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Understanding the biological mechanisms of polymeric DNA delivery is essential to develop vehicles that perform optimally. In this work, the cellular internalization mechanisms of poly(glycoamidoamine) (PGAA) DNA delivery polymers were investigated. Polymer:DNA complexes interact with cell-surface glycosaminoglycans (GAGs) in a manner independent of electrostatic interactions. Desulfation and GAG removal leads to decreased uptake. Individual polyplexes appear to have differing affinities for specific GAGs, as polyplex dissociation occurs in a charge-independent manner, and may influence binding. Internalization occurs through close interactions with GAGs, as GAGs accumulate on polyplex surfaces, resulting in negatively-charged polyplexes and decompaction of intact polyplexes is observed upon interaction with GAG. PGAA polyplexes enter cells via a complex, multifaceted internalization route. Pharmacological inhibition of endocytosis and visualization by confocal microscopy reveal that internalization occurs primarily through an actin and dynamin-dependent mechanism. Caveolae/raft-mediated endocytosis appears to be the predominant internalization mechanism, with clathrin-mediated endocytosis also significantly involved. Internalization occurs to a smaller degree via macropinocytosis and direct membrane penetration. Caveolae-mediated, but not clathrin-mediated, internalization leads to transgene expression, suggesting a targeting opportunity based on uptake mechanisms. PEGylation of PGAA polyplexes was achieved to minimize polyplex aggregation in serum. Polyplex size increased in serum, but PEGylation prevented further polyplex growth over time compared to non-PEGylated polymers. Specific targeting of hepatocytes through end-modification of PEG with galactose was unsuccessful, likely due to inaccessibility of targeting groups. Further hepatocyte targeting efforts focused on malonate-based polymers with clickable linkages for facile linkage of targeting groups. Despite favorable surface presentation of galactose, receptor-specific internalization of polyplexes was unsuccessful, as competitive inhibition in HepG2 cells resulted in significant polyplex internalization derived from nonspecific membrane interactions. Chemical modification of vehicles allows systematic study of structure-function properties leading to efficient intracellular delivery. Increasing G4 molecular weight generally increases toxicity and decreases transgene expression in HeLa cells. Incorporating galactose into a lanthanide-chelating polymer facilitated efficient cellular internalization that was visualized by two-photon microscopy. Increased gene expression was observed that correlated to increasing galactose, suggesting that polymer degradation increases gene expression. Also studied were branched peptides targeted to HIV-1 TAR, which displayed high biocompatibility and favorable internalization profiles in mammalian cells. / Ph. D.

The cysteine proteinases cathepsins B, L, and S are lysosomal enzymes responsible for the degradation of endocytosed proteins. Their presence in human cell monocytic lines THP1 and U937 was detected by the use of the membrane-permeable, irreversible, active-site directed inhibitor Fmoc-(¹²⁵I)Tyr- Ala-CHN₂ followed by immunoprecipitation of the enzymes, SDSPAGE, and autoradiography. All three enzymes were detected in THP1 cells; only after differentiation of U937 cells to macrophage-like cells were the enzymes detectable. Both cell lines show multiple forms of cathepsin S, at 35 kDa, 28 kDa, and 26 kDa, suggesting the presence of an active pro-form of cathepsin S as well as the processing of cathepsin S into single- and two-chain forms. This is the first evidence for an active pro-form of a cysteine proteinase and for the processing of cathepsin S to a two-chain enzyme form. Multiple forms of cathepsin L were analyzed by isoelectric focusing followed by denaturing polyacrylamide gel electrophoresis. The multiple forms are not due to the presence of carbohydrate chains on the protein. The inhibitor Fmoc-Tyr-Ala-CHN₂ synthesized and its inhibitory properties against cathepsins B, L, and S were determined. Both in vitro and in vivo studies show that this inhibitor is an effective reagent for studying lysosomal cysteine proteinases. In order to be useful in the study of the delivery of lysosomal enzymes to vesicles containing recently internalized compounds, the deblocked peptidyl diazomethane inhibitor NH₂-Tyr-Ala-CHN₂ was cross-linked to bovine serum albumin (BSA) using the heterobifunctional crosslinking agent sulfo-SANPAH. This non-reducible cross-linked complex was used to characterize the inhibitory properties of the protein-inhibitor complex against cathepsins B, L, S and papain in vitro and in vivo. / Ph. D.

Yes / The activity-regulated cytoskeleton associated protein (Arc/Arg3.1) plays a key role in determining synaptic strength through facilitation of AMPA receptor (AMPAR) endocytosis. Although there is considerable data on the mechanism by which Arc induction controls synaptic plasticity and learning behaviours, several key mechanistic questions remain. Here we review data on the link between Arc expression and the clathrin-mediated endocytic pathway which internalises AMPARs and discuss the significance of Arc binding to the clathrin adaptor protein 2 (AP-2) and to endophilin/dynamin. We consider which AMPAR subunits are selected for Arc-mediated internalisation, implications for synaptic function and consider Arc as a therapeutic target. / The work in S.A.L.C. laboratory is supported by the BBSRC (BB/H018344/1 and BB/J02127X/1) and Wellcome Trust 200646/Z/16/Z. The work in M.J.W. Laboratory is supported by ERUK.

Fibroblast growth factor (Fgf) signaling plays multiple inductive roles during development of vertebrates (Itoh 2007). Some Fgfs, such as Fgf8, are locally secreted and signal over a long range to provide positional information in the target tissue (Scholpp and Brand 2004). Fgf ligands signal in a receptor-dependent manner via tyrosine kinase receptors, four of which have been so far identified. Fgf8 signaling was shown to depend both on receptor activation as well as endocytosis. The specificity of Fgf ligands and receptors as well as the function of receptors in the control of the Fgf signaling range have been, however, largely unclear. In this study, we show that the putative Fgf8 receptor Fgfr1 is duplicated in zebrafish and that it acts redundantly in the formation of the posterior mesoderm. Also, in overexpression studies we confirm the notion that receptor endocytosis influences Fgf8 signaling range. Through TILLING mutant recovery and morpholino knockdown studies we also show that Fgfr2 is required for growth and skeletal development in zebrafish, whereas Fgfr4 is required for pectoral fin specification and growth.

<p>Class A scavenger receptors are multifunctional transmembrane glycoproteins that mediate macrophage functions like phagocytosis and endocytosis. The macrophage receptor with collagenous structure (MARCO) is one such receptor. It has been shown that the extracellular cysteine-rich domain of MARCO is responsible for ligand binding, but the role of the cytoplasmic domain in ligand uptake is unclear. The aim of the studies presented in this thesis is to characterize the role of the cytoplasmic domain of MARCO and to characterize the molecular pathway of MARCO-mediated endocytosis.</p> <p>Full-length human MARCO (hMARCO) and Δ1-34hMARCO, which lacks the first thirty-four amino acids were created in order to determine whether amino acids 1-34 contained residues required for receptor internalization and surface expression. The constructs were stably expressed in HEK293T cells and found to have similar levels of surface expression and same rate of internalization without ligand. Interestingly, hMARCO, but not Δ1-34hMARCO, surface expression was up-regulated upon ligand incubation.</p> <p>In order to ascertain the importance of clathrin, dynamin and actin in MARCO-mediated endocytosis, specific endocytic inhibitors were used. MARCO-mediated ligand uptake was inhibited when clathrin and actin polymerization and, dynamin functions were impaired by these inhibitors. Furthermore, ligand uptake by Δ1-34hMARCO-expressing HEK293T was insensitive to inhibitors of clthrin and dynamin but not inhibitors of actin.</p> <p>In conclusion, MARCO mediates endocytosis via a clathrin-mediated, dynamin-dependent pathway that involves actin. Amino acids 1-34, are required clathrin and dynamin but not actin functions during MARCO-mediated endocytosis. Additionally, amino acids 1-34 might also be important for MARCO recycling but not receptor internalization or surface expression.</p> / Master of Science (MSc)

Gemini surfactants are versatile gene delivery agents because of their ability to bind and compact DNA and their low cellular toxicity. The aim of my dissertation work was to develop non-invasive mucosal formulations of novel amino acid-substituted gemini surfactants with the general chemical formula C12H25(CH3)2N+-(CH2)3-N(AA)-(CH2)3-N+(CH3)2-C12H25 (AA= glycine, lysine, glycyl-lysine, lysyl-lysine). These compounds were formulated with a model plasmid DNA, encoding for interferon-γ and green fluorescent protein, in the presence of helper lipid, 1,2 dioleyl-sn-glycero-phosphatidyl-ethanolamine. Formulations were assessed in Sf 1 Ep epithelial cells. Among the novel compounds, plasmid/gemini/lipid (P/G/L) nanoparticles formulated using glycine- and glycyl-lysine substituted gemini surfactants achieved significantly higher gene expression than the parent unsubstituted compound. The key physicochemical properties, e.g. size, surface charge, DNA binding, and toxicity of P/G/L complexes were correlated with transfection efficiency. The presence of amino-acid substitution did not interfere with DNA compaction and contributed to an overall low toxicity of all P/G/L complexes, comparable to the parent gemini surfactant. A cellular uptake mechanistic study revealed that both clathrin- and caveolae-mediated uptake were major uptake routes for P/G/L nanoparticles. However, amino acid substitution in the gemini surfactant imparted high buffering capacity, pH-dependent increase in particle size, and balanced DNA binding properties. These properties may enhance endosomal escape of P/12-7NGK-12/L resulting in higher gene expression. Finally, the P/G/L complexes were incorporated into an in-situ gelling dispersion containing a thermosensitive polymer, poloxamer 407, and a permeation enhancer, diethylene glycol monoethyl ether (DEGEE). A 16% w/v poloxamer concentration produced a dispersion that gelled at body temperature and exhibited sufficient yield value to prevent formulation leakage from the vaginal cavity. The formulations were prepared with a model plasmid, encoding for red fluorescent protein, and administered topically to rabbit vagina. In agreement with our in vitro results, confocal microscopy revealed that glycyl-lysine substituted gemini surfactant exhibited higher gene expression compared to the parent unsubstituted gemini surfactant. This provided proof-of-concept for use of amino acid-substituted gemini surfactant in non-invasive mucosal (vaginal) gene delivery systems with potential therapeutic applications. These formulations will be developed with therapeutically relevant genes to assess their potential as genetic vaccines. In addition, new gemini surfactants will be developed by grafting other amino acids via glycine linkage to retain conformation flexibility and enhance endosomal escape of DNA complexes for higher transfection efficiency.