| The latest global cancer burden data released by the World Health Organization’s International Agency for Research on Cancer(IARC)in 2020 shows that China ranks first in the world in terms of cancer incidence and mortality.Chemotherapy remains the most widely used cancer treatment in China,however,despite the widespread use of traditional small molecule chemotherapy drugs,they all have non-specific tissue distribution and more serious side effects.In addition,relying on methods such as pathological analysis or structural imaging can only roughly assess the area of the lesion.Therefore,the development of more effective methods for cancer diagnosis and treatment is one of the urgent scientific problems that need to be solved.In the previous work,this project innovatively proposed and established in vivo in situ biosynthetic gold,zinc oxide,ferric tetroxide nanocluster probes,and achieved accurate targeting and precise labeling of tumors and other lesions.Based on the previous results of the group,this study discovered that in situ biosynthetic gold nanoclusters can inhibit tumor development and preliminarily elucidated the molecular mechanism of its cancer suppression effect;it proposed a method for precise imaging and suppression of tumors based on selfassembled fluorescent nanocluster-DNA complexes,which realized real-time monitoring of lesion sites while gene therapy.Finally,based on the mechanism that in situ biosynthetic nanoclusters can achieve nucleic acid delivery without introducing exogenous elements into the human body,we designed nanocapsules with multimodal therapeutic potential to not only achieve gene therapy of tumor cells but also target circulating cancer cells/exosomes in the blood of tumor patients.The results show that the in situ self-assembly method of fluorescent nanoclusters with nucleic acids is an effective and accurate method for targeting tumor imaging and therapy,providing a simple,safe and promising therapeutic strategy for tumor treatment.The specific results are as follows:(1)The molecular mechanism of in situ synthesized gold nanoclusters’ cancer-inhibiting effect was discovered and elucidated.By using in vitro assays,we found that the in situ biosynthesized gold nanoclusters could exert tumor suppressive effects,and this effect was verified in an orthotopic liver cancer mouse model.Then,with the help of microarray analysis and the combination of molecular biology experimental tools,we found that the molecular mechanism of the cancer inhibitory effect of the nanoclusters is to inhibit the activity of the PI3K-AKT signaling pathway by suppressing the expression of m TOR,AKT and P13 K.(2)A self-assembled gold nanocluster-PTEN complex was constructed in situ using PTEN,a core oncogene in the PI3K-AKT signaling pathway,which can be used for accurate imaging of subcutaneous tumors in mice.The fluorescent gold nanocluster-PTEN complex was demonstrated by atomic force microscopy imaging,Raman spectroscopy detection,laser confocal microscopy imaging,high-resolution transmission electron microscopy imaging and XPS analysis;with the help of mouse subcutaneous tumor model,it could be detected by small animal imager that the gold nanocluster-PTEN complex has a good fluorescent targeting effect on the tumor site;isolated organ experiments showed that the gold nanoclusters in the complex were mainly excreted through the kidney and liver;the results of blood cell analysis,blood biochemical analysis and H&E organ section showed that the complex had no significant organ and cytotoxicity.(3)The self-assembled gold nanocluster-PTEN complex can be used for targeted therapy of subcutaneous tumors in mice.In this study,with the help of q PCR and western blot assay,it was verified that the complex could achieve intracellular delivery of PTEN gene and promote the high expression of PTEN gene in cells.In addition,the complex was found to inhibit tumor growth in a subcutaneous tumor treatment model.Finally,with the help of RNA-seq analysis,the molecular mechanism of its oncogenic effect was by up-regulating oncogenes and down-regulating oncogenes,in addition to achieving delivery of oncogenes PTEN.(4)The self-assembled gold nanocluster-PTEN complexes can be used for targeted imaging and therapy in a mouse model of hepatocellular carcinoma in situ.Firstly,through in vitro cell phenotyping assays,we found that the gold nanocluster-PTEN complex could also inhibit the proliferation,invasion and metastasis of hepatocellular carcinoma cells in vitro;then,with the help of an orthotopic liver cancer mouse model,the complex could be detected by small animal imager to achieve in situ liver tumor fluorescence-targeted imaging,and the targeting effect was also verified in isolated organs of mice;finally,the enrichment of the complex in the liver was found to inhibit the growth of liver tumors.(5)Nanocapsules designed based on the nucleic acid delivery principle that in situ biosynthetic nanoclusters can enable nucleic acid delivery and the photothermal characteristics of melanin nanoparticles can enable multimodal therapy and targeted imaging of tumor cells.First,after bioinformatic analysis of several gene chips in the GEO database,this study revealed that KRAS was highly expressed in the tumors of pancreatic cancer patients,and this high expression was closely associated with poor prognosis of patients.This suggests that inhibition of KRAS expression has great potential in gene therapy for pancreatic cancer.Then,in this study,a nanocapsule was designed that could inhibit KRAS expression,and the successful construction of the nanocapsule was demonstrated by high-resolution projection electron microscopy,Fourier transform infrared spectroscopy,and ZETA potential analysis,among other characterization tools.Subsequently,the nanocapsules were validated by q PCR and western blot assays to achieve intracellular delivery of KARS sh RNA plasmid and suppress the high expression of KRAS gene in tumor cells;meanwhile,the nanocapsules were found to inhibit the proliferation,invasion and metastasis of tumor cells in vitro.By photo-thermal imaging and cell clone formation assay,we found that pancreatic cancer cells could be well killed under 808 nm laser irradiation.Finally,this study tested that the nanocapsules can easily enable the bioassay of cancer cells/exosomes in the blood of tumor patients. |
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