Preparation, Characterization And Bioapplication Of Multifunctional Organic/Inorganic Polymer Microspheres

With the research development in the field of nano-biotechnology, smart microstructures based on organic/inorganic composite polymer microspheres have attracted more and more attention owing to their broaden applications in the biology labeling, biomolecules purifications, cancer therapy and so on. Based on the research background, this thesis has focused on the preparation of multifunctional microspheres, which involves in the following parts. Firstly, the polymer microcapsules with the movable magnetic cores were prepared, and FITC, which is a common organic dye, was attached to the surface of magnetic cores to give the microcapsules with fluorescent function. Secondly, the high-quality thiol-capped CdTe nanocrystals were prepared by the hydrothermal route. As the ideal probes, the as-prepared CdTe nanocrystals were utilized in the cell labeling. Thirdly, the multifunctional microspheres, combining three advances of photoluminescence, magnetic and temperature responses into one single entity, have been prepared. Finally, for improvement of the biocompatibility of multifunctional microspheres, a robust and facile one-pot approach was presented to prepare luminescent/magnetic chitosan-poly(methacrylic acid) microspheres embedded with Fe₃O₄ and CdTe nanoparticles. The results of each part are listed as follows:(1) Based on the previous work, the magnetic and thermoresponsive microspheres were prepared by coating the Fe₃O₄@SiO₂ particles with the cross-linked poly(N-isopropylacrylamide) (PNIPAM) shells via precipitation polymerization. Since PNIPAM shells have a fast response to the temperature stimuli, the silica shells sandwiched by the PNIPAM shells and magnetic cores can be controllably etched by NaOH solution under the condition of an "open" or "close" state for PNIPAM shells, forming the PNIPAM microcapsules with mobile magnetic cores. For functionalizing the microcapsules, such smart hollow spheres also could serve as microreactors to introduce fluorescence property for them by post-modification of the residue silica on the cores. The organic dyes (FITC) were successfully coupled on the magnetite cores, resulting in forming the desired multifunctions of polymer microcapsules.(2) The high-quality thiol-capped CdTe nanocrystals were prepared by the hydrothermal technique. The growth and photoluminescence dependence of thiol-capped CdTe nanocrystals on the reaction conditions were systematically studied. Through the optimum conditions, including monomer-to-ligand molar ratio, two monomer ratios, precursor concentration and reaction temperatures, the maximum quantum yield of CdTe nanocrystals arrived up to 50%. Meanwhile, it was discussed that both of mono-thiol complexes and dual-thiol complexes significantly affected the nucleation and growth. In addition, it was well demonstrated that the synthesized CdTe nanocrystals have excellent photoluminescence properties and biology compatibleness in the cell labeling experiments. Although the as-prepared CdTe nanocrystals precipitated in a short period under the radiation of a UV lamp, the composite nanoparticles have the better stability with a silica-coating post-treatment. And the core-shell structures can be obtained by the second growth of silica shells on the post-treated CdTe nanoparticles, which is in favor of modification of biomolecules on the surface of silica for the targeted fluorescence probes.(3) Based on the excellent luminescence properties of CdTe nanocrystals, a rational design to prepare luminescent/magnetic composite microspheres with combining advances was discussed in detail. Firstly, the TGA-stabilized CdTe and silica particles can be co-precipitated by adding the Cd²⁺ ions through electrostatic interactions. Induced by it, magnetite nanoparticles (Fe₃O₄) were homogeneously incorporated into silica spheres using the modified Stober method, and thiol-capped CdTe nanocrystals were building up a luminescent shell on the surface of magnetic silica spheres. The obtained Fe₃O₄@SiO₂@CdTe particles were coated by an outer shell of silica for avoiding the corrosion of CdTe shells, while providing a robust platform for the covalent binding of specific ligands. The luminescent/magnetic microspheres maintained the photostability and allowed for the surface modification. The subsequent design is that these microspheres were used as seeds to be covered by the crosslinked poly(N-isopropylacrylamide) (PNIPAM). We characterized the particle morphology, size distribution, thermosensitive, magnetic and luminescent properties. For the potential application of intracellular drug delivery, we also demonstrated PNIPAM-covered luminescent/magnetic microspheres were able to be taken up by CHO cells without inducement of any specific ligands. As a result, the designed multifunctional microspheres have much promise in detecting and treating cancer at its earliest stages due to their advances of magnetic targeting, excellent imaging, and drugs-carried matrices, and will be extended to in-vivo systems in the next experiments.(4) In the current study, a facile one-pot approach was presented to prepare luminescent/ magnetic chitosan-poly(methacrylic acid) microspheres with the biodegradable property. The different kinds of -COOH modified nanoparticles (Fe₃O₄ and CdTe) can be designed to be encapsulated within the chitosan(CS)-based colloids based on the complexation between CS and nanoparticles and subsequent polymerization of methacrylic acid, resulting in formation of the multifunctional organic/inorganic hybrid vectors. The composite microspheres have the narrow size distribution and regular spheric shape. Due to the favorable molar ratio of [methacrylic acid]:[glucosamine units] (4:1), the high envelopment of Fe₃O₄ nanoparticles allows the controllable magnetic content by calculating the feed amount. Highly luminescent CdTe semiconductor nanocrystals encapsulated in the vectors presented the excellent photoluminescence property as pH was adjusted from 5 to 11. This method would be extended to prepare the various kinds of organic/inorganic composite microspheres in a large scale on the basis of the complexation between CS and inorganic nanoparticles and subsequent polymerization of cationic monomers. Also, PLC/PRF/5 cells were chosen to assess cellular uptake of these multifunctional vectors. The result shows such vectors can significantly improve the rate and extent of transfect into cells under the short-term exposure to the applied magnetic field. This suggests that the luminescent/magnetic CS-based vectors have a great potential to be used as bioactive molecule carriers for gene therapy.