Study On PH/H₂O₂-responsive Oxygen-Producing Nanosystem For Dignosis And Treatment Of Cancer

Objective:Hypoxia and vascular proliferation at the tumor site are the main causes of multidrug resistance?MDR?and cancer metastasis.In this study,an intelligent controlled-release delivery system?FA-BSA-MnO₂/DOX/siRNA?with oxygen production was constructed.The physicochemical properties,the reversal of chemotherapeutic resistance in vitro,anti-tumor metastasis in vivo and in vitro,and magnetic resonance imaging?MRI?characteristics were systematically studied.Methods:?1?Synthesis and characterization of FA-BSA-MnO₂/DOX/siRNA:bovine serum albumin?BSA?was used as a carrier to biosynthesize Mn²⁺under alkaline conditions to prepare oxygen-producing carrier BSA-MnO₂.The gene drug small interfering RNA?siRNA?and chemotherapeutic drug doxorubicin?DOX?were encapsulated in the carrier by desolvent cross-linking method,and finally folic acid?FA?was modified on the surface of carrier by the amide bond.A multifunctional FA-BSA-MnO₂/DOX/siRNA drug delivery system was constructed.The optical properties and chemical structure of nanocomposites were successfully characterized through ultraviolet-visible?UV-vis?,fourier transform infrared spectroscopy?FT-IR?and X-ray photoelectron spectroscopy?XPS?.The morphology and particle size of the nanocomposites were observed by transmission electron microscopy?TEM?and dynamic light scattering?DLS?.A portable dissolved oxygen analyzer was used to detect the oxygen generation characteristics of nanocomposites.The MRI characteristics of nanocomposites were detected by 3T MRI scanner.The serum stability of siRNA was investigated by gel electrophoresis.?2?In vitro anti-chemotherapy resistance study:Human breast cancer cell line?MCF-7/ADR?resistant to DOX was selected as the experimental cell line.Fluorescence microscopy and flow cytometry were used to observe and quantify the uptake of MCF-7/ADR cells to system.The inhibitory effect of system on the growth of MCF-7/ADR cells under normoxic or hypoxic conditions MTT assay.Western blot was performed to examine the effects of system on the expression of resistance-related protein.?3?In vitro anti-metastasis study:Murine breast cancer cells?4T1?were selected as experimental cell line.Cellular uptake and cell inhibition rate of nanocomposites were investigated.The effects of system on migration and invasion of cells were examination by scratch and transwell experiments.?4?In vivo distribution,imaging and pharmacodynamics study:The 4T1tumor-bearing mice were selected as animal models.The tissue distribution of nanocomposites was investigated by in vivo fluorescence imaging.The MRI characteristics of nanocomposites were studied by 3T MRI scanner.By observing tumor volume changes,mouse body weight and HE pathological sections,to investigate the inhibitory effect of system on tumors in situ.The effect of system on the expression of metastasis-associated proteins in tumor tissues was analyzed by western blot experiments;metastatic lung nodules were observed to evaluate the tumor metastasis inhibitory activity of the delivery system.Results:The results of UV-vis,FT-IR and TEM showed that FA-BSA-MnO₂/DOX/siRNA nanoparticles were successfully synthesized.It was spherical and had a particle size of about 80 nm.The encapsulation efficiency of DOX could reach 94%.Nanocomposites could rapidly generate large amounts of O₂and had good MRI characteristics.Gel electrophoresis experiments showed that the siRNA could be stably present in the serum.DOX was released at pH 5.0 and 100?M H₂O₂,and the cumulative release rate reached 86.3%.When MCF-7/ADR cells were used as a model,cell uptake rate to nanocomplexes was higher than that of free DOX.The cell inhibition rate experimental results showed that nanosystem could significantly inhibit the growth of cancer cells by chemotherapy combined with gene therapy.The results of western blot further demonstrated that the system significantly down-regulated the expression of HIF-1?,P-gp and VEGF,indicating that the delivery system could improve the therapeutic effect of drug-resistant tumor cells.In vitro anti-metastatic study,cellular uptake experiments demonstrated that the system increases the uptake of the drug by the cells.MTT assay showed that the inhibition rate of system on 4T1 cells was 83.9±1.3%.Compared with the control group,there was a significant difference?P<0.05?.Scratch and transwell experiments showed that nanocomposites could effectively prevent the migration and invasion of cancer cells.In vivo imaging results showed that the fluorescence signal at the tumor site was the strongest and reached the maximum at 12 h.MRI results showed that the nanocomposites had better T₁ nuclear magnetic imaging properties.In vivo studies have shown that the delivery system has strong tumor inhibition and anti-tumor metastasis effects.Conclusion:In this study,the prepared FA-BSA-MnO₂/DOX/siRNA drug delivery system has the characteristics of pH/H₂O₂ responsive drug release.By increasing oxygen content and vascular inhibition strategies,it could be used to reverse MDR and prevent cancer metastasis.