Popielarski, Stephen R (2005) I. Structural effects of carbohydrate-containing polycations on gene gelivery. II. Development of a nanoparticle-based model delivery system to guide the rational design of gene delivery to the liver. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-03302005-120327
Linear cationic beta-cyclodextrin (b-CD)-based polymers can bind with plasmid DNA to form colloid-sized composite particles that transfect cultured cells. In the first part of this thesis, synthetic variations of the b-CD structure are used to probe structure-function gene delivery properties. The type of cyclodextrin and its functionalization are investigated by synthesizing numerous 3A,3B-dideoxy-3A,3B-diamino-b- and g-CD monomers, which are polymerized with dimethyl suberimidate to yield amidine-based polycations. The nature of the spacer between the CD-ring and the primary amines of each monomer is found to influence both molecular weight and polydispersity of the polycations. When complexed with plasmid DNA, polycations with longer alkyl regions between the CD and the charge centers show increased transfection efficiency and toxicity in BHK-21 cells. More hydrophilic spacers resulted in lower toxicity, and g-CD-based polycations were less toxic than otherwise identical b-CD-based polycations.
In the second part of this thesis, a model delivery system is developed that can mimic the size and surface properties of the cyclodextrin-based gene-delivery particles, and this system is used to define design constraints that should be applied to next generation gene delivery particles targeted to the liver. Gal-50 and Gal-140 are galactosylated 50 nm and 140 nm nanoparticles that have the same surface galactose density, while MeO-50 and MeO-140 are methoxy-terminated 50 nm and 140 nm nanoparticles. All four particles have the same surface charge and resist aggregation in serum.
In freshly isolated hepatocytes, Gal-50 nanoparticles are taken up to a greater extent than are MeO-50, but both 50 nm beads are taken up to a much greater extent than are either of the 140 nm nanoparticles. TEM and immunohistochemistry confirm that Gal-140 nanoparticles are primarily internalized by Kupffer cells, though isolated examples of a few Gal-140 in hepatocytes can also be found. On the other hand, Gal-50 nanoparticles are overwhelmingly found in vesicles throughout the cytoplasm of hepatocytes, with only isolated examples of Kupffer cell uptake. As such, it is clear that slightly anionic, galactose-PEGylated nanoparticles should be about 50 nm in diameter to preferentially target hepatocytes while they should be about 140 nm in diameter to selectively target Kupffer cells.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||gene delivery; gene therapy; hepatocyte targeting; liver targeting; nanoparticle delivery system|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||4 March 2005|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||31 Mar 2005|
|Last Modified:||26 Dec 2012 02:36|
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