Construction Of Nanozyme Catalytic System That Responds To The Tumor Microenvironment For Enhanced Cancer Sonodynamic Therapy

Cancer has always been a difficult problem in human medical research.Hepatic carcinoma is a malignant tumor with high morbidity and mortality.Most patients have developed into the middle and late stages when they are discovered due to the lack of effective clinical early diagnosis methods.Traditional treatment methods such as surgery and liver transplantation can’t be used for treatment.Sonodynamic therapy（SDT）is a new type of treatment in which the sonosensitizer converts O₂into reactive oxygen species（ROS）under ultrasonic radiation to kill tumor cells.It is non-invasive,low toxic and side effects,and accurate treatment of tumors,which has attracted much attention.At present,SDT has shown great potential in tumor treatment.However,the hypoxia problem of the tumor microenvironment greatly limits the effect of SDT.Therefore,there is an urgent need to develop new strategies to alleviate the hypoxia in the tumor site and increase the efficiency of SDT.In this paper,we combined gold nanoparticles（Au NPs）with glucose oxidase activity and prussian blue nanoparticles（PBNPs）with catalase activity to design a composite nanoenzyme cascade catalytic system that can respond in the tumor microenvironment to solve the problem of hypoxia and insufficient endogenous H₂O₂.Au NPs can specifically catalyze the production of gluconic acid and H₂O₂from glucose in tumor tissues,cutting off the energy supply to the tumor site,and the generated H₂O₂is then catalyzed to produce O₂by PBNPs.Under ultrasound radiation,the O₂produced is converted into highly cytotoxic ROS by the sonosensitizer on the one hand,and on the other hand,it accelerates the consumption of glucose by Au NPs,which improves the therapeutic effect of sonodynamics and realizes the purpose of tumor starvation treatment.The main research contents and results are as follows:1.Construction and characterization of nanoenzyme catalytic delivery systemFirst,mercaptoethylamine was used as the bridging molecule of hyaluronic acid and nanocarrier to synthesize thiolated hyaluronic acid（HA-SH）,and its structure was confirmed by FT-IR and¹H NMR.The graft ratio of-SH to HA repeating unit is21.8%..Then,the mesoporous PBNPs protected by PVP were synthesized by hydrothermal method,and HAu Cl₄was reduced by Na BH₄and deposited in situ on PBNPs to prepare Au/PB composite nano-carriers.After physically encapsulating the sonosensitizer Ce6,HA-SH was modified onto Au/PB composite nanocarriers by Au-S,which was used as blocking agent and targeting agent,and finally Au/PB-Ce6-HA was obtained.The UV-Vis and XPS of the carrier indicate the successful preparation of Au/PB NPs.The nanoparticle size analyzer detected that the hydrated particle size of the preparation was 152±3.14 nm,and the potential was-32.7±1.27 m V.TEM showed that Au/PB-Ce6-HA presented an irregular cubic structure with a size of 70 nm.The UV-Vis of the preparation indicated that Ce6 was successfully loaded into Au/PB NPs,and the drug loading rate and encapsulation rate were 46.4%and 86.8%,respectively.The enzyme activity test shows that Au/PB-Ce6-HA has good oxygen production capacity and glucose mimic enzyme activity.2.In vitro anti-tumor activity of nanoenzyme-catalyzed delivery systemHuman hepatocellular carcinoma Hep G2 cells were used as an in vitro experimental research model.Fluorescence microscope and flow cytometer were used to investigate the cellular uptake of the nanozyme-catalyzed delivery system.The fluorescence images showed that the fluorescence intensity of the Au/PB-Ce6-HA group at 4 h was significantly stronger than that of the Ce6 group and the Au/PB-Ce6group.The uptake rate of Hep G2 cells to the preparation group was up to 89.5%measured by cytometer,indicating that HA played a tumor-targeting effect.The MTT method was used to determine the survival rate of cells in each group with or without ultrasound.After 1 HMz,1 W/cm²ultrasound irradiation for 1 min,the cell survival rate of Ce6 group（100μg/m L）was 47%,while that of preparation group was only18%.The DCFH-DA fluorescent probe was used to evaluate the generation of ROS in Hep G2 cells.Compared with the Ce6 group,Au/PB-Ce6-HA group showed obvious green fluorescence under ultrasonic irradiation,indicating that the Au/PB nanozyme carrier could effectively increase the intracellular ROS generation.3.In vivo anti-tumor activity of nanoenzyme-catalyzed delivery systemH22 tumor-bearing mices were used as in vivo experimental research models.The tumor targeting and distribution of the preparation in vivo were investigated by animal imaging instrument.The results showed that Au/PB-Ce6-HA could significantly increase the accumulation of the drug in the tumor site,and the tumor tissue still retains strong fluorescence at 24 h.According to the curve of relative tumor volume over time in mice,Au/PB-Ce6-HA group could significantly inhibit tumor growth under ultrasonic irradiation compared with other administration groups.This is nanozymes play a catalytic role in the tumor microenvironment to produce the result of starvation therapy synergistically enhancing SDT.The monitoring of the changes in body weight of the mices during the treatment and the H&E staining results of the main organs of each group showed that SDT is less harmful to other tissues,and Au/PB-Ce6-HA has good biological safety.In summary,the self-produced oxygen-enhanced nano-catalyst Au/PB-Ce6-HA has excellent tumor targeting ability.Two nanozymes with different catalytic properties make full use of the tumor microenvironment to produce H₂O₂and O₂in situ while consuming glucose,which significantly improves the effect of sonodynamics and realizes the synergistic treatment of tumor starvation therapy and SDT.It will have a good application prospect in the field of tumor treatment.