Study On Fluorescent Silica Nanoparticles For Highly Sensitivite Imaging In Vivo

Various non-invasive imaging methods are greatly required for the in vivo,quantitative, real-time, and visual investigation of life activities. Due to its uniqueadvantages, such as high sensitivity, rapid imaging, easy operation, no radioactivehazards and so on, the in vivo fluorescence imaging technology has been widelyapplied in the biomedical research field. For the high-sensitivity imaging in vivo, theimaging probe is a key factor influencing the imaging quanlity. In recent years, withthe development of nanotechnology, various functionalized nanomaterials have beendeveloped and used for in vivo imaging and tumor therapy. In particular, thedevelopment of nanomaterials with high signal to noise ratio, good biocompatibility,and excellent optical properties for in vivo fluorescence imaging holds greatsignificance. In this thesis, aiming at the highly sensitive fluorescence imaging in vivo,several imaging probes based on fluorescent silica nanoparticles have beenconstructed and utilized for fluorescence imaging in vivo. The main works aresummarized as follows.1. Methylene blue doped silica nanoparticles for cell labeling and in vivofluorescence imagingMethylene blue (MB) doped silica nanoparticles (MB-SiNPs) have beensynthesized in the water-in-oil microemulsion. By optimizing the concentration ofMB doped in the silica matrix, we obtained the near-infrared fluorescent MB-SiNPswith strong fluorescence. Then, the feasibility of MB-SiNPs for cell labeling andtracking in vivo has been studied. The cell viability assay of Hela cells in the presenceof MB-SiNPs was performed using MTT assay, and the result indicated that as theconcentration of MB-SiNPs was1mg/mL in medium, the cell survival percentage was80%. The distribution of MB-SiNPs in Hela cells was assessed by laser scanningconfocal microscopy and the result showed that MB-SiNPs entered into cells easilyand mainly located in lysosomes. In vivo fluorescence imaging of MB-SiNPs after tailvein intravenous injection showed that the whole body of the mice emitted NIRfluorescence and most of the MB-SiNPs were accumulated in some organs, such aslive and spleen. The imaging of isolated organs, MB-SiNPs and nude mice feedshowed that the fluorescence signal of feed was also very strong in the same condition,which interfered with the imaging sensitivity. All the results demonstrated that MB-SiNPs could be used for cell labeling and in vivo imaging. But, there is also achallenge to overcome the background fluorescence interference from mice tissuesand food.2. FRET mediated large stokes shifting near-infrared fluorescent silicananoparticles (LSS-NFSiNPs) for in vivo small-animal imagingIn order to develop an imaging probe which can overcome the backgroundfluorescence signal interference from mice tissues and food, we present the novellarge Stokes shifting NIR fluorescent silica nanoparticles (LSS-NFSiNPs) based onthe principle of fluorescence resonance energy transfer (FRET). Two highlywater-soluble dyes, Tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate (Rubpy)and methylene blue (MB), were chose as the donor-acceptor pair. The LSS-NFSiNPswere prepared by synchronously doping RuBpy and MB in the silica nanoparticles. Byoptimizing the molar ratio of RuBpy and MB for doping in the silica nanoparticles,the energy transfer from RuBpy to MB occurred in the silica matrix, resulting in thenear-infrared fluorescent silica nanoparticles with strong fluorescence and largeStokes shift (>200nm). As a result, it can effectively help to increase thediscrimination of fluorescence signal of interest over other background signals. Witha combination of excellent stability, large stokes shift and near-infrared spectralproperties, this novel LSS-NFSiNPs provides real-time, deep-tissue fluorescentimaging of live animals. In addition, the LSS-NFSiNPs can also be gradually clearedfrom the body through urinary clearance system. We anticipate this design conceptcan lay a foundation for further development of in vivo optical nanoparticulatecontrast toward clinical application.3. Study on the preparation of the acidic activatable fluorescent silicananoparticles probesActivatable probes with the “signal on” strategy hold the great potential toimprove the imaging contrast. This work presents an activatable fluorescent silicananoparticles probe which can be activated by acidic conditions. The RuBpy dopedcarboxyl-terminated silica nanoparticles have been synthesized in the water-in-oilmicroemulsion. Through EDC/NHS reaction, the acid-labile molecule ATU wascovalently modified to the particles surface. Then, the quenching molecule BHQ-3cross-linked to the particles surface by ATU to achieve the fluorescence quenching.The modification process was assessed by Zeta-Size, UV-visible spectrophotometer,and Fourier transform infrared spectroscopy. The results showed that ATU and BHQ-3were successfully conjugated to the particles surface. By optimizing the amount of ATU and BHQ-3on particles, we obtained an activatable probe with better quenchingeffect and the fluorescence quenching rate was about41.2%. When the probedispersed in acidic solution, the hydrolysis of ATU, making the BHQ-3away fromthe particle surface and the elevation of fluorescence was detected. The quenchingfluorescence signal could be restored by24.96%.This study lays a foundation for thefurther development of activatable probes based on fluorescent silica nanoparticles.