Construction Of Glycogen Nanoparticles Based On In-Situ Cascade Reaction For Antitumor Research

Chemodynamic therapy（CDT）is a new method of cancer treatment,which uses Fenton or Fenton-like reactions to convert hydrogen peroxide（H₂O₂）into more toxic hydroxyl radicals（·OH）to induce tumor cell apoptosisa.The choice of the Fenton reaction catalyst is particularly important.Hemin（He）is formed by the iron（II）center complexed with protoporphyrin,which can be used to simulate peroxidase.It has the advantages of high thermal stability and low cost compared with catalase.However,the tumor microenvironment（TME）insufficient endogenous hydrogen peroxide（50-100μM）,the resistance of reducing substances and the weak acidity of solid tumors（p H 6.0-7.0）also seriously affect the efficacy of CDT.In order to overcome the above shortcomings,this study selected Glycogen-NH₂（GN）nanoparticles with good biocompatibility and biodegradability to synergistically delivery of hemin and glucose oxidase（GOx）.GOx catalyzed the decomposition of glucose to produce a large amount of H₂O₂ and gluconic acid at the tumor,which provided substrates and favorable reaction conditions for the Fenton reaction,and enhanced the generation efficiency of·OH.In addition,the abnormally rapid proliferation of tumor cells requires adequate nutrition and energy support,so changing the metabolic pathway of tumors is also an important means of cancer treatment.GOx catalyzes the degradation of intracellular glucose and also causes tumor cells to not obtain enough energy to maintain their rapid growth,which also makes GOx a potential drug for the treatment of tumor starvation.In order to protect the activity of GOx and enhance the uptake of drug-loaded nanoparticles by tumor cells,hyaluronic acid（HA）targeted by the CD44 receptor was selected as the nanoparticles shell.Finally,a GN/He/GOx/HA tumor targeted delivery system was constructed to achieve synergistic anti-tumor therapy.（1）Construction and characterization of GN/He/GOx/HA nanoparticlesAminated glycogen was synthesized with a particle size of about 80 nm,and its structure and degree of amination were confirmed by nuclear magnetic,infrared and acid-base titration.The optimal drug loading ratio of Hemin,GOx,HA was screened by particle size,stability,drug loading and flow cytometry,and the loading efficiency of Hemin and GOx was verified.The GOx enzyme activity was tested by monitoring the p H change and the H₂O₂ produced of the solution.The results of electron spin resonance spectrum,ultraviolet absorption spectrum and fluorescence absorption spectrum proved that Hemin not only had catalase-like activity,but also had a cascade catalytic reaction with GOx.In addition,the results of protein adsorption experiments showed that nanoparticles coated with HA had better biocompatibility and stability.The most important thing was that glycogen had good stability in the blood circulation,but can be degraded byα-glycosidase in the lysosome to fast release the drugs.The above results showed that glycogen had a great application prospect in the delivery of macromolecular drugs.（2）Evaluation of antitumor effect of GN/He/GOx/HA nanoparticles in vivo and in vitroThe anti-tumor activity of GN/GOx/HA and GN/He/GOx/HA was evaluated in cytology and anti-tumor activity in vivo.The cell survival rates of blank carriers based on aminated glycogen was above 80%,indicating that they had excellent biological safety..Compared with normal cells,tumor cells need a lot of glucose to provide energy to maintain their abnormal proliferation,otherwise it will lead to starvation and death.The cytoxicity of drug-loaded nanoparticles on cells was glucose concentration dependent.The cytoxicity of GN/GOx/HA to normal cells was significantly less than that of Hela cells with high CD44 receptor expression,and GN/He/GOx/HA showed stronger cytoxicity than GN/GOx/HA on Hela cells.The results of flow cytometry and CLSM showed that GN/FITC-GOx/HA had CD44 receptor targeting and was endocytosed by clathrin-mediated endocytosis.After entering the cell,the nanoparticles can escape through the lysosome and release the drug.Moreover,GN/He/GOx/HA nanoparticles produced more ROS in tumor cells,which induced decrease of mitochondrial membrane potential and apoptosis of tumor cells.The in vivo safety evaluation showed that the drug-loaded nanoparticles had good safety,biocompatibility,and eminent anti-tumor effect.In summary,the above results indicated that the tumor-targeted drug delivery system based on the in-situ cascade catalytic reaction had great application prospects and was expected to be applied in clinical anti-tumor therapy.