Design Of Fluorescent Nano-probes Based On The Mechanism Of AIE Phenomenon And Applications In Tumor Cell-targeted Imaging

Section I Fabrication of Chitosan Nanoparticles with Aggregation-Induced Emission Characteristics and Their Applications in Long-Term Live Cell ImagingObjective:To fabricate chitosan (CS) nanoparticles with aggregation-induced emission (AIE) characteristics, assess the physical and chemical properties, AIE features, photostability and biocompatibility of these nanoparticles and explore the application value in long-term cell tracking of live cells.Methods:The TPE modified CS (TPE-CS) was synthesized through addition reaction between the isothiocyanate groups on TPEITC and amines on CS. The products were detected through NMR and FTIR, and the substitution degree of TPE was calculated. Then, the CS nanoparticles hybridized with TPE molecules (TPE-CS NPs) were fabricated by ionic gelation method. The hydrodynamic diameter and dispersity of TPE-CS NPs were measured by dynamical light scattering (DLS) and the zeta potential of these nanoparticles was detected by laser doppler electrophoresis. The diameter and morphology of TPE-CS NPs were observed using transmission electron microscope (TEM). The spectrofluorometer was performed to analyze the light property of TPE-CS NPs and effect of pH value on fluorescent intensity. The cytotoxicity of TPE-CS NPs was assessed using MTT assay. HeLa cells were stained with various concentrations of TPE-CS NPs to investigate the dose-dependent effect, and the cells were continuously scanned using confocal laser scanning microscope (CLSM) to assess the photostability of TPE-CS NPs. The cells were pretreated with NaN3, which reduced the cellular synthesis of adenosine triphosphate (ATP), to inhibit the uptake of nanoparticles, and then observed the fluorescent intensity using CLSM and flow cytometry (FCM). TPE-CS NPs labeled HeLa cells and unlabeled3T3cells were co-cultured and observed the distribution of fluorescence from the nanoparticles. The HeLa cells were stained with TPE-CS NPs and observed per day until the fluorescence disappeared, to assess the retention time of TPE-CS NPs in cells.Results:The TPE-CS was successfully synthesized through amidation reaction between the isothiocyanate groups and amines.1H NMR spectra showed that the new peaks present at the aromatic region emerged in the TPE-CS conjugates and the substitution degree of TPE is calculated to be4.13%. FTIR spectrum showed the isothiocyanate bond characteristic peak at2045cm-1in TPEITC disappeared in the TPE-CS conjugates. The TPE-CS NPs were fabricated through ionic gelation method. DLS revealed the average hydrodynamic diameter of TPE-CS NPs is170nm with good dispersity and TEM image showed TPE-CS NPs were spherical shapes, uniform in diameter with monodispersity. The zeta potential of TPE-CS NPs decreased with increase of pH value, but still kept positive at pH7.0. Nearly no fluorescence was recorded when TPE-CS was dissolved in pH2.5solution, while TPE-CS NPs emitted strong fluorescence owing to the restriction of intramolecular rotation of TPE by CS chain. When pH value was adjusted to7.0, the fluorescent intensity of TPE-CS and TPE-CS NPs was enhanced, but the solution of TPE-CS NPs was still stronger. The fluorescence from cells was dose-dependent. The cells treated with TPE-CS NPs were continuously scanned for30min, but the fluorescent signals only lost less than25%. The cells were pretreated with NaN3, blocking the synthesis of ATP, to inhibit the endocytosis. Compared with un-treated cells, the fluorescence of NaN3pretreated cells became weaker. After co-culturing TPE-CS NPs labeled HeLa cells and unlabeled3T3cells for24h, CLSM images showed the nanoparticles mainly distributed in HeLa cells and only scattered fluorescence is from3T3cell region. After labeled with TPE-CS NPs, the fluorescence became weaker with increase of subculture, but the cells still emitted light for7days.Conclusions:TPE-CS NPs possess spherical shape, uniform diameter, good dispersity and positive potential with typical AIE characteristics. TPE-CS NPs with low cytotoxicity enter cells through endocytosis. Only a small amount of TPE-CS NPs were excluded from cells and TPE-CS NPs can trace the live cells for long period of time. Therefore, TPE-CS NPs are promising for a long-term live cell nanoprobe and further applied in monitoring the loading and release of drug and gene. Section Ⅱ Biotin-Decorated Fluorescent Silica Nanoparticles with Aggregation-Induced Emission Characteristics:Fabrication, Biocompatibility and Tumor Cell-targeted ImagingObjective:To design and fabricate fluorescent silica nanoparticles (FSNPs) with aggregation-induced emission (AIE) characteristics, targeting to the tumor cells with over-expressed biotin receptors. To investigate the physical and chemical properties and biocompatibility, and assess the targeted imaging to the tumor cells with over-expressed biotin receptors.Methods:The FSNPs hybridized with silole derivative1were fabricated through sol-gel reaction, and then modified with amine groups on the surface of nanoparticles to obtain FSNP-1-NH2. The biotin molecules were decorated on the surface of FSNP-1-NH2by amidation reaction between amine groups and carboxyl groups of biotin, namely FSNP-1-biotin. The biotin groups on the surface of nanoaparticles were analyzed by IR spectrum. The diameter and morphology of nanoparticles were observed using transmission electron microscope (TEM) and scanning electron microscope (SEM). The chemical compose was analyzed X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDX). Thermogravimetric analysis was measured to assess the thermal stability and calculate the ratio of biotin. The biocompatibility of FSNP-1-biotin was investigated using cell morphological change, cell viability, cell apoptosis and intracellular reactive oxygen species (ROS) assays. Human cervical carcinoma HeLa cells and human hepatocellular carcinoma BEL-7402cells with over-expressed biotin receptors were stained with FSNP-1-biotin to observe the fluorescent intensity of cells. Human normal liver LO2cells with low-expressed biotin receptors were used as a negative control. The uptake of nanoparticles was investigated using TEM. HeLa cells labeled with FSNP-1-biotin were continuously cultured to observe the retention time of the nanoparticles inside the cells and tracked to study the process of tumor cell migration.Results:The FSNP-1were successfully fabricated through sol-gel reaction and modified with biotin molecules by amidation reaction. The IR spectrum showed the emergence of carbonyl group in the FSNP-1-biotin. TEM and SEM showed the nanoparticles were spherical in shape, uniform in diameters with narrow size distributions and possessed good dispersity. XPS and EDX analysis demonstrated a amount of sulfur atom in biotin molecules was detected in the FSNP-1-biotin. Nearly no fluorescence signals were recorded when the ethanol solution of precursor1was photoexcited, while the nanoparticles emitted strong fluorescence because the intramolecular rotation of1was restricted by silica network. After treating with FSNP-1-biotin, the cellular morphology did not remarkably change, only small amount of multinucleate cells emerged and a little amount of vacuole was observed in cytoplasmic region. Compared with control group, CCK-8and trypan blue assay showed low cytotoxicity at concentrations below100μg/mL, which is different significantly (P>0.05). At100μg/mL, the cytotoxicity of FSNP-1-biotin is remarkable (P<0.05). FSNP-1-biotin did not affect on the cell apoptosis and intracellular ROS at the concentration of40μg/mL (P>0.05), but accelerated apoptosis and increased the level of intracellular ROS at the concentration of80μg/mL (P<0.05) to a certain extent. FSNP-1-biotin could target to the tumor cells with over-expressed biotin receptor and selectively stain cytoplasmic region. These nanoparticles entered cells through receptor-mediated endocytosis pathway and stayed inside cells. The fluorescence was recorded from the labeled cells, even up to7days. After stained with FSNP-1-biotin, the cells were tracked to observe the migration process and analyzed quantitatively the ability of cell migration.Conclusions:The FSNP-1-biotin are promising for a novel tumor cell tracker, which target to the tumor cells with over-expressed biotin receptor, to investigate the biological properties of tumor and further apply in targeted imaging of tumor tissue in vivo.