condition of laser irradiation achieved excellent tumor photothermal therapy effect,which could effectively inhibit the growth of 4T1 tumor cells and significantly induce apoptosis.Finally,the female BALB/c mice with breast tumors(4T1 cells)were used to characterize the animal-level antitumor effects of IR820/ATO NPs.In vivo distribution experiments in mice proved that the nanodrug can be effectively accumulated at the tumor site.In vivo photothermal heating experiment further proved that the IR820/ATO NPs at the tumor site could achieve good photothermal heating effect under the condition of laser irradiation.Tumor suppression experiments proved that the tumor growth inhibition effect of mice in the IR820/ATO NPs group was the best,and good anti-tumor treatment effect could be achieved.Histological analysis showed that IR820/ATO NPs treatment could down-regulate the expression of HSPs in tumor sites during photothermal therapy with low toxicity and side effects.In summary,this project constructs a carrier-free self-assembled nanodrug that combines photothermal therapy with mitochondrial oxidative phosphorylation energy metabolism,which can inhibit ATP synthesis in cells,thereby reversing the thermal tolerance of tumor cells in the process of photothermal therapy.And this strategy shows high anti-tumor effect and low toxic side effects,which provides a new design idea for the application of oxidative phosphorylated drugs to adjuvant therapy of photothermal therapy.