Synthesis and evaluation of nanoparticle-polymer composites

This thesis describes the synthesis of functionalised polymeric material by variety of free-radical mediated polymerisation techniques including dispersion emulsion, seeded emulsion, suspension and bulk polymerisation reactions. Organic fluorophores and nanoparticles such as quantum dots were incorporated within polymeric materials, in particular, thiol-functionalised polymer microspheres, which were fluorescently labelled either during synthesis or by covalent attachment post synthesis. The resultant fluorescent polymeric conjugates were then assessed for their utility in biological systems as an analytical tool for cells or biological structures. Quantum dot labelled, thiol-functionalised microspheres were assessed for their utility in the visualisation and tracking of red blood cells. Determination of the possible internalisation of fluorescent microspheres into red blood cells was required before successful tracking of red blood cells could take place. Initial work appeared to indicate the presence of fluorescent microspheres inside red blood cells by the process of beadfection. A range of parameters were also investigated in order to optimise beadfection. Thiol-functionalised microspheres labelled successfully with organic fluorophores were used to image the tear film of the eye. A description of problems encountered with the covalent attachment of hydrophilic, thiol-reactive fluorescent dyes to a variety of modified polymer microspheres is also included in this section. Results indicated large microspheres were particularly useful when tracking the movement of fluid along the tear meniscus. Functional bulk polymers were synthesised for assessment of their interaction with titanium dioxide nanoparticles. Thiol-functionalised polymethyl methacrylate and spincoated thiouronium-functionalised polystyrene appeared to facilitate the attachment of titanium dioxide nanoparticles. Interaction assays included the use of XPS analysis and processes such as centrifugation. Attempts to synthesise 4-vinyl catechol, a compound containing hydroxyl moieties with potential for coordination with titanium dioxide nanoparticles, were also carried out using 3,4-dihydroxybenzaldehyde as the starting material

Synthesis and characterization of dual-functionalized core-shell fluorescent microspheres for bioconjugation and cellular delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins, as described in the accompanying paper

Confocal microscopy of microspheres.

<p>Confocal microscope images of DY-630 labelled microspheres conjugated to GFP <b>10a</b>: a) excited at 633 nm; b) excited at 488 nm; c) composite image showing co-localization of DY630 and GFP fluorescence. Scale bar = 4 µm.</p

Flow cytometry analysis of microspheres.

<p>Flow cytometry analysis of a) unlabelled microspheres <b>7a</b> (negative control); b) DY-630 labelled microspheres <b>8a</b>; c) DY-630 labelled microspheres <b>8b</b></p

Ceullular uptake of dual core/shell-labeled microspheres, assessed by FACS.

<p>Cellular uptake of shell-conjugated, DY-630 core-labelled microspheres by HeLa cells, as measured by flow cytometry: a) shell conjugated to fluoresceinamine 9a; b) shell conjugated to GFP 10a.</p

Composition and sizing data of microspheres <b>4</b> and <b>6a</b>–<b>h.</b>

a<p>ratio determined on the basis of mass</p><p>Mean diameter and standard deviation of thiouronium-functionalized microspheres <b>4</b> and <b>4a</b> and core-shell microspheres <b>6a</b>–<b>h</b>, as measured dispersed in water using a laser diffractometer.</p

Confocal microscopy of a single microsphere.

<p>An overlay confocal microscope image of a DY-630 labelled microsphere conjugated to GFP <b>10a</b>: excited at 633 nm (core, red) and at 488 nm (shell, green). Scale bar = 2 µm.</p

Cellular uptake of core-labeled microspheres, assessed by FACS.

<p>Percentage cellular uptake of DY-630 labelled microspheres <b>8</b> by HeLa cells, as measured by flow cytometry: a) <b>8a</b> (1 µm diameter); b) <b>8b</b> (500 nm diameter).</p

Fluorescent labeling of core-shell microspheres.

<p>Reaction conditions: i) DMF, MeOH, RT, 16 h; ii) DMF, RT, 2 h; iii) EDAC, MES pH 5.5, DMF, RT, 18 h; iv)EDAC, MES pH 6.5, NaOH, RT, 2.5 h.</p