Construction Of Intelligent Responsive Nanotheranostic Probe And Its Applications In Tumor Metastasis And Drug Resistance

Cancer is a serious threat to human health.Metastasis and drug resistance are the main reasons for poor postoperative prognosis and increased mortality.In order to improve the survival rate of patients,various methods,such as tumor tissue biopsy,biomarker detection,and emerging liquid biopsy,are currently used for tumor metastasis and drug resistance research.However,histopathological testing and molecular biological validation cannot reflect the true state of the tumor.Therefore,it is very important to establish a method for detection of tumor metastasis and drug resistance for individualized tumor therapy.The development of molecular imaging technology has brought new opportunities for the precise diagnosis and treatment of tumors.In vivo multimodal imaging can not only accurately locate the tumor,but also evaluate the drug resistance status through the change of tumor size after drug treatment.Although some research progress has been made,due to the lack of tumor-specific markers,it is impossible to accurately quantify tumor metastases in real time,and delayed tumor changes cannot reflect the real situation of drug resistance in real time.Therefore,it is necessary to continuously explore new methods for in vivo detection of tumor metastasis and drug resistance.Based on the above research background,a silica-based nanoprobe was constructed firstly to achieve tumor detection in vivo.On this basis,an orthotopic metastatic tumor model was constructed to achieve quantitative detection of orthotopic tumors and metastases.In order to further detect tumor drug resistance,a Mn₃O₄-based intelligent responsive nanoprobe for tumor microenvironment was constructed to detect orthotopic drug-resistant tumors.On this basis,photothermal and chemodynamic therapy of drug-resistant tumors are realized.The specific research contents include the following aspects:（1）A silica-based nanoprobe for tumor in vivo detection was constructed.Firstly,silica nanoparticles（SiO₂NPs）with good monodispersity were synthesized,and then the polypeptide RGD was cross-linked on the surface of SiO₂NPs by the photo-click method using tetrazole compound T1（SiO₂@T1-RGDkNPs）.Transmission electron microscope images show that the SiO₂NPs have a spherical structure with good monodispersity and a particle size of about 50 nm.The good stability of SiO₂@T1-RGDkNPs was verified by the change of particle size under different solvents and different temperatures.The successful synthesis of SiO₂@T1-RGDkNPs was further demonstrated by UV absorption spectroscopy,fluorescence spectroscopy and infrared spectroscopy.The results of cellular uptake and affinity analysis indicated that SiO₂@T1-RGDkNPs had good tumor cell targeting and accumulated in the cytoplasm.The in vitro and in vivo biosafety of SiO₂@T1-RGDkNPs was evaluated by cytotoxicity assay and in vivo acute toxicity assay.Taking prostate cancer as a tumor model,the tumor can be detected in vivo through fluorescence imaging.（2）A nanoprobe for detection of tumor metastasis based on mesoporous silica was constructed.First,biodegradable mesoporous silica nanoparticles（bMSNNPs）were synthesized by a water-oil two-phase reaction method,and their surfaces were modified with amino groups.The fluorescent dye Cy7.5 and folic acid（FA）were then cross-linked on the surface of bMSNNPs（bMSN@Cy7.5-FANPs）.Cy7.5 can be used as an in vivo fluorescence imaging agent,and FA can specifically target tumor cells.The particle size of the bMSNNPs is about 100 nm with clear mesoporous structure.The good stability of bMSN@Cy7.5-FANPs was verified by the change of particle size in different solvents and different temperatures.The successful synthesis of bMSN@Cy7.5-FANPs was further verified by UV absorption spectroscopy and fluorescence spectroscopy.The results of cytotoxicity experiments showed that bMSN@Cy7.5-FANPs had good biosafety.The results of cellular uptake and affinity analysis showed that bMSN@Cy7.5-FANPs could be taken up by cells and accumulated in the cytoplasm,and had good targeting to tumor cells.A pancreatic cancer metastasis model was constructed,and the in vivo distribution of bMSN@Cy7.5-FANPs was verified by in vivo fluorescence imaging to achieve quantitative detection of metastases.The distribution of bMSN@Cy7.5-FANPs in tumor and muscle was evaluated according to the three-compartment model,and the results showed that the fluorescence signal at the tumor site increased rapidly,indicating that bMSN@Cy7.5-FANPs had good tumor targeting.（3）An intelligent responsive nanoprobe for tumor microenvironment for the in vivo detection and synergistic treatment of drug resistance was constructed.Firstly,manganese tetroxide nanoparticles（Mn₃O₄NPs）were synthesized by thermal decomposition,and the surface was modified with polydopamine.The tetrazole compound T2 was used to cross-link the gastric cancer multidrug resistance-specific polypeptide GMBP1 on the surface of the nanoparticles by photo-click reaction（MPGNPs）.Transmission electron microscopy results showed that the MPGNPs had a particle size within 10 nm and had good stability in different solvents and temperatures.The UV absorption and fluorescence spectra further verified the successful synthesis of MPGNPs.Cellular uptake and affinity analysis indicated that MPGNPs had good targeting to gastric cancer multidrug-resistant cells.The MRI relaxation rates of MPGNPs were measured at different p H to verify their MRI performance.An orthotopic gastric cancer drug resistance model was established,and the drug resistance of gastric cancer was detected in vivo by MRI.The results of in vivo acute toxicity and hemolysis experiments indicated that MPGNPs had good biosafety.（4）The synergistic therapeutic effect of MPGNPs on drug-resistant gastric cancer was further verified.The good chemical kinetic activity of MPGNPs was verified by the reaction with glutathione（GSH）and hydrogen peroxide（H₂O₂）.The photothermal effect was verified by the temperature change of MPGNPs solution under different conditions.The tumor cell inhibitory effects of MPGNPs were verified by cytotoxicity,clone formation,flow cytometry and apoptosis staining.Establish an orthotopic gastric cancer drug resistance model,and the photothermal and chemodynamic synergistic treatment effects of MPGNPs were verified.The results showed that the tumors in the synergistic treatment group gradually became smaller or even disappeared,indicating that MPGNPs have a good synergistic therapeutic effect.