Hydrogels for Biomacromolecule Immobilization and Sensing

125 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.The main focus of this thesis centers on the development of stimuli-responsive hydrogels that have utility for pH-responsive valves, biomacromolecule sensing, irreversible biomacromolecule immobilization, and reversible biomacromolecule immobilization. Each system discussed has its function derived from a novel fabrication protocol or through the development of new hybrid polymer biomaterials. Molecular sensing was accomplished by preparing crosslink-cleavable hydrogels with progressively more complex recognition elements. Initial work investigated the swelling kinetics of disulfide crosslinked hydrogels as a model system for protease responsive hydrogels. More complex peptide-containing hydrogels that have utility for biomacromolecule detection, were then prepared using a novel disulfide-exchange protocol for the preparation of methacrylamide containing peptides. Utilizing this protocol, methacrylamide containing peptides that were responsive to alpha-chymotrypsin (CKYC) and botulism neurotoxin (CSNKTRIDEANQRATK{Nle}LC) were successfully synthesized and copolymerized into polyacrylamide hydrogel networks. When exposed to buffered solutions that contain their sequence specific protease, the hydrogels were completely dissolved. Hydrogels capable of covalent and reversible biomacromolecule immobilization were also developed. Covalent attachment of proteins and lipids to a pH-responsive scaffold was accomplished through a mild oxidation of glycerol monomethacrylate- co-acrylic acid hydrogels, followed by chemoselective ligation of aminooxy, hydrazide, or amine functionalized molecules. Using this strategy, fluorescently labeled proteins as well as lipids that can maintain a chemical gradient between the inside and outside of the hydrogel were prepared. Finally, design elements for surface-immobilized temperature responsive hydrogels that have utility for reversible biomacromolecule immobilization were investigated. Atom transfer radical polymerization was used to grow poly(N-isopropylacrylamide) polymer films from surfaces with varying initiator densities and polymer molecular weights. The lower critical solution temperature behavior for these films were quantified. Finally, the appendix describes a silica colloid system that undergoes a charge reversal once exposed to UV light. Such systems should be useful for in situ modification of colloidal particles, a technique not currently available.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Hydrogels for Biomacromolecule Immobilization and Sensing

125 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2005.The main focus of this thesis centers on the development of stimuli-responsive hydrogels that have utility for pH-responsive valves, biomacromolecule sensing, irreversible biomacromolecule immobilization, and reversible biomacromolecule immobilization. Each system discussed has its function derived from a novel fabrication protocol or through the development of new hybrid polymer biomaterials. Molecular sensing was accomplished by preparing crosslink-cleavable hydrogels with progressively more complex recognition elements. Initial work investigated the swelling kinetics of disulfide crosslinked hydrogels as a model system for protease responsive hydrogels. More complex peptide-containing hydrogels that have utility for biomacromolecule detection, were then prepared using a novel disulfide-exchange protocol for the preparation of methacrylamide containing peptides. Utilizing this protocol, methacrylamide containing peptides that were responsive to alpha-chymotrypsin (CKYC) and botulism neurotoxin (CSNKTRIDEANQRATK{Nle}LC) were successfully synthesized and copolymerized into polyacrylamide hydrogel networks. When exposed to buffered solutions that contain their sequence specific protease, the hydrogels were completely dissolved. Hydrogels capable of covalent and reversible biomacromolecule immobilization were also developed. Covalent attachment of proteins and lipids to a pH-responsive scaffold was accomplished through a mild oxidation of glycerol monomethacrylate- co-acrylic acid hydrogels, followed by chemoselective ligation of aminooxy, hydrazide, or amine functionalized molecules. Using this strategy, fluorescently labeled proteins as well as lipids that can maintain a chemical gradient between the inside and outside of the hydrogel were prepared. Finally, design elements for surface-immobilized temperature responsive hydrogels that have utility for reversible biomacromolecule immobilization were investigated. Atom transfer radical polymerization was used to grow poly(N-isopropylacrylamide) polymer films from surfaces with varying initiator densities and polymer molecular weights. The lower critical solution temperature behavior for these films were quantified. Finally, the appendix describes a silica colloid system that undergoes a charge reversal once exposed to UV light. Such systems should be useful for in situ modification of colloidal particles, a technique not currently available.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Controlled Regioregularity in Oligo(2-methoxy-5-(2′-ethylhexyloxy)-1,4-phenylenevinylenes

A series of three pentameric derivatives of 2-methoxy-5-(2′-ethylhexyloxy)-<i>p</i>-phenylenevinylenes (<b>2</b>–<b>4</b>), with varying degrees of side chain regioregularity, was prepared. The oligomerization chemistry was carried out using repetitive Horner–Wadsworth–Emmons (HWE) reactions of precisely substituted aryl rings with four different substituents. The resulting oligomers were characterized by nuclear magnetic resonance spectroscopy (NMR), cyclic voltammetry (CV) and absorption spectroscopy in solution and in thin films. Each of the oligomers gave discrete ¹H and ¹³C NMR spectra. The regioregular pentamer (<b>2</b>) displayed the most resolution between signals and suggests those nuclei reside in more unique chemical environments than the regiorandom pentamers (<b>3</b> and <b>4</b>). The solution phase electronic (CV) and absorption properties of each of the new oligomers were found to be essentially identical. In contrast, the thin film absorption spectra were not equivalent. The more regioregular pentamers (<b>2</b> and <b>3</b>) possessed a new, red-shifted shoulder structure that suggests the packing order is heavily influenced by side chain regioregularity even at the pentamer level

Orthogonal Functionalization of Cyclopenta[<i>hi</i>]aceanthrylenes

A synthetic strategy to prepare 2,7- or 4,9-functionalized cyclopenta[<i>hi</i>]aceanthrylenes that are capable of Suzuki cross-coupling reactions is demonstrated. This method has been utilized to create a series of thiophene derivatized compounds that were subsequently used to investigate the role of substitution pattern on the photophysical and electronic properties of cyclopenta[<i>hi</i>]aceanthrylenes. The orthogonal functionalization provides access to unique substitution patterns (e.g., cruciform-like architectures) and materials with small optical band gaps (1.22–1.97 eV)

The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine.

Intestinal epithelial cells (IECs) overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes) play an active role in the uptake (sampling) of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs), which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20-40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer's patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine). Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies

Directing the Conformation of Oligo(phenylene­vinylene) Polychromophores with Rigid, Nonconjugatable Morphons

The synthesis and morphological investigation of a series of polychromophore polymers composed of oligomeric bis­(2-ethyl­hexyl)-<i>p</i>-phenylene­vinylene (BEH-PPV) (trimer, pentamer, and heptamer) monomers copolymerized with rigid morphological directing groups (morphons) are described. The polymerization was carried out using a Sonogashira cross-coupling polymerization between monomers composed of an iodo-terminated BEH-PPV oligomer and a bis­(phenyl­acetylene)-containing morphon. The rigid morphons are prepared from adamantane and diamantane frameworks that are composed solely of sp³ carbons, inhibit conjugation between BEH-PPV oligomers, and direct the local polymer morphology in either a bent or linear vector, respectively. The morphological properties of the polychromophore polymers were interrogated via single molecule fluorescence spectroscopy, thin-film absorption and fluorescence spectroscopy, and atomic force microscopy. Significant morphological variation was found upon substituting the morphon, as well as the chromophore size, with the most ordered structures being accessed with diamantane morphons

Conjugated Ladder Polymers by a Cyclopentannulation Polymerization

We report a nontraditional synthesis of cyclopentafused-polycyclic aromatic hydrocarbon embedded ladder polymers using a palladium catalyzed cyclopentannulation polymerization followed by a cyclodehydrogenation reaction. Donor–acceptor type polymers containing a cyclopenta­[hi]­aceanthrylene acceptor groups can be synthesized by a palladium catalyzed copolymerization between 9,10-dibromoanthracene and a variety of bis­(arylethynyl)­arenes to give polymers with molecular weights (Mn) of 9–22 kDa. The bis­(arylethynyl)­arenes were composed of benzene, thiophene, or thieno­[3,2-<i>b</i>]­thiophene moieties, which provided access to a series of four donor–acceptor copolymers. The polymers were subjected to cyclodehydrogenation with FeCl₃ to access rigid ladder type polymers with the conversion investigated by ¹³C NMR of isotopically labeled polymers. The ladder polymers possess broad UV–Vis absorptions and narrow optical band gaps of 1.17–1.29 eV and are p-type semiconductors in organic field effect transistors

The presence of NPs in the IECs isolated from the mouse SI.

<p>40(red) were injected into the lumen of the SI and 30 minutes later the SI was excised, Peyer's patches were removed (discarded), and IECs were isolated from the SI sections. (A–D) Isolated IECs from mice that were administered NPs (A, B) or PBS (C, D) were fixed then placed on a glass slide and imaged with a fluorescent microscope at 630× magnification. (A, B) A patch of IECs isolated from NP-treated mouse imaged in the green channel (autofluorescence) (A) and the red channel (red: NPs) (B). Characteristic GAPs that are not highlighted by NPs appear as black holes in isolated IEC patches (white arrows), while IECs exhibit strong red fluorescence due to the presence of NPs (similar to images taken in vivo). (C, D) No red fluorescence was detected in IEC patches isolated from a control mouse. (E) Expression of E-cadherin (green) in isolated IECs imaged with a fluorescence microscope. (F) A confocal image of IECs isolated from NP-treated mouse showing strong red fluorescence in IEC cytoplasm. (G) Distribution of E-cadherin (green) in a section of SI. Actin staining with phalloidin-Alexa 350 (blue) highlights the tissue architecture. (H) Western blot analysis of E-cadherin (120 kDa) expression in isolated IECs (Lane 1) or spleen lymphocytes (Control, Lane 3). Lane 2: Spectra™ multicolor protein ladder. Each image is a representative of at least 3 experiments.</p

Distribution of NPs in the SI and MLNs of mice 30–40 minutes after administration in the SI.

<p>(A) A three color IFM image of a villus showing co-localization of NPs (red) with CD11c+ DCs (yellow) in the LP. Actin highlighted by phalloidin-Alexa 350 (blue). (B) A three color IFM image of a MLN 40 minutes after NP administration into the SI. NPs (red) seen in the capsule of the MLN. The lymphatics of MLN stained with Lyve-1 antibodies (green). Actin is highlighted by phalloidin-Alexa 350 (blue). (C) A higher resolution three color image of MLN capsule taken at 630×. Some NPs (red) appear to be cell-bound (arrows). (D) A three color IFM image of an intestinal section. NPs (red) colocalize with the lymphatics (Lyve-1 staining, green, circled) in the submucosa and highlight the serosa (white arrow). Actin is highlighted by phalloidin-Alexa 350 (blue). (E) A confocal image of the SI serosa taken in vivo 40 minutes after NP (red) and dextran-fluorescein (green) administration. (F) A confocal image of the SI serosa taken in vivo 40 minutes after administration of dextran-fluorescein (green, control). Each image is a representative of at least 3 experiments.</p