Polyethylenimine-graft-Poly(ethylene glycol) Copolymers:  Influence of Copolymer Block Structure on DNA Complexation and Biological Activities as Gene Delivery System

Abstract

For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology: PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 +/- 59 to 61 +/- 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 +/- 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 +/- 60 nm). The zeta-potential of complexes was reduced with increasing degree of PEG grafting if MW >or= 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution: all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.

Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes

“…PEI-DNA complexes had a disperse distribution of condensates with rounded, globular forms [54,55]. PEGylated PEI-DNA complexes analyzed by AFM had defined, spherical complexes [30][31][32], with less aggregation and smaller diameters than similar complexes without PEG, but were also demonstrated to be less uniform.…”

“…PEG incorporated into DNA complexes of cationic polymers can reduce their surface charge [25,26,31,34], cytotoxicity [25,27,31] and interaction with blood components [25], as well as prevent aggregation through steric stabilization [25,26,[29][30][31]33,34]. Because of its ability to modulate the properties of complexes, PEG has been reported to enhance transfection when attached to polymer-DNA complexes directly [24,27,33,34] or simply added to transfection media containing lipoplexes [49].…”

“…Polyethylene glycol (PEG), which has the monomeric repeat unit [-CH 2 -CH 2 -O]-, is widely used in drug and gene delivery and has been incorporated into DNA complexes of several cationic polymers, including polymethacrylate [23], PEI [24][25][26][27][28][29][30][31][32], PLL [33][34][35] and poly(amidoamine)s [36]. PEG reduces the surface charge of the complexes [25,26,31,34], which in turn reduces cytotoxicity [25,27,31]. The shielding effect of PEG also reduces the interaction between the complex and blood components (plasma proteins and erythrocytes) [25], and can prolong circulation of the complexes in the blood stream [25,32].…”

“…The shielding effect of PEG also reduces the interaction between the complex and blood components (plasma proteins and erythrocytes) [25], and can prolong circulation of the complexes in the blood stream [25,32]. Furthermore, polyethylene glycosylation (PEGylation) can prevent salt-induced aggregation through steric stabilization [25,26,[29][30][31]33,34]. Additionally, PEG is often used as a spacer for targeting ligands since the shielding effect of PEG is able to decrease nonspecific interactions with negatively charged cellular membranes, which results in reduction of nonspecific cellular uptake [37].…”

“…Additionally, PEG is often used as a spacer for targeting ligands since the shielding effect of PEG is able to decrease nonspecific interactions with negatively charged cellular membranes, which results in reduction of nonspecific cellular uptake [37]. While some PEGylation strategies have had no effect on transfection efficiency in vitro [25,31,33] or in vivo [25], or even enhanced transfection [27,34], others have reported that PEGylation resulted in poor transfection [28,29,32], presumably due to interference with complexation [36]. These effects due to PEGylation have been associated with the extent of PEGylation, which may shield the surface charge [24,26], thus reducing cell binding and transfection, or alternatively, induce membrane leakage, resulting in enhanced cytoplasmic release [33,34].…”