Investigations of interactions between membrane and PEI/siRNA nanoparticles and their implication for non-viral gene delivery

• Author / Creator

  Nademi, Yousef

• Cell entry of polynucleotide-based therapeutic agents can be promoted by nanoparticle (NP) mediated delivery. This dissertation investigates membrane penetration of polynucleotide NPs, using mainly computational approaches, accompanied by some experiments. Major emphasis was placed on computational approaches to explore configurational changes and stability of polymer-polynucleotide NPs upon penetration into lipidic membranes at the all-atom level. The first part of our studies explored the stability and configurational changes of NPs formed by 6 polyethylenimine (PEI) and 2 siRNA molecules during penetration into the zwitterionic 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) membrane. For the zwitterionic membrane, we found that hydrogen bond formation between the PEIs and the membrane did not lead to instability of the polymer-polynucleotide NPs during the internalization process. Instead, our results suggested adoption of a “self-protecting” configuration by the NP during membrane penetration, where the NP become more compact and polynucleotides become aligned, leading to more stable configurations while detaching from the membrane. The polymer-polynucleotide NP modified with linoleic acid (LA) showed the smallest structural change due to its strong intra-particle lipid associations and the resulting rigidity, while NP modified with caprylic acid showed the largest structural changes. Next, in addition of zwitterionic membrane of POPC, anionic membrane of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) were also utilized. For the anionic membrane, our experiments showed that POPS liposomes interacted strongly with NPs, which caused partial dissociation of the NPs. Consistent with the experiments, steered molecular dynamics simulations (SMD) showed a stronger interaction between the NPs and POPS membrane as compared to the POPC membrane. Lipid substitution on the PEIs enhanced the stability of the NPs during membrane crossing; lipid association between PEIs of the LA-bearing NPs as well as parallel orientation of the siRNAs provided protection against their dissociation (unlike NPs from native PEI). Later on, deformation of lipid membranes and pore closure during a NP penetration process was studied. POPC, dipalmitoylphosphatidylcholine (DPPC) and dilauroylphosphatidylcholine (DLPC) lipidic membrane models were utilized. Our results showed that different membrane lipids could lead to differences in pore formation (symmetric vs. asymmetric), and could undergo different levels of pore-mediated flip-flops during the closure. DLPC showed the largest number of flip-flops among the three lipid membranes. In addition, introduction of hydrophobic LA substitution onto the PEIs was found to facilitate pore formation, since the long LA tails could insert themselves into the hydrophobic region of the membrane where the lipid tails were less aligned. Compared with DPPC, POPC and DLPC membranes had less alignment of lipid tails in the bilayer, which promoted the insertion of LA tails and hence NP entry into the cell. At the end, machine learning algorithms were employed on the basis of quantitative structure activity relationship (QSAR) method to predict the cellular uptake of hydrophobically modified PEI/siRNA nanoparticles (NPs) into various cancer cell lines. A dataset consisting of 197 datapoints along with 3 different regression models, namely random forest (RF), multi-layer perceptron (MLP) and linear regression (LR), were used. The results of this modeling showed that RF and MLP regression methods had a better performance than the LR method, suggesting that non-linear models were better estimators when predicting the cellular uptake of PEI/siRNA NPs. Additionally, critical descriptors that had major contributions to cellular uptake were found to be PEI-to-siRNA weight ratio, type of hydrophobic substitution, as well as total numbers of Cs, unsaturated C and thioester groups on substitutions in each PEI. This dissertation provides valuable insight into the various aspects of polymer-polynucleotide NPs as well as lipidic membranes which will help in the design of more effective carriers for nucleic acid delivery.

• Subjects / Keywords

  • Molecular Dynamics Simulations
  • Machine Learning
  • PEI
  • Nanoparticle

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-qk6h-j780

• License

  This thesis is made available by the University of Alberta Libraries 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.