Cell Imaging Studies On Anthrapyridone Dyes And Fluorescent Polymer Nanoparticles

In recent years, with the continuous development of the bioimaging technology, fluorescent dyes are more and more often applied in cell labeling and imaging, protein analysis, DNA sequencing, medical diagnostics, biosensing, and other research fields. The requirements of fluorescent in modern biological fluorescence imaging technology to dyes are also high. Fluorescent dyes should have long (visible light)excitation and emission wavelength, high quantum yield and photostability to overcome the problem of short wavelength light penetration and light damage; at the same time, when used in vivo or live-cell imaging, dyes should also have low cytotoxicity. While most commercial fluorescent dyes used for bioimaging are under foreign monopoly. Therefore, study on own fluorescent dyes with high performance for biological imaging has an important practical significance.In research, we found the4-N substituted anthrapyridone dyes have good spectral performance, long wavelength (λex/λem:～530/570nm), large Stokes shift (～40nm), multi-wavelength excitation (365,488,514,543nm), good photostability (10%higher than Rhodamine B)and low cytotoxicity (incubation at10μM for12h, cell viability>80%). Such dyes have live-cell membrane permeability. Through counterstain with commercial dye, they can stain nucleus, golgi apparatus, nucleoli and lysosome of live cells respectively. The substituting groups decide which organelles can be stained by the anthrapyridone dyes. i-6, i-7are able to stain nucleolus and lysosome of live cells; Ⅱ-A can specific stain the Golgi apparatus of living cells;Ⅱ-C to the nucleus. We can use QSAR model to predict cells dyeing which provide reference for designing new organelles specific dyes.Organic fluorescent dyes have simple structure and are convenient for using, but simple structure will inevitably lead to a single role. But fluorescent nanoparticles prepared by traditional method usually leak fluorescence which lead to background interference. To solve the dye leakage of doping and absorption method in preparing of fluorescent nanoparticles, we introduced polymerizable groups into the dye structure. Those dyes can polymerize after free radical initiator, during the reaction process the chromophores of those dyes have not changed.Non-ion surfactant Triton X-100was used for emulsification, redox initiated at low temperature (NaHSO3/KPS,40℃)for preparation of fluorescent polymer nanoparticles by free radicals emulsion copolymerization. After copolymerization, dye molecules connect to the polymer chain through a covalent bond, preventing dye’s leakage.A new type of benzyl chloride group containing fluorescent nanoparticles obtained, followed by PEI (polyethylene imine,MW=25000) grafting, folic acid molecular labeled on the particle surface. Cellular uptake experiments show the folate receptor highly expressed of HeLa cells enhanced endocytic-uptake capacity compared with low expression of MCF-7, COS-7and HepG2cell lines. Also, the cellular uptake of the small particles is much easier than that of large size. The folic acid labeled fluorescence microspheres have potential application prospect in the early diagnosis and targeted therapy of cancer.