Study Of ZIF-90 Deliver Functional Nucleic Acid Cooperate With Photodynamic Therapy For Breast Cancer

Breast cancer is one of the most common cancers in the world.According to existing statistics,breast cancer accounts for 11.7%of new cancer cases.Chemotherapy is currently the main method of treating cancer,but it still has serious side effects,drug resistance and other issues that need to be resolved urgently.Therefore,the design and development of new cancer diagnosis and treatment methods have become the main research content of cancer treatment.Photodynamic therapy（PDT）irradiates the tumor site with a specific wavelength to activate the photosensitive drug that selectively accumulates in the tumor tissue to generate reactive oxygen species（ROS）,which triggers a photochemical reaction to destroy the tumor.Compared with traditional tumor therapy,PDT has low toxicity,low-invasiveness and many other advantages.Gene therapy is another new tumor treatment method besides conventional therapy.Among them,gene silencing therapy is the commonly used tumor gene therapy method.Antisense oligonucleotides are currently the most widely used nucleic acid drugs in clinical research.They can bind to target m RNA,inhibit its transcription and translation,and thereby weaken or inhibit protein expression.DNAzyme is an effective anti-cancer factor,which can effectively catalyze the cleavage of oncogene substrates to inhibit various tumorigenic processes.However,nucleic acid is negatively charged and repels the cell membrane,making it difficult to enter the cell.Therefore,a carrier is required to load it into the cell.The zeolite imidazole frameworks（ZIFs）structural material is a kind of metal organic frameworks（MOFs）.As a new type of drug delivery carrier,it has the advantages of porosity,large specific surface area and good biocompatibility.In this study,using the high loading capacity of the ZIF series to load DOX and functional nucleic acids into cells,two delivery systems based on ZIF-90combined with PDT were designed.The main research contents and results are as follows:（1）A drug delivery system based on ZIF-90 co-delivery of doxorubicin（DOX）and photosensitizer modified antisense oligonucleotide G3139 was designed and constructed.First,synthesize and characterize ZIF-90 nanoparticles,and explored the reasonable use range of the material.Then,the delivery system based on ZIF-90 was constructed and characterized,and its drug loading capacity was determined to be 19.2%and drug release capacity was 78%.The inhibition of tumor cells by the drug-loading system is determined by the MTT.The experimental results showed that when ZIF-90（180μg/m L）synergistically deliver low-dose DOX（0.4μg/m L）and photosensitizer-modified G3139（60 n M）,the inhibition rate of cancer cells reached 42%.After combined with PDT,the inhibition rate of cancer cells reached 51%.At the same time,the uptake of ZIF-90 delivery system by tumor cells was studied by laser confocal;secondly,it was confirmed by Western Blot（WB）that G3139 could down-regulate the expression of anti-apoptotic protein BCL-2,and it was dose-dependent with the concentration of G3139;finally,ROS kit was used to explore the ROS production of Ce6-G3139 by detecting the green fluorescence produced after irradiation at a specific wavelength of 660 nm.The results showed that the intensity of the green fluorescence produced and the concentration of Ce6-G3139 were also dose-dependent.（2）DOX has systemic toxicity and tends to have adverse effects on normal tissues.Therefore,a gene therapy system based on ZIF-90 loaded with antisense nucleic acid drugs（G3139）and nuclease（DNAzyme）combined with PDT was designed and constructed.The system down-regulates the expression of corresponding proteins by silencing BCL-2 and cutting EGR-1,respectively.Among them,the clever application of Zn²⁺produced by the acid decomposition of ZIF-90 promotes the cleavage of DNAzyme and achieves an effective anti-tumor effect without the use of DOX.First,constructed and characterized the gene therapy system;determined the reasonable range of its nucleic acid loading by fluorescence quenching.The release of nucleic acid in a weakly acidic environment was simulated in vitro by dialysis,and its released capacity was 81%.The inhibition of tumor cells by the gene therapy system was determined by the MTT.The experimental results showed that the inhibition rate of tumor cells reached 45%when ZIF-90（180μg/m L）loaded with 60 n M Ce6-G3139 and 200 n M Ce6-DNAzyme.After combined with PDT,the inhibition rate of tumor cells increased by 20%to65%,showing a good anti-tumor effect.At the same time,the uptake of tumor cells into the gene therapy system was studied by laser confocal.The results showed that by loading on ZIF-90,nucleic acid could be quickly and effectively delivered into MCF-7 cells and released.Then,it was verified by PAGE that the Zn²⁺produced by ZIF-90 nanoparticles after acid decomposition could assist DNAzyme to cleave EGR-1 in vitro.In addition,it was confirmed by WB that DNAzyme could down-regulate the expression of EGR-1 protein,and it was dose-dependent with the concentration of DNAzyme.Finally,the ROS kit was used to detect the green fluorescence generated after irradiation at a specific wavelength of 660 nm,which proved Ce6-G3139 and Ce6-DNAzyme can produce cytotoxic ROS.In summary,the two multifunctional delivery systems were designed and constructed in this study both exhibiting good anti-tumor effects and providing a new type of collaborative treatment strategy for achieving more efficient cancer treatment.