Study Of Ultrasound Responsive Self-oxygen Red Cell Carrier System For Dignosis And Treatment Of Cancer

Objective:Sufficient oxygen,ultrasound and sonosensitizer are the three necessary conditions for the treatment of sonodynamic therapy?SDT?.Lacking of oxygen in tumors can lead to an inadequate oxygen supply during SDT,which strengthens tumor resistance to sonodynamic efficacy.To overcome hypoxia in tumors and improve the treatment effection,we developed oxygen self-sufficient red blood cell?RBC?carrier system to decompose tumor endogenic H₂O₂ into O₂ and combine triplex cancer therapy:ferryl-hemoglobin?ferryl-Hb?,sonodynamic and chemical therapy.The physical and chemical properties,endothelial permeability,in vivo enhanced antitumor activity,MR imaging and pharmacokinetic characteristics were investigated systematically.Methods:?1?In this study,a hydrophilic sonosensitizer,together with a chemotherapy drug doxorubicin?DOX?,was encapsulated inside RBCs?DOX/Mn-TPPS@RBCs?.The composites were characterized by ultraviolet visible?UV-vis?,Optical and fluorescence microscope,flow cytometrya and 3 T MRI scaner.The catalase activity of DOX/Mn-TPPS@RBCs was detected by the Catalase Assay Kit.The hemoglobin?Hb?of DOX/Mn-TPPS@RBCs was quantified by a cyanide methemoglobin reagent kit.We have established high performance liquid chromatography?HPLC?to detect drug encapsulation efficiency and release rate.?2?In vivo pharmacokinetics and tissue distribution studies:SD rats were used as the model in vivo pharmacokinetic experiments.The plasma concentrations of DOX were determined by HPLC method after intravenous injection.Taking S180ascites tumor-bearing mice as an animal model,with IR783-labeled the drug devilery system,drug distribution in tissues of mice was determined using an in vivo imaging system.?3?Study of endothelial permeability and anti-tumor activity:The integrity of the human umbilical vein endothelial cell?HUVEC?monolayer in vitro was monitored by trans-endothelial electrical resistance?TEER?value during the experiments,which was typically above 200?cm².This value demonstrated that the cellular part was a monolaye.The ability of RBC carrier system to cross a reconstructed endothelial monolayer was studied in vitro using a trans-well technique.MCF-7 was used as the model in vitro anti-tumor activity.We used MTT method to detect effect of the system on cell proliferation inhibition efficiency.The uptake of the cells was observed by fluorescence microscopy and quantified by flow cytometry.Intracellular reactive oxygen species?ROS?generation was tested by using DCFH-DA fluorescent probe.?4?In vivo pharmacodynamics and MR imaging studies:The S180 tumor models were generated by subcutaneous injection of S180 cells into the right shoulder of female kunming?KM?mice.The synergistic therapy effect of chemotherapy,ROS and ferryl-Hb was studied by tumor volume.The toxicity of carrier system was analyzed by the body weight during treatment and HE staining after treatment.The magnetic imaging ability of carrier system was based on magnetic imaging efect of the carrier system in vivo.Results:DOX fluorescence was observed from fluorescence microscope images of DOX/Mn-TPPS@RBCs,suggesting that DOX were effectively encapsulated inside the RBCs,which was well consistent to our original design.Flow cytometry data further confirmed that most of DOX were loaded into RBCs.The loading of Mn-TPPS could be confirmed by UV-vis spectroscopy of the DOX/Mn-TPPS@RBCs,which showed the characteristic absorption of Mn-TPPS at around.MRI of DOX/Mn-TPPS@RBCs samples also evidenced that Mn-TPPS was successfully encapsulated into RBCs.The catalase activity was found to be around 9000?U/mL?,which was very high for DOX/Mn-TPPS@RBCs system.The value of ferryl-Hb dramatically increased to 60%with H₂O₂ and US.The results of in vitro drug release showed that the drug delivery system could release rapidly with H₂O₂ and US,and the release amount was up to 80%.The results of pharmacokinetic study showed that DOX/Mn-TPPS@RBCs could increase the retention time of DOX in vivo and slowed the clearance of the drug.The in vivo fluorescence signals of DOX/Mn-TPPS@RBCs at the tumor site could be detected after 6 h,and became strongest at 12 h post-injection,indicating DOX/Mn-TPPS@RBCs possessed efficient tumor accumulation.In addition,MR imaging in mice showed that DOX/Mn-TPPS@RBCs was able to pass through the tumor microvascular gap and act as a suitable T₁ contrast agent in MRI application.Pharmacodynamics in vivo results showed that DOX/Mn-TPPS@RBCs was able to combine ferryl-Hb,sonodynamic and chemical therapy,and significantly inhibit tumor growth.The result of endothelial permeability study suggested that the DOX/Mn-TPPS@RBCs could migrate efficiently across the blood vessels into the inflamed tumor.The results of cell cytotoxicity test showed that the engineered DOX/Mn-TPPS@RBCs after specific binding H₂O₂ could combine ferryl-Hb,sonodynamic and chemotherapy upon US irradiation and then induce effective cancer cell killing.The results of cellular uptake demonstrated that H₂O₂ and US-triggered drug release could greatly facilitate drug uptake by cancer cells.Conclusions:Overall,this drug delivery system is a promising therapeutic agent involving in situ production of oxygen inside the tumor,triplex therapy,and MRI.