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<p>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.</p><p>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.</p><p>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. </p><p>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.</p>
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