A New Biological Detection Method Based On PEG Nanomicelles And Its Application In Cancer Diagnosis And Treatment

The study on sensitivity of fluorescence probe is the key to the development of fluorescence imaging technology,the degradation and destruction of the fluorescence probe by”digester " of cell-lysozyme greatly limit the efficient detection of fluorescent probe.In this paper,the swelling effect of polymeric micelles was transplanted into the study of fluorescence detection,the phagocytosis and digestion of lysosomes were inhibited by the swelling effect of PEG nanomicelle-based fluorescence probe,and the probe was targeted and accumulated in the cytoplasmic matrix for improving the detection accuracy and sensitivity.In addition,this paper,from the perspective of reducing the prevention of physiological barrier and blocking the self-protection mechanism of tumor cells,regulates the contradiction between efficacy and safety,and enhances the sensitivity of tumor cells for therapeutic agents by PEG nanomicelle-based multi-functional platform,so as to achieve safe and efficient tumor therapy.This paper mainly includes the following work:1.Detection of breast cancer cells based on PEG-PEI/FAM-ssDNA nanocomposite probe.PEG-PEI/FAM-ssDNA nanocomposite probe was prepared by a non-surfactant precipitation method with the approach of PEG nanomicelles combined with PEI and FAM-ssDNA,and the morphology,particle size and composition of the nanocomposite probe were characterized.The fluorescence performance was evaluated.The results showed that the nanocomposite probe had the characteristics of strong fluorescence in neutral or alkaline environment and weak or non-fluorescence in acidic environment due to FAM fluorescein-labeled DNA.Further,laser confocal imaging was used to visually track the performance of the nanocomposite probe in tumor detection.The results showed that PEG-PEI/FAM-ssDNA nanocomposite probe can specifically bind mucin on cell membrane which was the tumor marker of breast cancer.The surface of the cell membrane took on fluorescence;furthermore,even if part of the nanocomposite probes can be internalized by cells,the fluorescence can be quenched due to phagocytosis by acidic lysosomes,so that non-specific fluorescence was effectively avoided.The experimental results showed that the detection limit was as low as 40 cells/mL.The PEG-PEI/FAM-ssDNA nanocomposite probe provided a new idea for tumor-specific detection.2.New method for detection of hydrogen sulfide in living cells based on lysosomal phagocytosis and swelling effects of polymeric micelles.PEG-PEI/DP-NBD nanocomposite probe was prepared by combining PEG nanomicelles with organic small molecule DP-NBD.Cytotoxicity experiments demonstrated that the nanocomposite probe had good cytocompatibility and can avoid the toxicity problems caused by organic solvents.Cell imaging showed that as the incubation time prolonged,PEG-PEI/DP-NBD nanocomposite probe with swelling effect can gradually escape from lysosomes to cytoplasmic matrix.Since the degradation and destruction by lysosomes were avoided,the detection efficiency of hydrogen sulfide in living cells was improved.The experimental results showed that PEG-PEI/DP-NBD nanocomposite probe for fluorescence detection of H2S had broad detection range(10-200 μM)and low detection limit(0.10μM)with good specificity.The PEG-PEI/DP-NBD nanocomposite probe provided a new perspective for the sensitive detection of tumors.3.Photothermal therapy at low-temperature based on PEG-PEI/CDs-E64d nanomicelles.PEG-PEI/CDs-E64d nanomicelles was prepared by complexing the photothermal agent carbon dots(CDs)and the autophagy inhibitor E64d with PEG-PEI nanomicelles.PEG-PEI/CDs-E64d nanomicelles integrated four functions which were fluorescence imaging,photothermal therapy,swelling effect and autophagy inhibition in one.Cell imaging showed PEG-PEI/CDs-E64d nanomicelles with swelling effect can escape from lysosome to cytoplasmic matrix to avoid the digestion and degradation by lysosomes,and the function of autophagy inhibition of PEG-PEI/CDs-E64d nanomicelles was verified by western blot.Further,PEG-PEI/CDs-E64d nanomicelles was applied for tumor therapy of tumor-bearing mice.The thermal imaging showed PEG-PEI/CDs-E64d nanomicelles had excellent tumor selectivity;when tumor area was irradiated with laser light,the tumor temperature could reach 42℃ which belonged to low temperature range,and the growth of tumor was effectively inhibited.Therefore,PEG-PEI/CDs-E64d nanomicelles had potential clinical application value which was expected to be personalized diagnostic reagents for inhibiting the growth of tumor at low temperature.4.Photothermal/photodynamic synergistic cancer therapy based on PEGPEI/CDs-Cys-E64d nanomicelles.Photosensitizer with 2,4-dinitrobenzenesulfonamide group(Cys)was introduced to prepare PEG-PEI/CDs-Cys-E64d nanomicelles and the therapeutic effect of the nanomicelles was evaluated.The photothermal performance shows that photodynamic effect of photosensitizer Cys can enhance photothermal conversion efficiency of photothermal agent CDs due to the same absorption wavelength range of Cys and CDs.Furthermore,cell imaging and western blot showed that the PEG-PEI/CDs-Cys-E64d nanomicelles had characteristic of swelling effect and autophagy inhibition.PEGPEI/CDs-Cys-E64d nanomicelles was applied for cancer therapy in vivo and found that can effectively inhibit the growth of tumor.Therefore,the swelling effect/autophagy inhibition-mediated photothermal/photodynamic co-therapy of PEG-PEI/CDs-CysE64d nanomicelles had potential prospect of clinical application.