Development Of An Intelligent Controlled-release For Diagnosis And Treatment Based On Nano-manganous Oxide And Preliminary Application

With the development of science and technology and the improvement of medical treatment,more and more people pay attention to tumor targeting therapy.The development of nano-carriers has opened a new door for the study of anti-tumor drug delivery systems,which solves the deficiencies of anti-tumor drugs in the water solubility and stability and improves the selectivity of drugs to tumor targets.The design of intelligent nano-carriers can also realize the specific response of drug delivery system in the tumor microenvironment,to reduce the toxic side effects and effectively enhance the anti-tumor effect of chemotherapy drugs.Artemisinin?ART?as a kind of Fe²⁺dependent anti-tumor drug,reacts with Fe²⁺,leading to the breaking of oxygen bridge and the production of reactive oxygen species?ROS?,which can kill tumor cells.while the poor water solubility and the large dosage required for treatment of ART derivatives greatly limit their later clinical application.In this paper,hollow mesoporous manganese trioxide?Mn₂O₃?was used to load ART to solve the above problems.Because the hollow mesoporous structure of Mn₂O₃ may lead to drug leakage in vivo delivery process,and as a foreign substance,Mn₂O₃ is easy to be recognized,swallowed and cleared by macrophages in vivo.Therfore,Mn₂O₃-ART was encapsulated by homologous erythrocyte membrane?RBCM?.On the one hand,the mesoporous surface of Mn₂O₃was blocked to avoid the leakage of drugs in vivo;on the other hand,erythrocyte membrane was used as endogenous substance,which can reduce or even avoid the phagocytosis of the immune system in vivo to realize the long circulation in vivo,and deliver more drugs to tumor sites.In addition,in order to increase the accumulation of the drug delivery system in the tumor site and achieve the effective uptake of tumor cells,we used TKD peptide to modify the surface of RBCm-Mn₂O₃-ART nanoparticles.The peptide was identified by heat shock protein?HSPs?over-expressed on the surface of breast cancer MCF-7 cells.Therefore,the modified drug delivery system can realize the active targeting of tumor by ligand-receptor pathway.In vitro characterization results showed that the particle size of TKD@RBCm-Mn₂O₃-ART was 403.4±15nm,the potential was-23.7±2.7eV,proving its good stability.Then we studied the reaction of Mn₂O₃ in tumor microenvironment.TEM results showed that the structure of carrier was disintegrated with time and produced a large number of Mn²⁺in vitro simulated tumor microenvironment,the particle size was about 50nm and 6nm after 1h and 4h reaction,respectively.The results of magnetic resonance imaging showed that Mn₂O₃ generated Mn²⁺in tumor microenvironment,and the relaxation rate was6.3113 mM^-1S^-1,exhibiting a good imaging capability.The results of drug release experiments showed that the cumulative drug release rate of 48h was 50.72%in the environment of PBS with pH=7.4,and the cumulative drug release rate of 48h was97.5%in the environment with pH=7.4 C_GSH=5mM.While the cumulative release rate of RBCm-Mn₂O₃-ART was 91.44%,indicating that tumor microenvironment promoted the release of drugs,and the encapsulation of erythrocyte membrane had no obvious effect on drug release.Human breast cancer MCF-7 cells were used as model in vitro.The inhibition rate showed that Mn₂O₃ had no obvious cytotoxicity.After loading ART,the anti-tumor effect of ART was significantly enhanced.The cell uptake experiment results showed that the encapsulation of erythrocyte membrane could reduce the phagocytosis of Mn₂O₃ by macrophages.The further modification of target polypeptide TKD significantly enhanced the ability of RBCm-Mn₂O₃-FITC to recognize cancer cells,reduced the uptake of normal cells and targeted tumor more efficiently.The cell confocal experimental results showed that TKD@RBCm-Mn₂O₃entered the lysosome first after entering the tumor cells,resulting in the dilatation of lysosomes,which was prolonged with time.The carrier moved towards the nucleus and then embedded into the nucleus.ROS assay,Calcein-AM/PI apoptosis assay,cell cycle assay and comet assay results showed that the cell cycle arrest effect of TKD@RBCm-Mn₂O₃-ART was not obvious.Which mainly induced apoptosis of tumor cells,DNA damage to achieve the purpose of killing tumor cells by producing a large amount of ROS.In vivo,nude mice bearing MCF-7 cells were selected as animal models.In vivo imaging results showed that the encapsulation of erythrocyte membrane could prolong the circulation time and decrease the metabolic rate in vivo.The modificationofTKDsignificantlyincreasedtheaccumulationof TKD@RBCm-Mn₂O₃-IR783 in tumor site.The results of tumor tissue osmotic experiment showed that the distance of drug permeation from tumor microvessel to deep tumor could be increased through the structural change of carrier in tumor tissue,and the uniform distribution of drug in tumor could be realized.HE pathological results of main tissues showed that the preparation had no obvious toxicity to normal tissues in vivo.According to the changes of tumor volume and HE staining of tumor tissue,compared with ART group,the tumor cells in the preparation group showed significant shrinkage and necrosis,and the tumor volume increased slowly,indicating that the nano-preparation could significantly improve the anti-tumor effect of ART in vivo.The results of magnetic resonance imaging?MRI?showed that TKD@RBCm-Mn₂O₃-ART can produce Mn²⁺by structural destruction in tumor microenvironment,which can be used as a contrast medium to realize the integration of diagnosis and treatment of tumor.There is a great prospect for development.