Engineering Gene Vectors: Synthesis of Thioether-Lipid Polyethyleneimine Conjugates for pDNA and siRNA Delivery

• Author / Creator

  Pontillas, Shienna Marie A

• Gene therapy offers a promising approach for treatment of a diverse array of diseases at their genetic root causes. The cationic polyethyleneimine (PEI) has been extensively used for successful non-viral nucleic acid delivery in experimental systems, but its clinical utility has been hampered by its severe cytotoxicity. Thus, hydrophobic modification of low molecular weight (LMW) PEIs has been used to enhance transfection efficiency along with acceptable toxicity. This study conjugated PEI1.2k with aliphatic lipids lauric, palmitic and stearic acid via thioether linkages and probed their in-vitro performance for nucleic acid delivery in suspension cells. The 1H-NMR and FTIR confirmed the desired structural characteristics of the synthesized lipid-substituted polymers. Particle sizes of the pDNA polyplexes varied from 140+1 to 330+10 nm, with positive ζ-potentials (+4 to +12 mV). The modified LMW polyplexes demonstrated negligible toxicity (cell viabilities ~97%) at a polymer-to-pDNA mass ratio of 10. The extent of stearic and lauric acid substitution improved the in vitro transfection, outperforming 25 kDa branched-PEI and matching the efficacy of the commercial transfection agent LipofectamineTM 2000, with over 80% pDNA-positive cells. Additionally, the inclusion of the Trans-Booster additive in the polyplexes further enhanced the transfection efficiency. Likewise, the potential for delivering other anionic biomacromolecules, such as small interfering RNA (siRNA) for gene silencing, was explored. Lauric acid-grafted polyplexes with therapeutic siBCR-ABL achieved approximately 58% maximum cell growth inhibition in drug-resistant chronic myeloid leukemia (CML) cells and maintained this effect for 9 days, which is comparable to or exceeds the duration reported in previous studies. These findings underscore the versatility of lipid-PEI conjugates via thioether linkage as delivery vectors for pDNA and siRNA in suspension-growing CML K562 and Jurkat T-cells, a strategy not previously reported in the literature and thus, merit further investigation.

• Subjects / Keywords

  • Polyethyleneimine
  • gene therapy
  • non-viral
  • synthesis
  • transfection
  • cancer
  • pDNA
  • siRNA

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-ka2a-3a45

• 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.