| Liver cancer is one of the most common malignant tumors in China,accounting for 9.42%of the new malignant tumors.At present,surgical resection or ablation treatment is still the first choice for the treatment of Ⅰ a liver cancer,but due to the concealment of early tumor,it is difficult to achieve early detection,early diagnosis and early treatment of liver cancer.For middle and advanced HCC,the recurrence rate of 5 years after surgical resection is as high as 40%-70%,and the 5-year survival rate is low.For middle and advanced HCC that cannot be surgically removed,the treatment of transhepatic artery chemoembolization,ablation,fofox4 systemic chemotherapy and solafini molecular targeted drug therapy,external radiotherapy or 125I interparticle tissue radiotherapy,immunotherapy can be selected.Among them,chemotherapy is one of the important treatment methods for liver cancer.However,the limitation of traditional small molecule chemotherapeutic drugs,such as side effects of chemotherapy drugs,limits the effective dosage of tumor treatment.The chemotherapy escape of tumor stem cells,and the formation of hypoxia and acid toxicity and interstitial high pressure in tumor tissue microring,the drugs cannot effectively penetrate into the tumor and other reasons lead to poor chemotherapy effect of liver cancer.Therefore,the development of an integrated drug loading system with active targeting,efficient tumor cell killing,low toxicity and side effects,real-time monitoring of drug distribution,metabolism and efficacy,with diagnostic functions,has become a current research hotspotFor the above requirements,the first chapter of this project designs and prepares a diagnosis and treatment integrated multifunctional hybrid mesoporous silicon nanoparticles(FA-Gd2O3@MSN-DOX),which with HCC drug targeting delivery,enhanced MRI contrast,and cancer cell microenvironment controllable drug release The FA-Gd2O3@MSN-DOX was characterized using transmission electron microscopy,scanning electron microscopy,x-ray diffractometer,fully automatic microporous physical adsorption and chemical adsorption analyzer,nuclear magnetic resonance spectroscopy,fourier transform infrared spectroscopy,thermogravimetric analyzer,fluorescence spectrophotometer,particle analyzer,inductively coupled plasma emission spectrometer,and dual beam uv/vis spectrophotometer.The synthesized FA-Gd2O3@MSN-DOX is spherical with an average particle size of about 50 nm,a specific surface area of 862.2 m2/g.The mesoporous is orderly,the pore diameter is about 3.8 nm,a loading of DOX about 4.2 wt%and the content of the loaded Gd2O3 nanoparticles on the mesoporous wall is 2.6 wt%.In a weakly acidic solution,the polymer degrades and releases the loaded anticancer drug,and its release rate accelerates with the decrease of pH.The relaxation rate of FA-Gd2O3@MSN-NH2 was about three times that of DTPA-Gd,while coating multilayer polymer membrane,the relaxation rate of Gd2O3@MSN decreased slightly lower than that of DTPA-Gd Utilizing weak acid to degrade the coated polymer,the MRI signal intensity gradually returned to high value,showing good pH sensing and T1WI enhancementIn chapter 2 of this project,the in vitro cytotoxicity results showed that Gd2O3@MSN/FA-Gd2O3@MSN without DOX are not toxic to HepG2 cells or normal cells,whereas the nanoparticle containing DOX have obvious cytotoxicity to HepG2 cells and other cancer cells,and the cytotoxicity of FA-Gd2O3@MSN-DOX was stronger than that of Gd2O3@MSN-DOX,but its toxicity to normal cells was less.It is suggeste that the system has less systemic toxicity and thus is beneficial to its biomedical application.The half maximal inhibitory concentration(IC50)of Gd2O3@MSN-DOX and FA-Gd2O3@MSN-DOX on HepG2 cells,Hela cells and A549 cells is approximately:52/30μg/mL,11/4μgmL,119/93μg/mL,respectively.The difference in the result may be due to the different efficiency of uptake nanoparticles by different tumor cells.FA-Gd2O3@MSN-DOX exhibits higher cell internalization performance due to the mediation of folate receptors in cancer cells.Apoptosis experiments further confirmed that the apoptosis rates of Gd2O3@MSN and FA-Gd2O3@MSN group without DOX were not significantly different from those of the control group at each time point of nanoparticles incubation with cells for 12 h,24 h and 48 h(p<0.05).The apoptosis rate of DOX load group FA-Gd2O3@MSN-DOX and Gd2O3@MSN-DOX gradually increased with the prolonged incubation time between nanoparticles and tumor cells.After 48 hours of incubation,the average apoptosis rate of Gd2O3@MSN-DOX group was(29.3133±0.67026)%.The average apoptosis rate of FA-Gd2O3@MSN-DOX group was(42.9100±6.22820)%,which was significantly higher than that of control group and Gd2O3@MSN and FA-Gd2O3@MSN group(p<0.01).Laser confocal microscopy confirmed that FA-Gd2O3@MSN-DOX and Gd2O3@MSN-DOX were mainly distributed in the lysosomes of cancer cells after transmembrane entry into cells.With the prolonged incubation time of nanoparticles and cancer cells,the accumulation of FA-Gd2O3@MSN-DOX or Gd2O3@MSN-DOX in lysosomes show a different.FA-Gd2O3@MSN-DOX group increased continuously Gd2O3@MSN-DOX group showed an upward trend within the first 24 hours,followed by a downward trend.This is due to the fact that the former is actively targeted by folic acid to enter cells,while the latter relied on passive targeting to enter the cell,the amount of nanoparticles into the cell mainly depended on the concentration gradient of nanoparticles inside and outside the cell.Further observation of the distribution of free DOX in the cells showed that free DOX diffusely distributed into the cells.It was can be seen that there were a lot of DOX in the nucleus,rather than confined to the lysosomes,which is attributed to the small molecules of free DOX mainly penetrate into the cell by means of diffusion.Laser scanning confocal fluorescence lifetime imaging conformed that DOX can be released from nanoparticles gradually with time prolonging in cells.From the long fluorescence lifetime of loading state to the short fluorescence lifetime of free state,tumor cells play a killing role.It was confirmed at the molecular level by western blot detection that FA-Gd2O3@MSN-DOX inhibited tumor cell autophagy and blocked autophagic flow,leading to apoptosis.Cell electron microscopy further confirmed that FA-Gd2O3@MSN-DOX inhibited tumor cell autophagy,blocked autophagic flow,and significantly increased and enlarged autophagosomes in the cytoplasm.In addition,in vitro MRI results showed that the FA-Gd2O3@MSN had better MRI contrast effect than the DTPA-Gd.The results suggest that the toxic effect of FA-Gd2O3@MSN-DOX on HepG2 HCC cells depends on the degradation of the pH-induced polymer p(DMA-co-TPAMA)/PAH coated on the surface of the nanoparticles and the release of DOX.The concentration of DOX in the lysosomes leads to the blocking of autophagy and ultimately the apoptosis of HCC cells.In chapter three of this topic,the distribution,metabolism and clearance process of FA-Gd2O3@MSN-DOX in vivo were proved by living animal experiments.FA-Gd2O3@MSN-DOX is mainly distributed in the lungs,liver and kidney after the tail vein injection.With the extension of time,the fluorescence signal of DOX in the above organs gradually weakened.After 72 hours of injection,except for a little DOX fluorescence signal residual in the lungs,the DOX fluorescence signal in the liver and kidney basically disappeared.The DOX fluorescence signal in the gastrointestinal tract increased significantly at 0.5 h and 6 h after the injection of the drug,and then the fluorescence signal was maintained at a high level.No obvious fluorescence signal was seen in the spleen and heart.It is worth noting that a strong fluorescence signal can be seen in the gallbladder 12h after the injection of the drug.This result suggests that the main metabolic pathways of FA-Gd2O3@MSN-DOX after intravenous injection are the renal and liver mononuclear phagocyte system as well as the bile-digestive tract pathway.Pathologically,the main organs n the body do not have the destruction of the tissue structure.The results of biosecurity experiments showed that transvenous injection of FA-Gd2O3@MSN-DOX had no significant effect on blood routine and liver and kidney function,showing good biocompatibility and biosecurity.By intratumoral administration,FA-Gd2O3@MSN-DOX can diffuse in the tumor and eventually kill HCC cells,and the effect of FA-Gd2O3@MSN-DOX is higher than that of Gd2O3@MSN-DOX.This result indicates that FA-Gd2O3@MSN-DOX has better active targeting and better tumor cell killing effect.MRI scans showed that the T1WI signal intensity gradually increased during one week of injection of FA-Gd2O3@MSN within the tumor,indicating its good intratumoral diffusion and targeting,which could be used to detect the distribution,release and real-time monitoring of the loaded drugsIn conclusion,the integrated diagnosis and treatment system of tumor microenvironment response was successfully synthesized,which can actively target HCC cells,and use the lysosomal weak acid environment of cancer cells to degrade coated pH-responsive polymers,release loaded DOX to kill tumor cells,and water molecules into the mesopores,causing MRI T1WI enhancement.It can be used to monitor the distribution,metabolism and efficacy of loaded drugs in tumor lesions in real time,with MRI contrast enhancement. |
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