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The Effect of Calcium Interaction and Mineralization on the Properties of Poly(Aspartic Acid) Modified Polyplexes for Gene Delivery

  • Author / Creator
    Dick, Teo Atz
  • Mineralization is frequently studied as a biomimetic process, owing to the sophisticated architectures and superior mechanical properties of the resultant materials. Bone is a classical example – a nanostructured material with a complex hierarchical architecture that is capable of resisting heavy loads and supporting the biological function necessary for its maintenance. Arguably, gene delivery could also be approached biomimetically, as viruses are the most efficient cell transfecting agents, selected by nature to deliver its genetic material inside cells. The study of synthetic gene carriers as virus inspired materials might therefore bring new ideas for nanoparticle fabrication in gene related applications.
    Recently, an unexpected property from viruses have been identified as an evolutionary advantage: the ability to become mineralized. A mineralized virus is more robust, with superior chance of survival when not infecting a host. When infecting a host, better adhesion and protection against the immune system are believed to confer superior infectivity. The new properties from mineralized viruses were proven to be translatable to therapeutic applications with success, generating viral vaccines and gene vectors with superior thermostability, immunization capability and transfection efficiency. However, the use of viral vectors is still unsafe, with many human deaths reported during clinical trials. For this reason, many believe that non-viral vectors could replace viral vectors as a safer option.
    In this thesis, a route for the fabrication of a novel non-viral mineralized delivery system was proposed. The non-viral vector used was a polyplex system produced by the self-assembly of a low molecular weight, lipid-modified version of poly(ethylenimine) (PEI), considered by many as the gold standard in non-viral gene delivery. However, polyplexes of this type are highly positively charged and, as a consequence, non-mineralizable. Due to this fact, poly(aspartic acid) (PAsp), a polyanion commonly used in the study of biomimetic bone mineralization was used to mediate the mineralization of the polyplexes. The mineralization strategy used was tailored for short incubation times necessary for effective gene delivery with polyplexes. Detailed physicochemical studies of every step of the fabrication method proposed were carried with the goal of providing a deep understanding of the phenomena responsible for particle properties. As a result of the approach taken, it was found that not only mineralized polyplexes are promising as new vectors, but also ‘calcium incubated polyplexes’, a particle that arose as an intermediated step of the fabrication route proposed.
    It was found that mineralization with calcium carbonate and calcium phosphate are efficient in promoting transfection efficiency in vitro and increased robustness as long as a certain Ca2+ excess is given. Calcium incubation can be used to achieve similar effects in transfection efficiency at higher Ca2+ concentrations with lower achievement of robustness, depending on polyplex composition. It is proposed that the improved robustness and transfection efficiency provided by means of mineralization and calcium incubation in the presence of PAsp can be used to expand the possible applications of polyplexes in gene therapy.

  • Subjects / Keywords
  • Graduation date
    Fall 2022
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-dwkt-yj51
  • License
    This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.