A Nano-delivery System Based On Converting The Degree Of Autophagy For Enhanced Tumor Starvation Therapy

Starvation therapy is a mature tumor treatment method which starves tumor cells by cutting off the supply of nutrients.However,mild protective autophagy can be induced during starvation treatment,which weakens the therapeutic effect of starvation therapy.If the degree of autophagy can be adjusted to overactivated stage,it can not only enhance the efficacy of starvation therapy,but also cause autophagic cell death,thus achieving optimal antitumor effects.Based on that,the Cur@MOF-GOx/HA nano-delivery system that converted the degree of autophagy was constructed in this paper.Hyaluronic acid（HA）could target to tumor cells by specifically recognizing highly expressed CD44 receptors on the surface of tumor.Glucose oxidase（GOx）catalyzed glucose into H₂O₂ and gluconic acids,which not only caused tumor starvation,but also provided reactant for the metal-organic framework（MOF）-mediated Fenton reaction,inducing chemo-dynamic therapy（CDT）,and generating highly toxic·OH.Most importantly,mildly protective autophagy caused by starvation could be converted into severe destructive autophagy by curcumin（Cur）,realizing augmented anticancer effects of tumor starvation therapy.The main research of this paper included following aspects:1.Synthesis and characterization of Cur@MOF-GOx/HA nano-delivery systemFirst,the MOF nanoparticles were synthesized by hydrothermal method.And the results of infrared spectrophotometer,X-ray photoelectron spectroscopy analysis and thermogravimetric analysis proved the successful synthesis of MOF.The nitrogen adsorption-desorption isotherm of the MOF results showed that the surface area of the MOF was approximately 26.4±0.5 m²/g and the average pore diameter was approximately 7.6±0.2 nm,allowing enzyme modification and Cur loading.Then,GOx was fixed to the surface of MOF through amide bonds,and the successful fixing of GOx was characterized by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The loading rate of GOx was about 52.6±4.3μg/mg measured by BCA protein quantification kit.Finally,Cur was loaded in the mesopores of MOF,and further decorated with HA to obtain the Cur@MOF-GOx/HA nano-delivery system.Particle size analysis and TEM image results showed that the Cur@MOF-GOx/HA nano-delivery system was bipyramidal hexagonal prism structure with a particle size of about 220.7±2.3 nm.The loading rate of Cur was about 20.5±0.6%,and the encapsulation rate was about 76.8±2.3%.2.In vitro properties of the Cur@MOF-GOx/HA nano-delivery systemFirst,different chromogenic agents showed that the MOF had peroxide-like activity and could catalyze the formation of·OH from hydrogen peroxide.Then,gluconic acid generation,hydrogen peroxide generation and glucose consumption experiments proved that GOx could still maintain its activity after fixing to MOF.In addition,the in vitro release behavior of Cur was investigated by dialysis,and the results showed that the Cur@MOF-GOx/HA nano-delivery system could release Cur in response to the acidic environment of tumor cells,and the cumulative release rate at72 h could reach 82.5±3.1%.Finally,stability experiments preliminarily showed that the nano-delivery system had good stability in both PBS solution and cell culture medium.In vitro hemolysis experiments showed that the nano-delivery system had good biocompatibility.3.In vitro antitumor activity study of Cur@MOF-GOx/HA nano-delivery systemIn vitro antitumor study was performed on breast cancer 4T1 cells.First,the uptake of the nano-delivery system was investigated by laser confocal scanning microscope and flow cytometry.The results showed that the nano-delivery system entered to tumor cells through CD44 receptor-mediated endocytosis,and the uptake rate reached 74.9±2.3%in 6 h.Then,the sulforhodamine B method was used to investigate the toxicity of different nanoparticles to 4T1 cells,and the results showed that MOF-HA had good safety to normal cells.However,MOF-HA had a certain inhibitory effect to tumor cells because of the toxic·OH generated by MOF-mediate Fenton reaction.The Cur@MOF-GOx/HA nano-delivery system could effectively kill4T1 cells.The ROS and H₂O₂ detection results demonstrated that the Cur@MOF-GOx/HA nano-delivery system was able to induce CDT and generate ROS in tumor cells.In addition,western blotting assays indicated that the nano-delivery system induces apoptosis through a mitochondria-dependent pathway.Finally,the degree of autophagy in tumor cells was investigated by western blotting assay,immunofluorescence assay,transmission electron microscopy and Ad-m Cherry-GFP-LC3B double fluorescence detection.The results showed that the nano-delivery system could induce severe autophagy in tumor cells.4.In vivo antitumor activity study of Cur@MOF-GOx/HA nano-delivery systemThe in vivo antitumor activity study was carried out in tumor-bearing BALB/c female mouse.First,in vivo fluorescence imaging experiments showed that the Cur@MOF-GOx/HA nano-delivery system has good tumor targeting ability in mice.Then,after 14 days of treatment,the results of H&E staining and relative volume of tumor showed that the Cur@MOF-GOx/HA nano-delivery system had good ability to inhibit tumor growth,and the tumor inhibition rate reached 70±2.3%.In addition,frozen section assay showed that the Cur@MOF-GOx/HA nano-delivery system could generate a large amount of ROS in tumor cells.The results of immunofluorescence and transmission electron microscopy showed that the nano-delivery system could induce severe autophagy in tumor cells.Finally,the results of mouse body weight change,histopathological section,blood routine and blood biochemistry preliminarily indicated that the nano-delivery system have good biosafety and biocompatibility.The above studies showed that the nano-delivery system successfully constructed in this paper could convert mildly protective autophagy induced by starvation therapy into severe destructive autophagy,enhancing the therapeutic effect of tumor starvation.At the same time,a large amount of ROS was generated in tumor cells through the CDT,which synergistically inhibited the growth of tumor cells.