| Multidrug resistance（MDR）continues to be the major limiting factor in the cure of patients with cancer.Studies have shown that tumor multidrug resistance may be closely related to the multidrug-resistant protein secreted by itself,such as breast cancer resistance protein（BCRP）.Therefore,inhibiting the production of multidrugresistant proteins through genetic intervention may reverse tumor drug resistance.Herein,we use layered double hydroxide nanoparticle（LDH）to co-deliver DOX and siRNA targeting BCRP RNA to reverse the resistance of MCF-7/ADR cells and enhance the anticancer activity of doxorubicin（DOX）.AimsWe used layered hydroxide nanoparticle（LDH）to co-deliver DOX and siRNA targeting BCRP RNA to investigate the effect of reversing drug resistance in MCF-7/ADR cells and enhancing the anticancer activity of doxorubicin（DOX）.Methods1.TEM detection of the basic structures of FeNi-LDH and DOX@FeNi-LDH nanoparticles.2.DLS measures all hydrodynamic diameters and PDI indices to determine if the nanoparticles diameter is within the nanoparticle range and if the dispersion is good.3.XRD detected whether FeNi-LDH and DOX@FeNi-LDH nanoparticles conformed to the hexagonal shape and whether FeNi-LDH was loaded with DOX.4.The absorption spectra of FeNi-LDH and DOX@FeNi-LDH nanoparticles were detected by FT-IR to determine if the preparation was successful.5.DSC detected whether LDH can protect nucleic acids and enzymes from being easily degraded under high heat.6.Due to the tumor-specific properties,whether LDH releases more DOX and siRNA under acidic conditions,the carrier is p H-dependent.7.Western blot was used to detect the difference in the expression levels of BCRP protein in MCF-7/ADR cells and MCF-7 cells,to observe whether the cause of drug resistance was due to BCRP protein and the following reversal experiments were performed with MCF-7/ADR cells8.The proportion of DOX@FeNi-LDH loaded with siRNA was detected by Western blot,which reduced the expression level of BCRP and reversed MDR.9.Flow cytometry results showed that siRNA-DOX@FeNi-LDH increased the uptake of siRNA by MCF-7/ADR cells and increased the concentration of siRNA in cells.10.Laser confocal results showed that siRNA-DOX@FeNi-LDH promoted the entry of siRNA into MCF-7/ADR cells,and the reversal effect was enhanced.11.The accumulation of intracellular DOX was detected by flow cytometry.The red-shift phenomenon appeared before and after the action of siRNA-DOX@FeNiLDH.The DOX in MCF-7/ADR cells increased significantly.12.LDH has no obvious intrinsic toxicity to MCF-7/ADR cells detected by MTT method,and LDH has high biocompatibility.13.MTT assay was used to detect the effect of siRNA-DOX@FeNi-LDH,and it was found that siRNA-DOX@FeNi-LDH had the best inhibitory effect on cells.14.The effect of siRNA-DOX@FeNi-LDH on apoptosis was detected by flow cytometry.The results showed that the apoptosis rate was significantly increased after siRNA-DOX@FeNi-LDH treatment.15.The effect of siRNA-DOX@FeNi-LDH on cell cycle was detected by flow cytometry.The results showed that the cycle arrest after siRNA-DOX@FeNi-LDH was obvious.16.The xenotransplantation of MCF-7/ADR cells in vitro was successful.17.After treatment with PBS,DOX,DOX@FeNi-LDH,siRNA-DOX@FeNiLDH,it was found that the tumor volume was the smallest after siRNA-DOX@FeNiLDH treatment.18.After removing the heart,liver,spleen,lung,and kidney from the treated mice,HE is staining showed that the heart and kidney tissues of the mice treated with siRNA-DOX@FeNi-LDH were normal,which alleviated the side effects of DOX.19.In vivo fluorescence detection of the heart,liver,spleen,lung,and kidney of mice showed that DOX accumulated in the tumor after siRNA-DOX@FeNi-LDH treatment enhanced the fluorescence and accumulated significantly more.Results1.TEM images showed that FeNi-LDH and DOX@FeNi-LDH nanoparticles had a uniform hexagonal layered structure2.DLS measurement of all hydrodynamic diameters and PDI indices of FeNiLDH,DOX@FeNi-LDH and DOX@FeNi-LDH nanoparticles.With a positive charge,its particle size is below 200 nm,which meets the requirements of nanoparticles.3.The XRD results showed that the interlayer strength of FeNi-LDH increased slightly,indicating that DOX was successfully loaded.4.The absorption spectrum of DOX@FeNi-LDH nanoparticle was detected by FT-IR,and a DOX peak appeared in the results,which confirmed the successful loading of DOX in FeNi-LDH.5.The results of DSC experiments showed that LDH successfully loaded DOX and siRNA,and LDH had the potential to protect drugs and nucleic acids from thermal and enzymatic damage.6.The release of DOX and siRNA in LDH was p H-dependent,and drugs and nucleic acids were more easily released in an acidic environment.7.Western blot detected the expression level of BCRP protein in MCF-7/ADR cells and MCF-7 cells.8.The proportion of DOX@FeNi-LDH loaded with siRNA was detected by western blot,which reduced the expression level of BCRP and reversed MDR.9.Flow cytometry results showed that siRNA-DOX@FeNi-LDH increased the uptake of siRNA by MCF-7/ADR cells and increased the concentration of siRNA in cells.10.Laser confocal results showed that siRNA-DOX@FeNi-LDH promoted the entry of siRNA into MCF-7/ADR cells,and the reversal effect was enhanced.11.The accumulation of intracellular ADR was detected by flow cytometry.The red-shift phenomenon appeared before and after the action of siRNA-DOX@FeNiLDH.The DOX in MCF-7/ADR cells increased significantly.12.LDH had no obvious intrinsic toxicity to MCF-7/ADR cells detected by MTT method,and LDH had high biocompatibility.13.MTT assay was used to detect the effect of siRNA-DOX@FeNi-LDH,and it was found that siRNA-DOX@FeNi-LDH had the best inhibitory effect on cells.14.The effect of siRNA-DOX@FeNi-LDH on apoptosis was detected by flow cytometry.The results showed that the apoptosis rate was significantly increased after siRNA-DOX@FeNi-LDH treatment.15.The effect of siRNA-DOX@FeNi-LDH on cell cycle was detected by flow cytometry.The results showed that the cycle arrest after siRNA-DOX@FeNi-LDH was obvious.16.The xenotransplantation of MCF-7/ADR cells in vitro was successful.17.After treatment with PBS,DOX,DOX@FeNi-LDH,siRNA-DOX@FeNiLDH,it was found that the tumor volume was the smallest after siRNA-DOX@FeNiLDH treatment.18.After removing the heart,liver,spleen,lung and kidney from the treated mice,HE is staining showed that the heart and kidney tissues of the mice treated with siRNA-DOX@FeNi-LDH were normal,which alleviated the side effects of DOX.19.In vivo fluorescence detection of the heart,liver,spleen,lung,and kidney of mice showed that DOX accumulated in the tumor after siRNA-DOX@FeNi-LDH treatment enhanced the fluorescence and accumulated significantly more.ConclusionsIn conclusion,DOX is encapsulated between the nanoparticles,which have the characteristics of high drug loading and strong controlled-release,and siRNA is tightly adsorbed on the surface of the nanoparticles through electrostatic action to avoid the degradation of siRNA.Moreover,FeNi-LDH shows excellent ability to deliver siRNA and DOX into cells and enhance the internalization of siRNA and DOX.The experiment further confirm that siRNA-DOX@FeNi-LDH（1:1）shows an effective gene silencing effect and down-regulated the expression of BCRP.In vitro experiments show that siRNA-DOX@FeNi-LDH inhibits cell proliferation by affecting the cell cycle and inducing apoptosis.In in vivo experiments,MCF-7/ADR xenotransplantation therapy also reveals that siRNA-DOX@FeNi-LDH has a significant antitumor effect.We believe that this work has great potential for reversing the drug resistance of breast cancer and improving the effect of chemotherapy. |
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