Event Abstract Back to Event A delicate balance when substituting a small hydrophobe onto low molecular weight polyethylenimine to improve its nucleic acid delivery efficiency Deniz Meneksedag-Erol1, 2*, Remant Bahadur Kc2, Tian Tang1, 3* and Hasan Uludag1, 2, 4* 1 University of Alberta, Department of Biomedical Engineering, Canada 2 University of Alberta, Department of Chemical & Materials Engineering, Canada 3 University of Alberta, Department of Mechanical Engineering, Canada 4 University of Alberta, Faculty of Pharmacy and Pharmaceutical Sciences, Canada Introduction: High molecular weight polyethylenimine (PEI) is an effective non-viral gene carrier extensively studied in gene therapies. However, its considerable amount of toxicity encouraged a search for non-toxic and non-immunogenic alternatives. Due to the low toxicity profile of low molecular weight (LMW) PEI, research has focused on improving its performance via hydrophobic modifications. In this study, we systematically investigated, via experiments and molecular dynamics simulations, how the hydrophobic modification of LMW (1.2 kDa) PEI with a short propionic acid (PrA) influences the complexation mechanism of short interfering RNA (siRNA), as well as transfection efficacy and silencing capability of polyplexes. Materials and Methods: 1.2 kDa PEI was modified with PrA via N-acylation, a similar procedure described in[1]. Composition of modified PEIs was analyzed by 1H-NMR. The PrA substitution amounts were α = 0.26, 0.69, 1.14 and 1.59 PrA/PEI. A chronic myeloid leukemia K562 cells was used as a cell model. siRNA delivery efficacy was assessed by flow cytometry with FAM-labeled siRNA. Green fluorescence protein (GFP) silencing in GFP-expressing K562 cells was investigated for silencing activity. For computational studies, a branched PEI (MW: 1205 Da) and an siRNA sequence against myeloid cell leukemia 1 (Mcl-1) were used. Four systems were designed with varying PrA substitutions; α = 0.25, 0.75, 1 and 2. Simulations were performed with NAMD[2] for 200 ns, and trajectories were analyzed with Visual Molecular Dynamics (VMD). Results and Discussion: Substitution of PrA at low amounts, α = 0.26 and 0.69, resulted in substantially higher (>10 fold) delivery than native PEI. Increasing the substitution amount α further to 1.14 and 1.59 abolished the uptake efficiency of siRNA (Figure 1a). Native PEI (α = 0) showed ~5% silencing, whereas α = 0.26 and α = 0.69 yielded ~10% and ~33% silencing, respectively. Further increase in the substitution extent decreased siRNA silencing down to 4% (Figure 1b). MD simulations on radial distribution function of the PrA Cs from the center of mass of polyplexes revealed a new assembly mechanism: preferential mobilization of PrA moieties to the core at high substitution. This caused a non-monotonic change in the surface PrA density of the polyplexes (Figure 2a), with α = 1 having the most hydrophobic surface. Migration of PrAs into the core dragged the associated PEIs, and caused higher cationic surface charge at intermediate substitutions (α = 0.75), while further increasing the substitution significantly reduced the surface cationic charge density (Figure 2b). Conclusions: Small amount of hydrophobic modification is beneficial to enhance the surface hydrophobicity of the polyplexes; while excess hydrophobic groups resulted in preferential mobilization of these groups to the polyplex core, lowering the surface hydrophobicity as well as cationic charge. This phenomenon results in less effective polyplexes for siRNA delivery. Compute Canada; National Institute for Nanotechnology, Edmonton, Canada; Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research; NSERC CREATE Program for Regenerative Medicine (NCPRM); Alberta Innovates-Technology Futures; Canada Foundation for Innovation
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Feb 16, 2017
Robert C Deller + 9
Open Access
