Nanoscaled Red Blood Cells Combine Photothermal/Photodynamic Therapy And Chemotherapy To Synergetically Treat Breast Cancer

Background and Objective:Breast cancer is one of the malignant tumors which seriously threatens women’s health.Currently,the main treatments for breast cancer include surgery,chemotherapy,radiotherapy,endocrine therapy and immunotherapy.However,conventional treatments have their own shortcomings,thus it is urgent to develop new strategies to treat breast cancer.Recently,phototherapy has been widely studied for cancer treatments,which includes two major types,photothermal therapy(PTT)and photodynamic therapy(PDT).In PTT,the photosensitizer converts the light energy of near-infrared laser into heat energy which directly causes thermal death of tumor cells.In PDT,the photosensitizer interacts with oxygen to generate a large amount of reactive oxygen species(ROS)that can directly killing tumor cells and indirectly regulate tumor microenvironment.In this study,we designed and constructed a simple and biomimetic nanocarrier system called nanoscaled red blood cells(DIRAs).Indocyanine green(ICG)and doxorubicin(DOX)were firstly complexed in aqueous solution containing ammonia bicarbonate(ABC)via hydrophobic interaction and π-π conjugation,and then wraped by red blood cells(RBCs)through repeatable extrusion to form DIRAs.Next,we investigated the loading efficiencies of DIRAs for ICG,DOX and gas generating agent ABC,thermal-responsive burst release of DOX,and PDT efficiency improved by hemoglobin(Hb)-based oxygen.Besides,the synergistic effects of PTT/PDT and chemotherapy on breast cancer and the related mechanisms were also studied both in vitro and in vivo.Methods:1.The ICG/DOX nanocomplexes were synthesized by complexation of ICG and DOX in ABC solution under sonication.Then,DIRAs were prepared by extruding RBCs with these ICG/DOX nanocomplexes repeatedly.The morphology,diameter,zeta potential,in vitro stability of DIRAs were analyzed with transmission electron microscopy(TEM),dynamic light scattering(DLS),respectively.Whole protein mapping was used to analyze their protein characteristics.The retention effiency of Hb in DIRAs was assessed by a Hb concentration detector.2.The temperatures of the DIRAs solution during laser irradiation were monitored by an infrared thermal imaging camera.The thermal-responsive gas production performance of ABC was studied by thermogravimetric analysis and gel experiment.The morphology of DIRAs after laser irradiation was observed by the TEM.Dynamic dialysis method and ultra-high performance liquid chromatography were used to evaluate the in vitro thermal-responsive release behavior of DOX.3.Ultraviolet spectrometry was used to detect the oxygen consumption of Hb protein during laser irradiation.The SOSG was used as a fluorescent probe to detect the production of singlet oxygen in DIRAs solution under laser irradiation.4.The killing and long-term inhibitory effects of various treatments on breast cancer 4T1 cells were evaluated using the CCK-8 assay.The intracellular distributions of ICG and DOX,the production of ROS,the mitochondrial damage,and the subcellular location of cytochrome C(Cyt c)in the cells with various treatments were observed by the confocal microscopy.The flow cytometry was used to quantitatively detect the cellualr uptake of DOX,the intracellulae generation of ROS,and the cell apoptosis in the treated cells.The Live/Dead Cell Staining Kit was conducted to visually monitor the killing efficiencies of different treatments on 4T1 cells.Besides,the expressions of proteins in caspase-dependent mitochondrial apoptosis pathway were detected by the western blotting technique.5.The 4T1 xenografted breast cancer mouse model was established to investigate the tumor-retention,PTT and PDT efficiencies of DIRAs in vivo.The tumor-bearing mice were intratumorally injected with PBS solution,free ICG and DIRAs,and then some of these mice were treated with local tumor laser irradation.Next,the tumor-retention efficiencies of ICG were monitored by the in vivo imaging technique,the tumor temperature changes upon laser irradiation were detected by an infrared thermal imaging camera,and the generations of singlet oxygen in tumor tissues were assessed after laser irradiation using the SOSG assay and cyosections observation.6.4T1 tumor-bearing mice were treated with normal saline,free DOX,free ICG ICG/RAs mixture and DIRAs combined with and without laser irradiation through intatumoral injection.During the treatment period,the tumor growth and reoccurrence were detected by measuring the tumor size.After treatments,the histopathological changes of tumor tissues and tumor metastases were observed using hematoxylin & eosin(H&E)staining and the tumor metastases in major organs were quantatively detected using the bioluminescence and the in vivo imaging technique.Moreover,the survival of these mice was recorded during a continuous 100-day observation period.7.Healthy mice were treated with normal saline,RBCs,RAs and DIRAs via administration of tail vein injection,and afterwards the body weights of these mice were recorded every 2 days.The blood routine blood routine parameters of the mice were examined by a blood analyzer.The flow cytometry was used to detect the proportions of myeloid-derived suppressor cells(MDSCs)in the spleen tissues.The liver and kidney function parameters of thsese mice were quantitatively assessed by the enzyme linked immunosorbent assay(ELISA).The histopathological damages of the major organs were observed using the H&E staining method.Results:1.The prepared DIRAs had a regular spherical morphology and an average size around 100 nm(PDI=0.203).The membrane of RBCs was clearly observed on the surfaces of the ICG/DOX nanocomplexes.The zeta potential of DIRAs was-21.6 mV that was almost similar to that of RBCs membrane.DIRAs retained approximately 52.7% of Hb in RBCs and no protein changes were observed in the whole protein mapping between DIRAs and RBCs.2.DIRAs exhibited a strong PTT effciciency in vitro with a maximum temperature up to 60°C during laser irradiation.ABC was decomposed with the temperature-resposive effect and triggered the obvious production of gas bubbles on the gels.The release profiles of DOX from DIRAs were increased by 40% and 30% separately in pH 6.5 and pH 7.4 PBS solutions at 24 h after pretreated laser irradiation.The obvious disintegration and drug release of DIRAs were observed by the TEM.3.Upon laser irradiation,the oxygen carried by Hb in DIRAs was significantly consumed due to the PDT activity of ICG and consequently the production of singlet oxygen was increased notably.4.DIRAs with laser irradiation showed strong killing efficacy and significantly induced the apoptosis of 4T1 cells.After laser irradiation,the cells located in and outside the laser spot were both efficiently killed by DIRAs as compared to free ICG.Furthermore,DIRAs were effectively uptaken by 4T1 cells,and then DOX was successfully released to enter the cell nucleus.The cellular uptake rate of DOX was increased by about 30% after laser irradiation.5.Compared with the free ICG,the ICG/RAs mixture and DIRAs increased the intracellular generation of ROS to a higher level and more seriously damaged the mitochondrial membrane after laser irradiation.The more amount of Cyt c was observed to release from the mitochondrial to the cytoplasm,and moreover the expression levels of cleaved caspase 9 and 3 poteins were significantly enhanced in the cells treated with ICG/RAs mixture and DIRAs with laser irradiation.These results revealed that the mitochondrial apoptotic pathway was activated to a large extent by the PDT effect of DIRAs.6.Compared to free ICG,DIRAs significantly prolonged the retention time of ICG in the tumors in 4T1 tumor-bearing mice even after laser irradiation,improved the temperature rise,and induced the generation of more amount of singlet oxygen,thus demonstrating their stronger PTT/PDT effiencies in vivo.7.After treatment of DIRAs with laser irradiation,the tumors in the mice were almost completely ablated,and the tumor recurrence and metastasis were notably inhibited as compared to the other tratments.80% of the mice were survived even at 100 th day after starting treatment and the survival time of the mice was greatly prolonged by DIRAs-medaied combination therapy.8.The blood parameters,MDSCs,body weights,liver and kidney functions in healthy mice with treatments of RBCs,RAs and DIRAs showed showed no differences as compared with the control mice.And furthermore,no detectable pathological changes or injuries were observed in the major organs of these mice.These results indicated that DIRAs had an excellent biocompability.Conslusion:In this study,we successfully prepared a nanoscaled red blood cells system DIRAs,which characterized with heat-responsive drug controlled release and oxygen-carrying enhanced PDT effect.DIRAs exhibited excellent biocompatibility and synergistic effects of PTT/PDT and chemotherapy on breast cancer both in vitro and in vivo,and successfully prevented breast cancer recurrence and metastasis.In summary,our study provides the theoretical basis and data support for developing a new strategy for clinical breast cancer treatment.