Stimuli-responsive Polymer Nanospheres With Fluorescent Properties For Bioimaging

Bioimaging technology can make cells visible, which plays an important role in biological research. As a sensitive, convenient and non-invasive analytic technique, fluorescence detection has been widely used in terms of molecular recognition, bio-labeling, cell imaging and many other fields. It is becoming an essential imaging method for biological applications. In the past decades, researchers have synthesized various fluorescent probes, including semiconductor quantum dots, carbon quantum dots, rare-earth doped nano-materials, photoluminescent silicon nanospheres, Au nanoclusters, organic fluorescent molecules and fluorescent proteins and so on.Among these materials, responsive polymers based fluorescent nanoparticles(FNPs) have drawn much attention because of their advantages:(1) Responsive polymer nanoparticles can be fabricated with controllable sizes, different surface charges and surface morphologies through various synthetic methods.(2) Responsive polymer nanoparticles have excellent dispersity, stability and biocompatibility in aqueous systems.(3) There are many functional groups on the surface of the responsive polymer nanoparticles, which can be integrated with some specific molecules(fluorophores, drugs and targeting molecules) through chemical or physical binding. In addition, stimuli-responsive materials are able to change their physical structures and chemical properties reversibly or irreversibly responsing to the external environment.At present, there are various organic dyes. Conventional organic dyes often emit intense fluorescence in dilute solutions, but the fluorescence will be quenched on aggregation state or in high concentration. This phenomenon was called aggregation-caused quenching(ACQ) which limits their application in biological applications. In 2001, a new category of organic dyes with aggregation-induced emission(AIE) effect was developed by Tang’s group. AIE fluorogens are non-emission in dilute solutions, but can emit intense fluorescence in aggregation state. Since the AIE effect allows a higher concentration of fluorophores to be loaded in FNPs, it makes the AIE-based FNPs more emissive with stronger photobleaching resistance.In Chapter 2, we successfully synthesized a kind of positively charged fluorescent nanoparticles(PFNPs) with intense emission and aggregation-induced emission(AIE) properties. The positively charged nanoparticles(PNPs) are synthesized through a surfactant-free copolymerisation using styrene and a positively charged quaternary amine(ar-vinylbenzyl) trimethylammonium chloride(VBTAC) as monomers. During polymerization process, the amphiphilic monomer VBTAC serves as a surfactant, which makes the surface of nanoparticles positively charged. Furthermore, fluorescent molecule sulfonated 9,10-distyrylanthracene derivatives(SDSA) with two negatively charged sites are immobilized in the PNPs by coulomb interaction. After modified on plymer chains, these SDSA molecules exhibit intense fluorescent emission. This phenomenon is known as an immobilized induced emission(IIE). Thus positively charged fluorescent nanoparticles(PFNPs) were obtained. PFNPs possess spherical morphology, positively charged surface and narrow size distribution with controllable diameter of 100 nanometers. PFNPs display remarkable fluorescence in water solution. The emission intensity of SDSA modified on PFNPs is improved by 25 times compared with that in the SDSA aqueous solution. The emission properties of PFNPs are stable in a wide p H range from 3 to 10. In addition, the positively charged surface of PFNPs enhances cellular uptake of the nanoparticles due to electrostatic interaction between positively charged particles the negatively charged cell surfaces. These advantages together with the low cytotoxicity of PFNPs imply that the prepared PFNPs have potential applications in bioimaging applications.In Chapter 3, in order to extend the study on responsive polymer nano-systems and improve fluorescent probe with targeting and responsive properties, a new kind of p H-sensitive fluorescent core-shell hydrogel(VANPs-1:2-FA-SDSA) particles has been synthesized through the emulsifier-free copolymerization method and further modified with folic acid(FA). Subsequently, fluorescence molecule SDSA is attached to the particles through electrostatic attraction, leading to a dramatic increase of its fluorescence. Moreover, the fluorescent intensity remains stable in biological conditions with different p H. At physiological p H=7.4, VANPs-1:2-FA-SDSA are stably dispersed, which are beneficial to permeating into tumor tissues through EPR effect. Once the particles arrive at the acidic tumor microenvironment(p H=6.5), they aggregate into large size structures with low migration, which prevents polymer nanospheres from re-entering into bloodstreams. The p H-induced aggregation promotes the accumulation and retention time in tumors and FA can accelerate the cancer cell uptake. Both of these improve the bioimaging efficiency and effectiveness. Furthermore, VANPs-1:2-FA-SDSA exhibits good biocompability and low cytotoxicity. CLSM illustrates that VANPs-1:2-FA-SDSA show an effective targeting effect on He La cells for imaging applications. Considering the outstanding properties of VANPs-1:2-FA-SDSA, including enhanced accumulation, long retention time in tumor tissues and high cell uptaking amount, it is highly promising as a candidate for efficient tumor diagnosis.