Abstract
The tight binding of pDNA with a cationic polymer is the crucial requirement that prevents DNA degradation from undesired DNase attack to safely deliver the pDNA to its target site. However, cationic polymer-mediated strong gene holding limits pDNA dissociation from the gene complex, resulting in a reduction in transfection efficiency. In this study, to control the decomplexation rate of pDNA from the gene complex in a hard-to-transfect cell or an easy-to-transfect cell, either α-poly(l-lysine) (APL) or ε-poly(l-lysine) (EPL) was incorporated into branched polyethylenimine (bPEI)-based nanocomplexes (NCs). Compared to bPEI/pDNA NCs, the addition of APL or EPL formed smaller bPEI-APL/pDNA NCs with similar zeta potentials or larger bPEI-EPL/pDNA NCs with reduced zeta potentials, respectively, due to the different characteristics of the primary amines in the two poly(l-lysine)s (PLs). Interestingly, although both bPEI-APL/pDNA NCs and bPEI-EPL/pDNA NCs showed similar pDNA compactness to bPEI/pDNA NCs, the addition of APL or EPL resulted in slower or faster pDNA release, respectively, from the bPEI-PL/pDNA NCs than from the bPEI/pDNA NCs. bPEI-EPL/pDNA NCs with a decomplexation enhancer (i.e., EPL) improved the transfection efficiency (TE) in both a hard-to-transfect HepG2 cell and an easy-to-transfect HEK293 cell. However, although a decomplexation inhibitor (i.e., APL) reduced the TE of bPEI-APL/pDNA NCs in both cells, the degree of reduction in the TE could be compensated by PL-mediated enhanced nuclear delivery, particularly in HepG2 cells but not HEK293 cells, because both PLs facilitate nuclear localization of the gene complex per its cellular uptake. In conclusion, a decomplexation rate controller could be a potential factor to establish a high TE and design clinically available gene complex systems.
Highlights
As the significance of the application of genetic material for treating various disorders has increased, nanoscale nonviral gene complexes have been extensively and intensively investigated for the last three decades by using newly synthesized or naturally found gene carriers and by introducing functional moieties [1,2,3,4]
Prior to the evaluation of decomplexation of the branched polyethylenimine (bPEI)-PL/Plasmid DNA (pDNA) NCs, the different decomplexation rates of the PL-based NCs were reconfirmed because the experimental conditions in this study were different from those in a previous study [24]
a decomplexation inhibitor (APL)/pDNA NC did not show any decomplexed pDNA from the NC at 50 μg/mL heparin, whereas either α-poly(l-lysine) (APL) or ε-poly(l-lysine) (EPL)/pDNA NC partially released pDNA at 30 μg/mL heparin and substantial pDNA (>75%) was released at heparin concentrations greater than 35 μg/mL. These results indicate that APL has a strong electrostatic attraction to pDNA, unlike the weak pDNA electrostatic attraction of EPL, which is in agreement with previous results [24]
Summary
As the significance of the application of genetic material for treating various disorders has increased, nanoscale nonviral gene complexes have been extensively and intensively investigated for the last three decades by using newly synthesized or naturally found gene carriers and by introducing functional moieties [1,2,3,4]. The complexes (hydrophobic or cationic complexes) can bind with plasma proteins (e.g., albumin and opsonins) or can be scavenged by phagocytic cells in blood These rate-limiting steps consecutively occur at the cellular and intracellular levels and are comprised of noncontact or contact cellular interactions, cellular internalization (e.g., endocytosis, membrane penetration), endosomal/endolysosomal/lysosomal escape, intracellular trafficking, nuclear or mitochondrial translocation (depending on the subcellular gene delivery target), and unpacking of gene complexes and gene release from the complexes [2,4,5]. After the resultant ternary bPEI-APL/pDNA and bPEI-EPL/pDNA complexes were constructed, their physicochemical characteristics (e.g., size, zeta potentials, gene condensation) and biological characteristics (e.g., cellular uptake, nuclear uptake, transfection efficiency) in HEK293 or HepG2 cells were investigated
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