Synthesis and performance of molecules for protein crystallization and drug & gene delivery
Abstract
Membrane proteins are essential to biological systems, however, there are fewer than 250 membrane protein structures that have been atomically-resolved, largely due to limitations of expression and crystallization of these biomacromolecules. We have developed new strategies for protein crystallization using water-soluble nucleating agents that are amenable to membrane protein applications. A small library of conformationally-restricted, 2-, 3- and 4-fold multivalent nitrilotriacetic (NTA) ligands have been developed to bind proteins bearing polyhistidine tags in a symmetry-guided manner in the presence of Ni(II). These reagents seek to control protein:ligand speciation and promote self-assembly of the protein:ligand complexes in a manner that provokes his-protein crystal nucleation and growth. Several lines of evidence using his6-GFP as a model system suggest that these reagents are capable of achieving these design goals. Dendritic cells (DC) are potent professional antigen presenting cells in the immune system and play a central role in adaptive immunity against a variety of pathogens and tumors. For targeting DCs, mannose-PEG3000-DSPE, and BCAT were synthesized and a series of liposomes containing different molar ratios of mannosylated lipid and DOPE were prepared. The gene delivery efficiency of these liposomes in DC was examined via mixing with Plasmid gWIZ GFP. Based on the transfection study, the obtained result suggested that gene delivery efficiency in dendritic cells could be affected not only by mannose density but also by BCAT and DOPE ratio. A change in pH environmental occurs during endosomal trafficking and tumor growth; it also plays a crucial role of endosomal uptake of drugs by tumor cells. We developed a novel class of pH-sensitive linkers, phenyl substituted vinyl ether linkers (PIVEs), which were designed for use as heterobifunctional crosslinkers to create biodegradable carriers with tunable pH-sensitivity. In this study, a series of mPEG-lipids molecules with PIVE linkers was prepared and the acid-triggered release of liposomal cargo via liposome dePEGylation of the PIVE mPEG-lipid conjugates was examined. Bladder cancer is one of the most prevalent cancers in men in the US. Mycobacterium bovis bacillus Calmette-Guerin (BCG) therapy is widely used to treat malignant bladder cancer. A recent report showed that Fibronectin Attachment protein (FAP) inhibits bladder tumor growth and can be an important mediator of BCG anti-tumor activity. To understand the mechanisms of attachment and internalization of FAP in bladder carcinoma cells, novel tris-nitrilotriacetic acid (NTA)-Alexa probes were prepared and tested for their ability to promote bladder cell internalization of his-tag FAP. In addition, fluorescently-labeled liposomes containing trisNTA-PEG-modified lipids were constructed for confirming target specificity and localization of his-tag target liposomes in bladder tumor cells.
Degree
Ph.D.
Advisors
Thompson, Purdue University.
Subject Area
Pharmacology|Biochemistry|Organic chemistry
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