Delivery and Scavenging of Nucleic Acids by Polycationic Polymers

Electrostatic interaction is a strong force that attracts positively and negatively charged molecules to each other. Such an interaction is formed between positively charged polycationic polymers and negatively charged nucleic acids. In this dissertation, the electrostatic attraction between polycationic polymers and nucleic acids is exploited for applications in oral gene delivery and nucleic acid scavenging. An enhanced nanoparticle for oral gene delivery of a human Factor IX (hFIX) plasmid is developed using the polycationic polysaccharide, chitosan (Ch), in combination with protamine sulfate (PS) to treat hemophilia B. For nucleic acid scavenging purposes, the development of an effective nucleic acid scavenging nanofiber platform is described for dampening hyper-inflammation and reducing the formation of biofilms.

Non-viral gene therapy may be an attractive alternative to chronic protein replacement therapy. Orally administered non-viral gene vectors have been investigated for more than one decade with little progress made beyond the initial studies. Oral administration has many benefits over intravenous injection including patient compliance and overall cost; however, effective oral gene delivery systems remain elusive. To date, only chitosan carriers have demonstrated successful oral gene delivery due to chitosan’s stability via the oral route. In this study, we increase the transfection efficiency of the chitosan gene carrier by adding protamine sulfate to the nanoparticle formulation. The addition of protamine sulfate to the chitosan nanoparticles results in up to 42x higher in vitro transfection efficiency than chitosan nanoparticles without protamine sulfate. Therapeutic levels of hFIX protein are detected after oral delivery of Ch/PS/phFIX nanoparticles in 5/12 mice in vivo, ranging from 3 -132 ng/mL, as compared to levels below 4 ng/mL in 1/12 mice given Ch/phFIX nanoparticles. These results indicate the protamine sulfate enhances the transfection efficiency of chitosan and should be considered as an effective ternary component for applications in oral gene delivery.

Dying cells release nucleic acids (NA) and NA-complexes that activate the inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation related to autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease. Studies have shown that certain soluble, cationic polymers can scavenge extracellular nucleic acids and inhibit RNA-and DNA-mediated activation of Toll-like receptors (TLRs) and inflammation. In this study, the cationic polymers are incorporated onto insoluble nanofibers, enabling local scavenging of negatively charged pro-inflammatory species such as damage-associated molecular pattern (DAMP) molecules in the extracellular space, reducing cytotoxicity related to unwanted internalization of soluble cationic polymers. In vitro data show that electrospun nanofibers grafted with cationic polymers, termed nucleic acid scavenging nanofibers (NASFs), can scavenge nucleic acid-based agonists of TLR 3 and TLR 9 directly from serum and prevent the production of NF-ĸB, an immune system activating transcription factor while also demonstrating low cytotoxicity. NASFs formed from poly (styrene-alt-maleic anhydride) conjugated with 1.8 kDa branched polyethylenimine (bPEI) resulted in randomly aligned fibers with diameters of 486±9 nm. NASFs effectively eliminate the immune stimulating response of NA based agonists CpG (TLR 9) and poly (I:C) (TLR 3) while not affecting the activation caused by the non-nucleic acid TLR agonist pam3CSK4. Results in a more biologically relevant context of doxorubicin-induced cell death in RAW cells demonstrates that NASFs block ~25-40% of NF-ĸβ response in Ramos-Blue cells treated with RAW extracellular debris, ie DAMPs, following doxorubicin treatment. Together, these data demonstrate that the formation of cationic NASFs by a simple, replicable, modular technique is effective and that such NASFs are capable of modulating localized inflammatory responses.

An understandable way to clinically apply the NASF is as a wound bandage. Chronic wounds are a serious clinical problem that is attributed to an extended period of inflammation as well as the presence of biofilms. An NASF bandage can potentially have two benefits in the treatment of chronic wounds by reducing the inflammation and preventing biofilm formation. NASF can prevent biofilm formation by reducing the NA present in the wound bed, therefore removing large components of what the bacteria use to develop their biofilm matrix, the extracellular polymeric substance, without which the biofilm cannot develop. The NASF described above is used to show the effect of the nucleic acid scavenging technology on in vitro and in vivo biofilm formation of P. aeruginosa, S. aureus, and S. epidermidis biofilms. The in vitro studies demonstrated that the NASFs were able to significantly reduce the biofilm formation in all three bacterial strains. In vivo studies of the NASF on mouse wounds infected with biofilm show that the NASF retain their functionality and are able to scavenge DNA, RNA, and protein from the wound bed. The NASF remove DNA that are maintaining the inflammatory state of the open wound and contributing to the extracellular polymeric substance (EPS), such as mtDNA, and also removing proteins that are required for bacteria/biofilm formation and maintenance such as chaperonin, ribosomal proteins, succinyl CoA-ligase, and polymerases. However, the NASF are not successful at decreasing the wound healing time because their repeated application and removal disrupts the wound bed and removes proteins required for wound healing such as fibronectin, vibronectin, keratin, and plasminogen. Further optimization of NASF treatment duration and potential combination treatments should be tested to reduce the unwanted side effects of increased wound healing time.

Distribution and Conservation of the Antillean Manatee in Hispaniola

Antillean manatees (Trichechus manatus manatus) were heavily hunted in the past throughout the Wider Caribbean Region (WCR), and are currently listed as endangered on the IUCN Red List of Threatened Species. In most WCR countries, including Haiti and the Dominican Republic, remaining manatee populations are believed to be small and declining, but current information is needed on their status, distribution, and local threats to the species.

To assess the past and current distribution and conservation status of the Antillean manatee in Hispaniola, I conducted a systematic review of documentary archives dating from the pre-Columbian era to 2013. I then surveyed more than 670 artisanal fishers from Haiti and the Dominican Republic in 2013-2014 using a standardized questionnaire. Finally, to identify important areas for manatees in the Dominican Republic, I developed a country-wide ensemble model of manatee distribution, and compared modeled hotspots with those identified by fishers.

Manatees were historically abundant in Hispaniola, but were hunted for their meat and became relatively rare by the end of the 19th century. The use of manatee body parts diversified with time to include their oil, skin, and bones. Traditional uses for folk medicine and handcrafts persist today in coastal communities in the Dominican Republic. Most threats to Antillean manatees in Hispaniola are anthropogenic in nature, and most mortality is caused by fisheries. I estimated a minimum island-wide annual mortality of approximately 20 animals. To understand the impact of this level of mortality, and to provide a baseline for measuring the success of future conservation actions, the Dominican Republic and Haiti should work together to obtain a reliable estimate of the current population size of manatees in Hispaniola.

In Haiti, the survey of fishers showed a wider distribution range of the species than suggested by the documentary archive review: fishers reported recent manatee sightings in seven of nine coastal departments, and three manatee hotspot areas were identified in the north, central, and south coasts. Thus, the contracted manatee distribution range suggested by the documentary archive review likely reflects a lack of research in Haiti. Both the review and the interviews agreed that manatees no longer occupy freshwater habitats in the country. In general, more dedicated manatee studies are needed in Haiti, employing aerial, land, or boat surveys.

In the Dominican Republic, the documentary archive review and the survey of fishers showed that manatees still occur throughout the country, and occasionally occupy freshwater habitats. Monte Cristi province in the north coast, and Barahona province in the south coast, were identified as focal areas. Sighting reports of manatees decreased from Monte Cristi eastwards to the adjacent province in the Dominican Republic, and westwards into Haiti. Along the north coast of Haiti, the number of manatee sighting and capture reports decreased with increasing distance to Monte Cristi province. There was good agreement among the modeled manatee hotspots, hotspots identified by fishers, and hotspots identified during previous dedicated manatee studies. The concordance of these results suggests that the distribution and patterns of habitat use of manatees in the Dominican Republic have not changed dramatically in over 30 years, and that the remaining manatees exhibit some degree of site fidelity. The ensemble modeling approach used in the present study produced accurate and detailed maps of manatee distribution with minimum data requirements. This modeling strategy is replicable and readily transferable to other countries in the Caribbean or elsewhere with limited data on a species of interest.

The intrinsic value of manatees was stronger for artisanal fishers in the Dominican Republic than in Haiti, and most Dominican fishers showed a positive attitude towards manatee conservation. The Dominican Republic is an upper middle income country with a high Human Development Index. It possesses a legal framework that specifically protects manatees, and has a greater number of marine protected areas, more dedicated manatee studies, and more manatee education and awareness campaigns than Haiti. The constant presence of manatees in specific coastal segments of the Dominican Republic, the perceived decline in the number of manatee captures, and a more conservation-minded public, offer hope for manatee conservation, as non-consumptive uses of manatees become more popular. I recommend a series of conservation actions in the Dominican Republic, including: reducing risks to manatees from harmful fishing gear and watercraft at confirmed manatee hotspots; providing alternative economic alternatives for displaced fishers, and developing responsible ecotourism ventures for manatee watching; improving law enforcement to reduce fisheries-related manatee deaths, stop the illegal trade in manatee body parts, and better protect manatee habitat; and continuing education and awareness campaigns for coastal communities near manatee hotspots.

In contrast, most fishers in Haiti continue to value manatees as a source of food and income, and showed a generally negative attitude towards manatee conservation. Haiti is a low income country with a low Human Development Index. Only a single dedicated manatee study has been conducted in Haiti, and manatees are not officially protected. Positive initiatives for manatees in Haiti include: protected areas declared in 2013 and 2014 that enclose two of the manatee hotspots identified in the present study; and local organizations that are currently working on coastal and marine environmental issues, including research and education on marine mammals. Future conservation efforts for manatees in Haiti should focus on addressing poverty and providing viable economic alternatives for coastal communities. I recommend a community partnership approach for manatee conservation, paired with education and awareness campaigns to inform coastal communities about the conservation situation of manatees in Haiti, and to help change their perceived value. Haiti should also provide legal protection for manatees and their habitat.