Investigation On A Mitochondrial Temperature Induced Free Radical Burst Drug Delivery System With Stepwise Targeting Function

Breast cancer is currently the highest incidence of cancer in women.Traditional surgical treatment,radiotherapy and chemotherapy can easily bring serious psychological and physical harm to patients.Photodynamic therapy and sonodynamic therapy based on free radicals are limited in clinical application because of the hypoxic environment of tumors and the necessity of using physical stimulation.Therefore,how to construct an efficient and low toxicity therapeutic method without oxygen and physical stimulation is very important to improve the treatment effect of breast cancer.Among many free radicals,besides singlet oxygen,hydroxyl radical and superoxide anion radical,alkyl radicals also have the anti-tumor activity,which can be generated without oxygen and only by thermal stimulation.The high temperature characteristics of mitochondria provide a new idea for the application of alkyl radicals in cancer therapy.Based on this,we constructed a mitochondrial temperature induced free radical burst drug delivery system with stepwise targeting function?AIPH/MSN-TPP@Lipo/DTX-FA?,which has the following advantages:?1?2,2'-azobis[2-?2-imidazolin-2-yl?propane]dihydrochloride?AIPH?could rapidly decompose under the thermal stimulation to produce cytotoxic alkyl radicals,which is independent of oxygen;?2?The generation of alkyl radicals was trigged by utilizing the organelle's own characteristics,overcoming the limitation of physical stimulus responsive therapy strategies based on the free radicals;?3?Combining two drugs with different mechanisms in the treatment of tumors:docetaxel?DTX?promoted the AIPH-induced mitochondrial apoptotic pathway by down-regulating the expression of Bcl-2 anti-apoptotic protein;?4?The step-by-step targeting nanoparticles have two targeted groups:folic acid?FA?and triphenylphosphine?TPP?,which were helpful to realize the targeted release of DTX and AIPH,to maximize their antitumor effects and to reduce the side effects of the drugs.Specific studies are as follows:1.The preparation and characterization of AIPH/MSN-TPP@Lipo/DTX-FAFirstly,the decomposition of AIPH at different temperatures,different pH and different physiological conditions were investigated by detecting the generation of alkyl radicals.Then,mesoporous silica nanoparticles?MSNs?were prepared by sol-gel method and modified by the mitochondria-targeted group?MSN-TPP?,AIPH was loaded into the MSN-TPP?AIPH/MSN-TPP?.Finally,the FA-targeted pH-sensitive lipid films containing DTX were prepared by thin-film dispersion method,AIPH/MSN-TPP@Lipo/DTX-FA were formed by self-assembly after the addition of AIPH/MSN-TPP into the liposomes during the hydration process.In the experiment,we found that the AIPH had both temperature and acid sensitivity,and the decomposition process of AIPH was time-dependent.The experimental results of N₂ adsorption-desorption determination,fourier transform infrared?FT-IR?and transmission electron microscope?TEM?showed that the products in every stage during the synthesis process of nanoparticles were prepared successfully.The results of particle size distribution and zeta potential suggested that the average particle size of the prepared AIPH/MSN-TPP@Lipo/DTX-FA was 87.8±3.1 nm,and the zeta potential was-19.3±1.7 mV.The in vitro release experiment showed that AIPH/MSN-TPP@Lipo/DTX-FA had the characteristic of pH-sensitive release,which can prevent DTX and AIPH from leaking before reaching the tumor site.2.The anti-tumor efficiency of AIPH/MSN-TPP@Lipo/DTX-FA in vitroThe MCF-7 cells were used to investigate the in vitro anti-tumor activity of AIPH/MSN-TPP@Lipo/DTX-FA.Cell uptake analysis showed that the step-by-step targeting drug delivery system had the good targeting ability to MCF-7 cells.The results of cytotoxicity experiments suggested that the prepared carrier system was safe,AIPH and DTX had the synergistic anti-tumor effect,and AIPH/MSN-TPP@Lipo/DTX-FA could effectively inhibit the growth of MCF-7 cells.The fluorescence imaging of lysosomes and the co-localization imaging of mitochondria showed that AIPH/MSN-TPP@Lipo/DTX-FA could escape from lysosome and accumulate effectively in mitochondria.The detection of intracellular alkyl radicals,the detection of mitochondrial membrane potential,the release of cytochrome C and the single cell gel electrophoresis showed that AIPH/MSN-TPP@Lipo/DTX-FA could produce alkyl radicals in MCF-7 cells and damage the mitochondria,causing the decrease of mitochondrial membrane potential,leading to the release of cytochrome C and other substances into the cytoplasm,resulting in the damage to DNA.The results of cell cycle and apoptosis experiments showed that AIPH/MSN-TPP@Lipo/DTX-FA could block cell cycle at S phase and G2/M phase,which played an effective anti-tumor role by inducing cell apoptosis.The anti-tumor mechanism of DTX and AIPH was verified by Western blotting.These results suggested that AIPH and DTX had the synergistic anti-tumor effects,and the AIPH/MSN-TPP@Lipo/DTX-FA could effectively activate the mitochondrial apoptosis pathway,resulting in the growth inhibition of cancer cells.3.The anti-tumor efficiency of AIPH/MSN-TPP@Lipo/DTX-FA in vivoIn order to evaluate the pharmacodynamics and pathological characteristics of AIPH/MSN-TPP@Lipo/DTX-FA delivery system,the BALB/c nude mice were used as the animal models to observe the distribution of step-by-step targeting drug delivery system in nude mice,the changes of relative tumor volume,the changes of relative body weight,the result of H&E staining and TUNEL apoptosis staining.The in vivo distribution suggested the prepared drug delivery system could accumulate at the tumor site quickly and persistently.Pharmacodynamic and pathological analysis results showed that the step-by-step targeting drug delivery system had good biocompatibility,and AIPH/MSN-TPP@Lipo/DTX-FA could inhibit the growth of tumors by inducing apoptosis/necrosis of tumor cells.The above results indicated that AIPH/MSN-TPP@Lipo/DTX-FA had the good targeting ability and inhibition effect on tumor in mice.