Investigation On The Construction Of Copper-based Metal Organic Framework Nano-drug System For Anti-tumor Therapy

In recent years,studies have shown that the alcohol-abuse drug disulfiram（Disulfiram,DSF）approved by the U.S.Food and Drug Administration has potential anti-tumor activity.The dithiocarbamate（Dithiocarbamate,DTC）of DSF partially can chelate divalent metal ions,especially copper ions(Copper ions,Cu²⁺),to form the DSF/Cu complex.According to reports,the complex can strongly bind to nucleoprotein location 4（Nuclear protein localization 4,NPL4）and induce its aggregation,causing severe damage to the p97-NPL4-ubiquitin fusion degradation protein 1（Ubiquitin fusion degradation protein 1,UFD1）pathway,leading to the accumulation of the wrong protein in tumor cells and cell death.Considering that the anti-cancer activity of DSF significantly depends on the concentration of Cu²⁺,and the endogenous biodistribution of Cu²⁺is very small,it can be considered to use the Cu²⁺supplementation strategy to maximize the effect of DSF.However,this systemic copper administration may cause an imbalance in the body’s copper content and lead to heavy metal poisoning in cancer patients who are not lacking in copper.In addition,low water solubility,poor stability and rapid metabolism in the body hinder DSF application in the body.Therefore,the co-delivery of DSF and Cu²⁺to the tumor site and simultaneous release to generate DSF/Cu complex in situ is a potential strategy to improve the efficacy of DSF chemotherapy.In this study,the metal organic framework（Metal-organic framework,MOF）composed of Cu²⁺and 2-methylimidazole（2-Methylimidazole,2-MI）was used as the carrier.During the formation of the MOF,DSF is encapsulated in its cavity structure,and then the surface is wrapped with a layer of hyaluronic acid（Hyaluronic acid,HA）by electrostatically adsorbed,and finally DSF@HA/Cu-MOF nanoparticles（DSF@HA/Cu-MOF NPs）are obtained.This nano drug delivery system has the ability to actively target the tumor site due to the presence of HA,and can remain stable to avoid degradation during the blood circulation process.Once it reaches the tumor site,it will be taken up by tumor cells through endocytosis mediated by the CD44 receptor on the surface of tumor cells and degraded by lysosomal hyaluronidase（Hyaluronidase,HAase）and low p H conditions.Eventually,DSF and Cu²⁺are released simultaneously in the intracellular tumor microenvironment,and the DSF/Cu complex is formed,which will bind to the NPL4 target in the p97-NPL4-UFD1 pathway of tumor cells,thereby ultimately inducing cell death.DSF@Cu-MOF and DSF@HA/Cu-MOF were characterized by transmission electron microscope（Transmission electron microscope,TEM）,scanning electron microscope（Scanning electron microscope,SEM）,UV-Visible spectrum（UV-Visible spectrum,UV-Vis）and nano laser particle size analyzer.The results confirmed that DSF@Cu-MOF was successfully synthesized with an average particle size of～300nm,infrared absorption visible light spectrum（Fourier transform infrared resonance,FT-IR）and energy dispersive spectrometer（Energy dispersive spectrometer,EDS）confirm the successful loading of DSF,and the UV spectrophotometer determined that the encapsulation rate and drug loading rate of DSF were 86.67%and 54.17%,respectively.DSF@HA/Cu-MOF was investigated by TEM,nano laser particle size analyzer and other methods,and the results showed that the HA-coated nanoparticles were uniform in size,with a particle size of～300 nm and a potential of-25.4±1.5m V.The results of in vitro preparation degradation and drug release showed that HA/Cu-MOF nano drug delivery system has strong acid responsiveness and HAase sensitivity with the ability to release DSF and Cu²⁺from tumor sites and form DSF/Cu complexes in situ.It also proves that HA has a good protective effect on DSF@HA/Cu-MOF.The mouse breast cancer cells 4T1 overexpressing the CD44 receptor were used as the model.The SRB method proved that DSF@HA/Cu-MOF has a strong killing effect on tumor cells,and its IC₅₀ value is the lowest in each experimental group,indicating that the preparation has the best inhibitory effect on cell proliferation,while the same dose of blank carrier HA/Cu-MOF is almost non-toxic to cells,which proves that the nano-preparation has good safety.The uptake and distribution of the preparation by the cells were observed by confocal laser microscope and flow cytometer.The results show that DSF@HA/Cu-MOF can quickly enter the cell through the endocytosis pathway mediated by the over-expressed CD44 receptor on the surface of 4T1 cells and achieve lysosomal escape through the proton sponge effect and gradually release the loaded drug,thereby exerting anti-tumor effect.4T1 tumor-bearing mice were used as the model,the in vivo tumor targeting and pharmacodynamics of DSF@HA/Cu-MOF were further investigated.The results of X-ray and fluorescence images of mice collected by the small animal live imaging instrument show that the HA/Cu-MOF labeled with IR783 has a good targeting and retention effect at the tumor site.The content of DSF/Cu complex in mouse tumor tissues at different times after administration was detected by HPLC.The results showed that the intratumoral concentration of DSF/Cu complex in DSF@HA/Cu-MOF group was prolonged with the administration time.It rises significantly and reaches the maximum value～12 h after administration,which fully confirms that the preparation can degrade at the tumor site in vivo and form the DSF/Cu complex with anti-tumor effect.Pharmacodynamic experiments showed that the tumor inhibition rate of DSF@HA/Cu-MOF reached 86%,which was significantly higher than that of DSF/Cu complex（52%）and DSF@Cu-MOF（61%）.The results of H&E staining and TUNEL staining showed that the tumor cells in the preparation group had a large area of plasmolysis,necrosis,and obvious apoptosis.In addition,the body weight of the mice in each group did not change significantly during the treatment period,and the liver and kidney function indicators were all within a reasonable range,but the histomorphological results of the main organs showed that the free Cu Cl₂,the DSF/Cu complex,and DSF@Cu-MOF groups have certain cardiotoxicity due to the presence and leakage of Cu²⁺.At the same time,all groups except the preparation group have a certain degree of alveolar collapse.In contrast,all results of the DSF@HA/Cu-MOF drug delivery system are normal,and it has excellent biological safety in cancer treatment.In summary,the DSF@HA/Cu-MOF nano drug delivery system has the characteristics of good tumor targeting and biological safety.It can achieve the fixed-point release of DSF and Cu²⁺,and produces anti-tumor effects by generating DSF/Cu complex in situ,which has broad application potential in the future tumor treatment.