| In recent years,the morbidity and mortality of malignant tumors have been increasing,which has become one of the important reasons that threaten human health.Free radical-based tumor therapy has been widely used in clinical practice.Free radicals mainly include peroxides,superoxides,hydroxyl radicals and singlet oxygen,etc.They can cause oxidative damage to lipids,proteins,and DNA,have strong cytotoxicity,and then induce apoptosis.Commonly used free radical treatments include radiotherapy(RT)and photodynamic therapy(PDT).Among them,photodynamic therapy(PDT)is widely used clinically for the treatment of various tumors due to its non-invasive treatment,high selectivity,and low toxicity and side effects.However,the hypoxic environment of tumors greatly reduces the generation of oxygen-dependent free radicals of PDT,and the highly resistant internal environment of tumor cells such as over-expressed glutathione(GSH),further weakens the efficiency of free radical treatment.However,for PDT,most photosensitizers currently have high phototoxicity and poor laser penetration ability,which limits their clinical application.Therefore,the construction of a therapeutic system that does not rely on oxygen and can break the reduction barrier of tumor cells is the current demand for efficient free radical therapy of tumors.Based on the above analysis,this subject constructed a spatiotemporal controllable free radical treatment system that can efficiently produce free radical in an hypoxic environment and down-regulate the intracellular reduction microenvironment.The system has the following characteristics:(1)the use of oxygen-independent alkyl groups to treat tumors,2,2’-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride(AIPH)can be quickly decomposed under thermal stimulation to generate alkyl radicals that have nothing to do with oxygen.It has high antitumor activity and achieves high-efficiency treatment in the hypoxic microenvironment of tumors;(2)Constructing both photothermal conversion characteristics and The "nanoactive carrier" that adsorbs intracellular GSH,while breaking the tumor reducing microenvironment,opens up the generation of alkyl radicals with spatial and temporal resolution.Hollow mesoporous copper sulfide nanoparticles(CuS)have super near-infrared absorption,high light-to-heat conversion efficiency,and high tissue penetration ability.They can efficiently load AIPH,reduce intracellular GSH levels,and break the tumor reduction barrier.Not only can it be used as a photothermal therapeutic agent under infrared light irradiation,it can also activate AIPH to generate alkyl radicals to achieve high space-time resolution and control free radical generation;(3)CuS can release a small amount of Cu2+ ions under near-infrared light irradiation conditions,and generate Fenton-like reaction to generate hydroxyl radicals,which enhances the effect of free radical treatment.(4)The surface of the drug-loading system is coated with albumin(BSA),which reduces the early leakage of AIPH,reduces toxic and side effects,and improve the biocompatibility.In short,the free radical treatment system has the ability to generate alkyl radicals by photothermal activation while breaking the tumor reduction barrier,and finally achieve efficient free radical cascade therapy for hypoxic tumors.The specific research content are as follows:1.Preparation and characterization of CuS/AIPH@BSA nanoparticlesHollow mesoporous copper sulfide nanoparticles were first synthesized by a template method.Transmission electron microscopy(TEM),X-ray diffraction(XRD),etc.proved that the hollow mesoporous copper sulfide was successfully prepared with a particle size of about 100 nm.Secondly,AIPH was successfully loaded into the nanoparticles by physical stirring,and its encapsulation efficiency was 21.53 ± 1.71%.Finally,the surface of CuS/AIPH was sealed with albumin(BSA)to obtain the CuS/AIPH@B S A free radical generator.The successful preparation of CuS/AIPH@BSA was confirmed by transmission electron microscopy and UV-visible spectroscopy,and it had good dispersibility.The final particle size measured by the nanometer particle size analyzer was 117.4±0.16 nm,and the potential was-23.2 ±1.5 mV.In addition,the properties of CuS/AIPH@BSA were investigated.First,by analyzing the temperature recorded by the infrared thermal imager,the result was proved that CuS has concentration,laser irradiation time,and power dependence.Electron spin resonance experiments and ABTS+· color reaction proved that photothermal can activate the generation of alkyl radicals.Finally,the GSH content measurement results also showed that the GSH content decreases with increasing CuS concentration.2.Antitumor activity of CuS/AIPH@BSA nanoparticles in vitroMouse breast cancer 4T1 cells were used as model cells to evaluate the antitumor activity of CuS/AIPH@BSA in vitro.In the cell uptake experiment,flow cytometry and laser confocal were used to investigate the uptake of CuS@BSA nanoparticles loaded with rhodamine B by 4T1 cells.The results showed that the CuS@BSA nanoparticles can effectively promote the uptake of AIPH compared with the free group,and the uptake efficiency is time-dependent.The detection of intracellular alkyl radicals showed that CuS/AIPH@BSA free radical generator can generate a large number of alkyl radicals in both normal and hypoxic conditions.At the same time,the intracellular GSH content measurement results showed that the nanoparticles reduced the intracellular GSH level to about 27%under laser irradiation.According to the results of cytotoxicity experiments,the survival rate of cells in the CuS/AIPH@BSA group was above 85%,while in the CuS/AIPH@BSA+NIR group,the survival rates of both normoxic and hypoxic cells were below 15%,which proved that the prepared carrier system has low toxicity,AIPH and CuS have a synergistic antitumor effect,and CuS/AIPH@BSA can effectively inhibit the growth and proliferation of 4T1 cells.According to the results of apoptosis experiments,CuS/AIPH@BSA can effectively induce the apoptosis of hypoxic cells under laser irradiation,with an apoptosis rate of up to 88%,to achieve efficient free radical treatment.The results of Western blot showed that regardless of the conditions of normoxia or hypoxia,the expression of y-H2AX in the cells of the CuS/AIPH@BSA+NIR treatment group increased significantly,and the DNA tailing phenomenon was obvious in the nucleus,indicating that the free radical generator has a strong nuclear DNA damage ability.3.Antitumor activity of CuS/AIPH@BSA nanoparticles in vivo4T1 cell tumor-bearing mice were used as animal models.By observing the distribution of the drug delivery system in mice,changes in relative tumor volume,body weight of mice in each administration group,H&E staining and TUNEL apoptosis staining of mice in each administration group evaluates the in vivo kinetic distribution and pharmacodynamic properties of the drug delivery system.The in vivo distribution results showed that CuS@BSA nanoparticles loaded by IR783 can effectively aggregate at the tumor site,and the fluorescence intensity of the tumor site reached its maximum at 6 h,which provided a basis for determining when to give laser irradiation.The relative tumor volume of mice after 14 days of treatment showed that compared with other groups,the relative tumor volume of the CuS/AIPH@BSA+NIR group had a tendency to decrease significantly.After treatment,the relative tumor volume became 1.5 times,and the tumor suppression rate was 67%;The results of free radical immunofluorescence staining of tumor tissue showed that compared with other groups,the free radicals in the tumor site of the CuS/AIPH@BSA+NIR group increased significantly by 3.32 times that of PBS group;H&E pathological section results showed that the main organs of mice in each group such as heart,liver,spleen,lung and kidney had no obvious pathological changes after treatment,indicating that the prepared free radical generator has good biocompatibility in vivo.Pathological analysis of tumor tissue showed that the tumor sections of mice treated with CuS/AIPH@BSA+NNIR showed a significant decrease in the number of cells and nuclear shrinkage;TUNEL apoptosis staining results also showed CuS/AIPH@BSA+NIR-treated mice had the highest degree of apoptosis.In this project,a space-time-controllable free radical therapy system that combines the efficient production of hypoxic environment and the down-regulation of the intracellular reduction microenvironment is established.This system has photothermal conversion characteristics,efficiently delivers AIPH,and reduces GSH levels to break intracellular barrier,control the generation of free radicals with high spatiotemporal resolution,and achieve efficient free radical cascade therapy for hypoxic tumors.The results of in vivo and in vitro treatments showed that the free radical generator has a strong induction effect on the apoptosis of hypoxic cancer cells and effectively inhibit tumor growth,which has broad application prospects in high-performance tumor treatment to overcome the hypoxic tumor environment,and provides a new idea for free radical treatment. |
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