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.

Beam-target double-spin asymmetry A{LT} in charged pion production from deep inelastic scattering on a transversely polarized {3}He target at 1.4

We report the first measurement of the double-spin asymmetry A{LT} for charged pion electroproduction in semi-inclusive deep-inelastic electron scattering on a transversely polarized {3}He target. The kinematics focused on the valence quark region, 0.16

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells.

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an "inclusion". Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.

Measuring the $^{127}$I, $\nu_{e}$ Charged-Current Cross-Section of NaI[Tl]

The primary objective of this experiment is to measure the cross-section of $\nu_{e}$ charged-current neutrino interactions on $^{127}$I. To measure this interaction, an array of twenty-four, 7.7 kg sodium iodide (NaI[Tl]) scintillating detectors will be deployed to the Spallation Neutron Source at Oak Ridge National Laboratory. The design of the detector array is presented here along with preliminary characterization and background measurements conducted at Duke University.

Phase behavior and far-from-equilibrium gelation in charged attractive colloids

In this Rapid Communication we demonstrate the applicability of an augmented Gibbs ensemble Monte Carlo approach for the phase behavior determination of model colloidal systems with short-ranged depletion attraction and long-ranged repulsion. This technique allows for a quantitative determination of the phase boundaries and ground states in such systems. We demonstrate that gelation may occur in systems of this type as the result of arrested microphase separation, even when the equilibrium state of the system is characterized by compact microphase structures.