Aminoglycoside antibiotics such as kanamycin and neomycin have been known to cause severe hearing loss in some patients. The Rubel Lab in the UW Department of Otolaryngology has recently reported a molecule, termed PROTO1, which has been shown to protect cochlear hair cells from aminoglycoside induced degradation. However, due to the poor vascularization of the cochlea, it is difficult to deliver ototherapeutic drugs systemically, and thus other methods of delivery must be explored. This study reports on the development of a PROTO1-loaded hydrogel based on the amphiphilic triblock copolymer poly(ethylene oxide)-poly[(r)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO). In this study, two cross linking mechanisms were explored. The first uses the oligosaccharide α-cyclodextrin to thread α-cyclodextrin onto the terminal PEO chains of the micelles resulting in a supramolecular structure termed Pluronic/aCD pseudorotaxanes. Hydrogels formulated in this manner showed controlled linear release of PROTO1 over 25 days but are difficult to manipulate and requires sonication and heating to achieve gelation. An ideal hydrogel would be able to undergo in situ gelation, in which the hydrogel components remain liquid prior to being introduced to the active site, upon which they immediately cross-link to form a hydrogel. In this work, we synthesize materials for an alternative approach that is based on crosslinked cyclodextrin-modified polymers. The synthesis and characterization of the polymer components are reported.

The field of gene therapy relies on vehicles that can introduce exogenous genetic material into target cells in order to affect the production of a desired therapeutic protein. Viruses have long been used as delivery vehicles, but concerns over immunogenicity have fueled efforts to develop alternative nonviral delivery vehicles. Current polycationic polymers used in gene delivery methods generally exhibit poor transfection efficiencies and also dose-dependent cell toxicity. This project seeks to address these parameters through the synthesis of a novel copolymer of 2-hydroxypropyl methacrylate (HPMA) and a methacryated oligoarginine peptide (MaAhxR10). In phase one, the HPMA-oligoarginine copolymer was synthesized and tested for basic functionality. Transfections of HeLa cell populations demonstrated that the transfection capabilities of this polymer were comparable to those of the oligolysine standard, but the toxicity was significantly enhanced. Phase two consisted of modifying the peptide with neutral amino acid residues (such as glycine) to reduce the charge density of the polycationic peptides. Further, a more efficient approach to preparing arginine peptides was developed by chemically guanidinylating lysine residues. This approach was used to generate a lysine and a homoarginine version of each polymer. The resulting copolymers were tested for transfection efficiency and cell toxicity in cultured mammalian cells. Copolymers made from the resulting ‘spaced’ peptides (MaAhx(GK)5 and MaAhx(GhR)5) demonstrated reduced cytotoxicity compared the MaAhxR10 and PEI standards, but were still comparable in transfection efficiency to the oligolysine standard. While the delivery vehicle has not performed as expected, the project may have revealed a relationship between charge density (and therefore accessibility of charge) and cytotoxicity. The next step will be to generate another panel of polymers, this time with even more spacing between positive charge residues. This will allow for the identification of a correlation between charge density and cytotoxicity.

Gene therapy is the treatment of disease by inserting genes or altering them in a subject’s cells and tissues. The Pun lab develops cationic polymer-based delivery vehicles. Our agenda was to determine the intracellular fate of drug delivery vehicles carrying the therapeutic genes in order to better understand cellular barriers that limit efficient delivery. For this project, we applied two methods to study the intracellular trafficking of polyplexes (polymer-DNA complexes). The first approach is a quantitative method using subcellular fractionation to separate organelles and determine the distribution of polyplexes in various cellular compartments after internalization. We hypothesized that by using methods of centrifugation, organelle-specific enzyme assays, and radiolabeling, polyplex distribution could be quantitatively analyzed. We demonstrated successful separation and quantification of organelles using a combination of differential and density-gradient centrifugation. The polyplex distribution in each fraction was determined through radiolabeling. In this way, a quantitative “snapshot” was taken of the distribution of polyplexes inside the cell, and yielded information for the identification of potential bottlenecks in trafficking to the nucleus. Our second approach is to study the intracellular trafficking of drug delivery vehicles by time-lapse fluorescence imaging combined with single-particle tracking. This complementary approach reveals both the time-course and behavior of particles as they are trafficked within the cell. By fluorescently -labeling the polymer and applying the polyplexes to cells, we successfully observed their motion within a single cell. We further optimized the quantity of polyplexes delivered and the image capture techniques to robustly track polyplexes within a single cell. Our work therefore provides improved methods for determining the mechanism and efficacy of gene delivery systems and potentially any particle-based drug delivery system. For future work, we plan to investigate the process of unpackaging of the polyplexes inside cells.