Preparation Of Celastrol Loaded Fe₃O₄@MnO₂-Ce6/CSL Nanoprobe And Its Application In Diagnosis And Treatment Of Liver Neoplasms

Objective:To realize the early diagnosis and treatment of liver neoplasms,In this paper,an intelligent nano drug delivery platform,Fe₃O₄@MnO₂-Ce6/CSL,which integrates imaging and therapeutic functions,was prepared.Through the study of the characteristics of this nano drug delivery platform and its anti liver cancer efficacy,it provided a preliminary theoretical research for the combination of diagnosis and treatment of liver cancer.Methods:Firstly,a layer of MnO₂ was grown on the surface of Fe₃O₄ nanoparticles and then Ce6 and CSL were loaded on by electrostatic adsorption.Finally,the cancer therapostic nanoplatform（Fe₃O₄@MnO₂-Ce6/CSL）was prepared.The morphology and size of the nanoplatform were characterized by TEM and DLS.The components were identified by UV/fluorescence spectra and X-ray photoelectron spectroscopy（XPS）.The specific UV absorption of celastrol and Ce6 were used to calculate the drug loading rate and drug release rate in different p H values.The oxygen production and singlet oxygen production efficiency in different p H values were also measured.MRI was used to obtain the imaging of the nanoplatform in different p H values and calculate its r₁ and r₂ relaxation rate.In this study,Bel-7402 cells were used as the research object.The uptake and distribution of Fe₃O₄@MnO₂-Ce6/CSL in Bel-7402 cells were investigated by laser confocal microscopy.The cytotoxicity of Fe₃O₄@MnO₂-Ce6/CSL to Bel-7402 cells was studied by CCK-8,Flow cytometry and Calcein-AM/PI staining.The 3D Multicellular tumor spheroid（MCTS）with Bel-7402 cell was constructed to simulate solid tumor and assess its infiltration in solid tumor.In animal experiment,we constructed a nude mouse subcutaneous tumor transplantation model,and then MRI and NIRF were used to monitor the distribution and metabolism of this nanoplatform in vivo in real time.The oxygen deficiency in the solid tumor of the model mice was evaluated by photoacoustic imaging（PA）.Finally,the mice were irradiated by 633 laser to evaluate the antitumor activity in vivo.Results:1.The TEM showed that this study had successfully synthesized a multi-functional nano drug delievery system with uniform particle size——Fe₃O₄@MnO₂-Ce6/CSL.The nano platform exihibited good stability in aqueous solution,its hydrated particle size is 150 nm,and the zeta potential is+26 m V.The UV and fluorescence spectra,high performance liquid chromatography（HPLC）and XPS experiments proved the successful synthesis of manganese dioxide and the successful loading of the drugs.The UV spectra showed that the loading rates of Ce6 and CSL were 11.9%and8%,respectively.Drug release experiments showed that the nanoplatform had p H and H₂O₂ dual sensitivity,which can promote the drug release induced by the tumor microenvironment.It can be inferred from the above experimental results that the nanoplatform has the property of drug release induced by tumor microenvironment.Oxygen detection experiment and and singlet oxygen（¹O₂）detection experiment revealed that the nanoprobe had the ability to catalyze H₂O₂ to produce oxygen and promote photosensitive drugs to produce more singlet oxygen.Therefore,when the nanoplatform is loaded with photodynamic drugs,it can enhanc PDT and improved the over acid and hypoxia environment of the tumor microenvironment.In vitro MRI experiments showed that Mn²⁺and Fe₃O₄ nanoparticles released from the nanoplatform under the stimulation of p H and H2O2 could be used for T₁/T₂ weighted MRI imaging.Moreover,the MRI relaxation rate of the nanoplatform increased significantly in p H=5.0 and H₂O₂.This part of the experiment showed that the multifunctional nanoplatform we synthesized had good dispersion stability,biodegradability and high drug loading rate.It can generate oxygen induced by p H/H₂O₂ to promote photodynamic therapy,and has good magnetic resonance imaging performance,which lays a foundation for further cell experiment in vitro.2.The cellular uptake experiments showed that Bel-7402 cells had a high uptake rate of the nanojprobe,reaching the maximum uptake at 8 h,and the nanoprobe were distributed in the cytoplasm after uptaken.The CCK-8 experiments proved the single nano carrier Fe₃O₄@MnO₂ had negligible affect to the activity of Bel-7402 cell.However,when the concentration of Ce6 reached 8ug/ml,the cell survival rate decreased significantly after incubating with Fe₃O₄@MnO₂-Ce6/CSL nanoparticle and irradiating with 633nm laser（cell survival rate was 15.5%）.Compared with single photodynamic therapy or chemotherapy,the combination of photodynamic therapy and chemotherapy has obvious inhibitory effect on tumor cells.3D MCTS experiment results showed that the small molecular substances produced by the decomposition of the nanoplatform induced by the TME could penetrate into the solid tumor very well.These results showed that the penetrating efficacy of the nanoplatform to the 3D MCTS reached the maximum peak after the cell spheres were incubated with the nanoplatform for 8 h,which provided a reference for our subsequent treatment in vivo.3.The in vivo MRI and fluorescence imaging of mice showed that the nanoparticles were first deliveried to the tumor due to the EPR effect,triggering the degradation of nanocarriers in the tumor area and releasing drug molecules.Finally,after 633nm laser irradiation,compared with the growth rate of tumor volume in the control group and single drug treatment group,the nanomaterial group growed slowly.At the end of treatment,TUNEL staining showed that there was obvious apoptosis in the tumor site in the nanodrug group.The above experiments confirmed that Fe₃O₄@MnO₂-Ce6/CSL could promote tumor cell apoptosis and inhibit the tumor growth to a certain extent.The photoacoustic imaging showed that the oxygen content in tumor area increased significantly,which suggested that Fe₃O₄@MnO₂-Ce6/CSL could improve the hypoxia in tumor area.No obvious pathological changes were found in other organs（heart,liver,spleen,lung and kidney）,indicating that this nanoplatform has no obvious toxicity to other organs and has good biological safety.Conclusion:1.In this experiment,the Fe₃O₄@MnO₂-Ce6/CSL nanoprobe was successfully synthesized by a simple two-step method.The nanoprobe had characteristics of good dispersion stability,uniform particle size distribution,high drug loading rate,and could be degraded under different p H values and different concentrations of H₂O₂,which could promote drug release and produce oxygen and singlet oxygen.After degradation,it had better magnetic resonance imaging effect.2.The Fe₃O₄@MnO₂-Ce6/CSL had good targeting to Bel-7402 cells and good permeability to 3D cell sphere model.Under the irradiation of 633 nm laser,the combined photodynamic/chemotherapy effect of the nanoprobe on tumor cells was better than that of single therapy.3.When entering the tumor tissue,the drug release could be explained by the p H and H₂O₂,which could realize the specific release of drug molecules in the tumor site.At the same time,it had good in vivo magnetic resonance imaging and fluorescence imaging performance,which could realize the integration of diagnosis and treatment of hepatocellular carcinoma.