Preparation Of Functionalized Fluorescent Nanoparticles And Their Biological Imaging

In order to realize a sensitive in vitro imaging of cancer cells,and a sensitive,long-life in vivo imaging of in-situ tumor,three parts of research work were carried out in this paper,including:（1）preparation of fluorescent silver nanoclusters protected by protein,surface functionalization of the silver nanoclusters and it’s use in tumor cell imaging;（2）in vitro and in vivo imaging of fluorescent nanoparticles conjugated by fluorophores and polymer;（3）preparation and in vitro and in vivo imaging of fluorescent nanoparticles conjugated by ruthenium complexes and polymer.In the first part,silver nanoclusters（BSA-AgNCs）protected by bovine serum albumin（BSA）were prepared with BSA as template.BSA-AgNC has strong fluorescence emission in the excitation wavelength range from 400 nm to 500 nm.It shows red fluorescence at the maximum emission wavelength of 600 nm where the biological matrix only has weak absorption and weak fluorescence.After that,the BSA-AgNCs-PEI nanoparticles were prepared by modifying the as-prepared silver nanoclusters with a small molecular weight of PEI,to enhance the structural stability and its transmembrane action on cells.The particle size of these nanoparticles ranged from 30 nm to 100 nm after the surface modified with PEI,and most of them are spherical.Zeta potential increased obviously comparing with the unmodified particles and the charge of nanoparticles is close to neutrality which is-2.13±0.9 mV,thus enhancing the affinity of the nanoparticles to cell membranes.Meanwhile,a low negative charge will also help to avoid the cytotoxicity caused by the high positive charge of PEI.For imaging analysis of tumor cells,the results showed that the BSA-AgNCs-PEI nanoparticles enhanced the detection sensitivity significantly.The BSA-AgNCs-PEI nanoparticles can quickly achieve the transmembrane movement on cells and prevent the hydrolysis of carrier protein from intracellular enzymes effectively compared with non-crosslinked nanoparticles.The fluorescence of intracellular nanoparticles could be observed after 24 h,hence realizing a long-life fluorescent cell imaging with the as-prepared fluorescence nanoparticles and BSA as template.In the second part,we have prepared the fluorescent nanoparticles with a novel structure suitable for detection of in vitro and in vivo cells.Firstly,a strong hydrophilic polymer with rich branch amino groups was synthesized,and it was conjugated with FAM as much as possible to further synthesize a hydrophobic polymer with a strong fluorescence.After that,by using a self-assembling approach of hydrophobic interaction and a PAA surface cross-linked one,the nanoparticle PAA-FPNP with a strong fluorescence and a good hydrophilicity was prepared.It has not only a fluorescent“core-cover-type”rigid structure but also a non-fluorescent flexible structure（i.e.the residual chains of the surface cross-linked PAAs）.It is because of this novel structure that the PAA-FPNP has four excellent properties as following:（1）a small particle size,a narrow particle distribution and rich negative surface charges;（2）a large number of the fluorophores in the core,a very thin cover and the numerous PAA residual chains cross-linked on the surface of the nanoparticle;（3）a good protection of the embedded fluorophores against in vivo enzymatic hydrolysis,in addition,a suitable pH sensitiveness of fluorescence for cell detection;（4）a weak steric hindrance for the nanoparticle-labeled aptamer recognizing a huge-size target cell.So the prepared PAA-FPNP has both a strong fluorescence and a suitable nanoparticle structure for cell imaging.Based on the aptamers labeled by PAA-FPNP,we realized a sensitive in vitro imaging of cancer cells and a long-life,in vivo imaging of in situ tumor.Ruthenium complexes show red fluorescent at long wavelength absorption of 610nm where the biological matrix only have weak absorption and weak fluorescence,a significant advantage for bio-imaging comparing with fluorescent dyes.That is,there is still a longer excitation wavelength（460 nm）that gives rise to the strongest fluorescence under the condition of large stocks shift（155 nm）.Based on these,two fluorescent nanoparticles,Ru-1-PAA and Ru-1-PAA/PEI,were prepared using the graft polymer Ru-1-PAA synthesized by using fluorophor Ru（bpy）₂（phen-epoxide）（PF₆）₂,Ru-1,as phosphor in the third part.In vitro imaging of cancer cells and in vivo imaging of in situ tumors were studied using Ru-1-PAA/PEI nanoparticle labeled with aptamer.Firstly,the fluorescent polymer,Ru-1-PAA,was synthesized with a large number of ruthenium complexes Ru-1 immobilized on PAA by chemical grafting in DMF solvent with anionic polymer PAA as carrier.Then the fluorescent nanoparticles,Ru-1-PAA,were prepared by hydrophobic interaction in aqueous solution.Their size distribution was 10-50 nm.Most of them were small particles around 10 nm and few have large particle size.Zeta potential was-15.07±0.6 mV.The Ru-1-PAA/PEI nanoparticles were prepared by following in order to improve the structural stability and size distribution:Ru-1-PAA were cross-linking accumulated with PEI and a small molecular weight cationic PEI（Mw=1800）was used as a crosslinking agent.The hydrophobic interaction of nanoparticles accumulated by Ru-1-PAA polymer,chemical cross-linking and electrostatic interaction based on PEI,providing a smaller particle size and a more uniform distribution for the nanoparticle,producing a more significant fluorescence enhancement at the same time.All these characteristics are more favorable for imaging analysis.The results of in vitro and in vivo imaging showed that we realized the high sensitivity and long-life fluorescence imaging by Ru-1-PAA/PEI nanoparticle labeled with aptamer.