SYNTHESIS AND ANTICANCER ACTION OF NITRIC OXIDE-RELEASING LIPOSOMES Public Deposited

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  • March 21, 2019
Creator
  • Suchyta, Dakota
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • The implementation of nitric oxide (NO)-based therapeutics has been met with formidable challenges relating to NO’s gaseous, reactive nature and difficulties associated with controlled delivery. Although macromolecular vehicles have been developed for applications in NO release, a common limitation associated with these systems is exposure of the NO donor to the surrounding medium, resulting in unintended NO release. To overcome this issue, liposomes were investigated as new vehicles for NO delivery whereby the NO donor is encapsulated within the aqueous core, protected from the external solution by a lipid membrane. Liposomes with encapsulated N-diazeniumdiolate NO donors were first synthesized using a reverse-phase evaporation protocol. Encapsulation efficiencies for several molecular NO donors were in the range of 33–41%. Relative to the unencapsulated (free) NO donor, NO-release half-lives at pH 7.4 were up to 7-times greater upon encapsulation, yet the NO-releasing liposomes still exhibited their unique pH-sensitive release properties. The liposomes retained ~80% of the encapsulated NO concentrations after 3 months of storage at 4°C, indicating excellent stability. In order to determine if the liposomes held merit as therapeutic agents, cytotoxicity against human pancreatic cancer cells were performed that demonstrated the liposomal NO donors required less NO to kill versus the free NO donor (183 μM and 2.4 mM, respectively). The ability to tune NO-release kinetics of these liposomes was further studied. It was possible to vary the NO-release kinetics by altering the encapsulated NO donor molecule or the phospholipid composing the bilayer (independently or in combination). Phospholipid headgroup surface area was determined to be a main factor in controlling NO-release half-lives. As the surface area of the lipid headgroup was decreased from 0.660 nm2 to 0.420 nm2, a concomitant increase in NO-release half-life was also observed. The composition of the lipid bilayer is known to affect in vivo properties, so NO-release kinetics were also measured in serum and whole blood. Half-lives in serum were equivalent to those measured in buffer, while those measured in blood were ~60% faster. An investigation into the cytotoxicity of slow (t1/2 > 72 h) versus fast (t1/2 ~ 2.5 h) NO-releasing liposomes demonstrated how the biological consequences were dependent on the NO-release rate. Fast NO-releasing liposomes yielded consistently higher LD50 values (>230 μM NO), relative their slow-releasing counterparts (<230 μM NO), across 9 different cancer cell lines encompassing 3 different types of cancer (breast, colorectal, and pancreatic). The fast-release system was able to eradicate 50% of the cells much quicker (~36 h vs. 72 h for slow-release system). Flow cytometry studies suggest that this faster killing is due to a more rapid intracellular build-up of NO, which was observed for both the free and encapsulated NO donors. Western blotting revealed that both the slow and fast NO-release systems could induce apoptosis, albeit to different degrees.
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  • In Copyright
Advisor
  • Schoenfisch, Mark H.
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2017
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